blister and blebbistatin

Optical mapping is an imaging technique that is extensively used in cardiovascular research, wherein parameter-sensitive fluorescent indicators are used to study the electrophysiology and excitation-contraction coupling of cardiac tissues. Despite many benefits of optical mapping, eliminating motion artifacts within the optical signals is a major challenge, as myocardial contraction interferes with the faithful acquisition of action potentials and intracellular calcium transients. As such, excitation-contraction uncoupling agents are frequently used to reduce signal distortion by suppressing contraction. When compared with other uncoupling agents, blebbistatin is the most frequently used, as it offers increased potency with minimal direct effects on cardiac electrophysiology. Nevertheless, blebbistatin may exert secondary effects on electrical activity, metabolism, and coronary flow, and the incorrect administration of blebbistatin to cardiac tissue can prove detrimental, resulting in erroneous interpretation of optical mapping results. In this "Getting It Right" perspective, we briefly review the literature regarding the use of blebbistatin in cardiac optical mapping experiments, highlight potential secondary effects of blebbistatin on cardiac electrical activity and metabolic demand, and conclude with the consensus of the authors on best practices for effectively using blebbistatin in optical mapping studies of cardiac tissue.

Topics: Action Potentials; Animals; Artifacts; Biomedical Research; Cells, Cultured; Excitation Contraction Coupling; Heart Rate; Heterocyclic Compounds, 4 or More Rings; Humans; Myocardial Contraction; Myocytes, Cardiac; Time Factors; Voltage-Sensitive Dye Imaging

Memories associated with substance use disorders, or substance-associated cues increase the likelihood of craving and relapse during abstinence. There is a growing consensus that manipulation of synaptic plasticity may reduce the strength of substance abuse-related memories. On the biological front, there are new insights that suggest memories associated with substance use disorder may follow unique neurobiological pathways that render them more accessible to pharmacological intervention. In parallel to this, research in neurochemistry has identified several potential candidate molecules that could influence the formation and maintenance of long-term memory. Drugs that target these molecules (blebbistatin, isradipine and zeta inhibitory peptide) have shown promise at the preclinical stage. In this review, we shall provide an overview of the evolving understanding on the biochemical mechanisms involved in memory formation and expound on the premise that substance use disorder is a learning disorder.

Topics: Cues; Heterocyclic Compounds, 4 or More Rings; Humans; Isradipine; Memory, Long-Term; Molecular Targeted Therapy; Neuronal Plasticity; Substance-Related Disorders

( S)-Blebbistatin, a chiral tetrahydropyrroloquinolinone, is a widely used and well-characterized ATPase inhibitor selective for myosin II. The central role of myosin II in many normal and pathological biological processes has been revealed with the aid of this small molecule. The first part of this manuscript provides a summary of myosin II and ( S)-blebbistatin literature from a medicinal chemist's perspective. The second part of this perspective deals with the physicochemical deficiencies that trouble the use of ( S)-blebbistatin in advanced biological settings: low potency and solubility, fluorescence interference, (photo)toxicity, and stability issues. A large toolbox of analogues has been developed in which particular shortcomings have been addressed. This perspective provides a necessary overview of these developments and presents guidelines for selecting the best available analogue for a given application. As the unmet need for high-potency analogues remains, we also propose starting points for medicinal chemists in search of nanomolar myosin II inhibitors.

Topics: Animals; Chemistry, Pharmaceutical; Drug Discovery; Heterocyclic Compounds, 4 or More Rings; Humans; Myosin Type II

Blebbistatin is a widely used inhibitor of myosin 2 that enables the study of a broad range of cytoskeleton-related processes. However, blebbistatin has several limitations hindering its applicability: it is fluorescent, poorly water soluble, cytotoxic, and prone to (photo)degradation. Despite these adverse effects, being the only available myosin 2-specific inhibitor, blebbistatin is rather a choice of necessity. Blebbistatin has been modified to improve its properties and some of the new compounds have proven to be useful replacements of the original molecule. This review summarizes recent results on blebbistatin development. We also discuss the pharmacological perspectives of these efforts, as myosins are becoming promising drug target candidates for a variety of conditions ranging from neurodegeneration to muscle disease, wound healing, and cancer metastasis.

Topics: Animals; Drug Delivery Systems; Heterocyclic Compounds, 4 or More Rings; Humans; Muscular Diseases; Myosins; Neoplasm Metastasis; Neoplasms; Neurodegenerative Diseases; Wound Healing

Topics: Actin Cytoskeleton; Animals; Heterocyclic Compounds, 4 or More Rings; Muscle Contraction; Muscle, Smooth; Ultrasonography; Vascular Diseases

Fluorescent reporters of cardiac electrophysiology provide valuable information on heart cell and tissue function. However, motion artifacts caused by cardiac muscle contraction interfere with accurate measurement of fluorescence signals. Although drugs such as blebbistatin can be applied to stop cardiac tissue from contracting by uncoupling calcium-contraction, their usage prevents the study of excitation-contraction coupling and, as we show, impacts cellular structure. We therefore developed a robust method to remove motion computationally from images of contracting cardiac muscle and to map fluorescent reporters of cardiac electrophysiological activity onto images of undeformed tissue. When validated on cardiomyocytes derived from human induced pluripotent stem cells (iPSCs), in both monolayers and engineered tissues, the method enabled efficient and robust reduction of motion artifact. As with pharmacologic approaches using blebbistatin for motion removal, our algorithm improved the accuracy of optical mapping, as demonstrated by spatial maps of calcium transient decay. However, unlike pharmacologic motion removal, our computational approach allowed direct analysis of calcium-contraction coupling. Results revealed calcium-contraction coupling to be more uniform across cells within engineered tissues than across cells in monolayer culture. The algorithm shows promise as a robust and accurate tool for optical mapping studies of excitation-contraction coupling in heart tissue.

Topics: Artifacts; Calcium; Calcium, Dietary; Coloring Agents; Humans; Induced Pluripotent Stem Cells; Myocytes, Cardiac; Software

Topics: Artifacts; Computers; Humans; Myocytes, Cardiac; Paralysis; Software

Photocages can provide spatial and temporal control to accurately release the various chemicals and bioactive groups when excited by light. Although the absorption spectra of most photocages are in the ultraviolet absorption region, only a few absorb in the visible or near-infrared region. Blebbistatin (

Topics: Halogens; Heterocyclic Compounds, 4 or More Rings; Humans; Infrared Rays; Photolysis

Acute lung injury (ALI) or its most advanced form, acute respiratory distress syndrome (ARDS), is a severe inflammatory pulmonary process triggered by varieties of pathophysiological factors, among which endothelial barrier disruption plays a critical role in the progression of ALI/ARDS. As an inhibitor of myosin II, blebbistatin inhibits endothelial barrier damage. This study aimed to investigate the effect of blebbistatin on lung endothelial barrier dysfunction in LPS induced acute lung injury and its potential mechanism. Mice were challenged with LPS (5 mg/kg) by intratracheal instillation for 6 h to disrupt the pulmonary endothelial barrier in the model group. Blebbistatin (5 mg/kg, ip) was administrated 1 h before LPS challenge. The results showed that blebbistatin could significantly attenuate LPS-induced lung injury and pulmonary endothelial barrier dysfunction. And we observed that blebbistatin inhibited the activation of NMMHC IIA/Wnt5a/β-catenin pathway in pulmonary endothelium after LPS treatment. In murine lung vascular endothelial cells (MLECs) and human umbilical vein endothelial cells (HUVECs), we further confirmed that Blebbistatin (1 μmol/L) markedly ameliorated endothelial barrier dysfunction in MLECs and HUVECs by modulating NMMHC IIA/Wnt5a/β-catenin pathway. Our data demonstrated that blebbistatin could inhibit the development of pulmonary endothelial barrier dysfunction and ALI via NMMHC IIA/Wnt5a/β-catenin signaling pathway.

Topics: Acute Lung Injury; Animals; beta Catenin; Endothelium; Heterocyclic Compounds, 4 or More Rings; Human Umbilical Vein Endothelial Cells; Humans; Lipopolysaccharides; Lung; Mice; Mice, Inbred C57BL; Myosin Type II; Respiratory Distress Syndrome; Wnt-5a Protein

Ex-vivo simple models are powered tools to study cardiac hypertrophy. It is possible to control the activation of critical genes and thus test the effects of drug therapies before the in vivo tests. A zebrafish cardiac hypertrophy developed by 500 μM phenylephrine (PE) treatment in ex vivo culture has been demonstrated to activate the essential expression of the embryonal genes. These genes are the same as those described in several previous pieces of research on hypertrophic pathology in humans. The efficacy of the chemical drug Blebbistatin (BL) on hypertrophy induced ex vivo cultured hearts is studied in this research. BL can inhibit the myosins and the calcium wave in counteracting the hypertrophy status caused by PE. Samples treated with PE, BL and PE simultaneously, or pre/post-treatment with BL, have been analysed for the embryonal gene activation concerning the hypertrophy status. The qRTPCR has shown an inhibitory effect of BL treatments on the microRNAs downregulation with the consequent low expression of essential embryonal genes. In particular, BL seems to be effective in blocking the hyperplasia of the epicardium but less effective in myocardium hypertrophy. The model can make it possible to obtain knowledge on the transduction pathways activated by BL and investigate the potential use of this drug in treating cardiac hypertrophy in humans.

Topics: Animals; Cardiomegaly; Heterocyclic Compounds, 4 or More Rings; Humans; Pericardium; Phenylephrine; Zebrafish

The formation of neuron networks is a complex phenomenon of fundamental importance for understanding the development of the nervous system, and for creating novel bioinspired materials for tissue engineering and neuronal repair. The basic process underlying the network formation is axonal growth, a process involving the extension of axons from the cell body towards target neurons. Axonal growth is guided by environmental stimuli that include intercellular interactions, biochemical cues, and the mechanical and geometrical features of the growth substrate. The dynamics of the growing axon and its biomechanical interactions with the growing substrate remains poorly understood. In this paper, we develop a model of axonal motility which incorporates mechanical interactions between the axon and the growth substrate. We combine experimental data with theoretical analysis to measure the parameters that describe axonal growth on surfaces with micropatterned periodic geometrical features: diffusion (cell motility) coefficients, speed and angular distributions, and axon bending rigidities. Experiments performed on neurons treated Taxol (inhibitor of microtubule dynamics) and Blebbistatin (disruptor of actin filaments) show that the dynamics of the cytoskeleton plays a critical role in the axon steering mechanism. Our results demonstrate that axons follow geometrical patterns through a contact-guidance mechanism, in which high-curvature geometrical features impart high traction forces to the growth cone. These results have important implications for our fundamental understanding of axonal growth as well as for bioengineering novel substrates that promote neuronal growth and nerve repair.

Topics: Animals; Biomechanical Phenomena; Cell Culture Techniques; Cell Movement; Cells, Cultured; Growth Cones; Heterocyclic Compounds, 4 or More Rings; Neurons; Paclitaxel; Rats

Myosin IIs, actin-based motors that utilize the chemical energy of adenosine 5'-triphosphate (ATP) to generate force, have potential as therapeutic targets. Their heavy chains differentiate the family into muscle (skeletal [SkMII], cardiac, smooth) and nonmuscle myosin IIs. Despite the therapeutic potential for muscle disorders, SkMII-specific inhibitors have not been reported and characterized. Here, we present the discovery, synthesis, and characterization of "skeletostatins," novel derivatives of the pan-myosin II inhibitor blebbistatin, with selectivity 40- to 170-fold for SkMII over all other myosin II family members. In addition, the skeletostatins bear improved potency, solubility, and photostability, without cytotoxicity. Based on its optimal

Topics: Animals; Heterocyclic Compounds, 4 or More Rings; Mice; Molecular Structure; Muscle, Skeletal; Myosin Type II

Recently, there have been great advances in cardiovascular channelopathy modeling and drug safety pharmacology using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The automated patch-clamp (APC) technique overcomes the disadvantages of the manual patch-clamp (MPC) technique, which is labor intensive and gives low output. However, the application of the APC platform is still limited in iPSC-CM based research, due to the difficulty in maintaining the high quality of single iPSC-CMs during dissociation and recording. In this study, we improved the method for single iPSC-CM preparation by applying 2.5 µM blebbistatin (BB, an excitation-contraction coupling uncoupler) throughout APC procedures (dissociation, filtration, storage, and recording). Under non-BB buffered condition, iPSC-CMs in suspension showed a severe bleb-like morphology. However, BB-supplement led to significant improvements in morphology and I

Topics: Heterocyclic Compounds, 4 or More Rings; Humans; Induced Pluripotent Stem Cells; Myocytes, Cardiac; Patch-Clamp Techniques

Organization of intracellular content is affected by multiple simultaneous processes, including diffusion in a viscoelastic and structured environment, intracellular mechanical work and vibrations. The combined effects of these processes on intracellular organization are complex and remain poorly understood. Here, we studied the organization and dynamics of a free Ca

Topics: Adenosine Triphosphate; Aniline Compounds; Heterocyclic Compounds, 4 or More Rings; Humans; Jurkat Cells; Myosin Type II; Organelles; Xanthenes

During an infection, Cauliflower mosaic virus (CaMV) forms inclusion bodies (IBs) mainly composed of viral protein P6, where viral activities occur. Because viral processes occur in IBs, understanding the mechanisms by which they are formed is crucial. FL-P6 expressed in N. benthamiana leaves formed IBs of a variety of shapes and sizes. Small IBs were dynamic, undergoing fusion/dissociation events. Co-expression of actin-binding polypeptides with FL-P6 altered IB size distribution and inhibited movement. This suggests that IB movement is required for fusion and growth. A P6 deletion mutant was discovered that formed a single large IB per cell, which suggests it exhibited altered fusion/dissociation dynamics. Myosin-inhibiting drugs did not affect small IB movement, while those inhibiting actin polymerization did. Large IBs colocalized with components of the aggresome pathway, while small ones generally did not. This suggests a possible involvement of the aggresome pathway in large IB formation.

Topics: Actin Cytoskeleton; Caulimovirus; Cell Membrane; Coiled Bodies; Diacetyl; Heterocyclic Compounds, 4 or More Rings; Inclusion Bodies, Viral; Microfilament Proteins; Mutation; Nicotiana; Plant Leaves; Protein Domains; Trans-Activators

Benign prostatic hyperplasia (BPH) is a common disease among aging males with the etiology remaining unclear. We recently found myosin II was abundantly expressed in rat and cultured human prostate cells with permissive roles in the dynamic and static components. The present study aimed to explore the expression and functional activities of myosin II isoforms including smooth muscle (SM) myosin II (SMM II) and non-muscle myosin II (NMM II) in the hyperplastic prostate. Human prostate cell lines and tissues from normal human and BPH patients were used. Hematoxylin and Eosin (H&E), Masson's trichrome, immunohistochemical staining, in vitro organ bath, RT-polymerase chain reaction (PCR) and Western-blotting were performed. We further created cell models with NMM II isoforms silenced and proliferation, cycle, and apoptosis of prostate cells were determined by cell counting kit-8 (CCK-8) assay and flow cytometry. Hyperplastic prostate SM expressed more SM1 and LC17b isoforms compared with their alternatively spliced counterparts, favoring a slower more tonic-type contraction and greater force generation. For BPH group, blebbistatin (BLEB, a selective myosin II inhibitor), exhibited a stronger effect on relaxing phenylephrine (PE) pre-contracted prostate strips and inhibiting PE-induced contraction. Additionally, NMMHC-A and NMMHC-B were up-regulated in hyperplastic prostate with no change in NMMHC-C. Knockdown of NMMHC-A or NMMHC-B inhibited prostate cell proliferation and induced apoptosis, with no changes in cell cycle. Our novel data demonstrate that expression and functional activities of myosin II isoforms are altered in human hyperplastic prostate, suggesting a new pathological mechanism for BPH. Thus, the myosin II system may provide potential new therapeutic targets for BPH/lower urinary tract symptoms (LUTS).

Topics: Adult; Aged; Apoptosis; Case-Control Studies; Cell Line; Cell Proliferation; Gene Expression Regulation; Heterocyclic Compounds, 4 or More Rings; Humans; Male; Muscle, Smooth; Myosin Heavy Chains; Myosin Type II; Nonmuscle Myosin Type IIB; Prostate; Prostatic Hyperplasia; Protein Isoforms; Signal Transduction

Blebbistatin,

Topics: Absorption, Physicochemical; Animals; Drug Discovery; Heterocyclic Compounds, 4 or More Rings; Humans; Molecular Dynamics Simulation; Myosins; Protein Conformation; Rats; Tissue Distribution

Correlated cell migration in fibrous extracellular matrix (ECM) is important in many biological processes. During migration, cells can remodel the ECM, leading to the formation of mesoscale structures such as fiber bundles. However, how such mesoscale structures regulate correlated single-cells migration remains to be elucidated. Here, using a quasi-3D in vitro model, we investigate how collagen fiber bundles are dynamically re-organized and guide cell migration. By combining laser ablation technique with 3D tracking and active-particle simulations, we definitively show that only the re-organized fiber bundles that carry significant tensile forces can guide strongly correlated cell migration, providing for the first time a direct experimental evidence supporting that matrix-transmitted long-range forces can regulate cell migration and self-organization. This force regulation mechanism can provide new insights for studies on cellular dynamics, fabrication or selection of biomedical materials in tissue repairing, and many other biomedical applications.

Topics: Actins; Animals; Cell Movement; Collagen; Dogs; Extracellular Matrix; Heterocyclic Compounds, 4 or More Rings; Humans; Madin Darby Canine Kidney Cells; Mechanotransduction, Cellular; Myosins; Paxillin; Tensile Strength

Non-muscle myosin II (NMII) plays a role in many fundamental cellular processes including cell adhesion, migration, and cytokinesis. However, its role in mammalian vascular function is not well understood. Here, we investigated the function of NMII in the biomechanical and signalling properties of mouse aorta. We found that blebbistatin, an inhibitor of NMII, decreases agonist-induced aortic stress and stiffness in a dose-dependent manner. We also specifically demonstrate that in freshly isolated, contractile, aortic smooth muscle cells, the non-muscle myosin IIA (NMIIA) isoform is associated with contractile filaments in the core of the cell as well as those in the non-muscle cell cortex. However, the non-muscle myosin IIB (NMIIB) isoform is excluded from the cell cortex and colocalizes only with contractile filaments. Furthermore, both siRNA knockdown of NMIIA and NMIIB isoforms in the differentiated A7r5 smooth muscle cell line and blebbistatin-mediated inhibition of NM myosin II suppress agonist-activated increases in phosphorylation of the focal adhesion proteins FAK Y925 and paxillin Y118. Thus, we show in the present study, for the first time that NMII regulates aortic stiffness and stress and that this regulation is mediated through the tension-dependent phosphorylation of the focal adhesion proteins FAK and paxillin.

Topics: Actins; Animals; Biomarkers; Cells, Cultured; Cytoskeleton; Fluorescent Antibody Technique; Focal Adhesions; Heterocyclic Compounds, 4 or More Rings; Immunohistochemistry; Male; Mice; Muscle Contraction; Myocytes, Smooth Muscle; Myosin Type II; Phosphorylation; Protein Binding; Protein Isoforms; Protein Multimerization; Stress, Mechanical; Vascular Stiffness

Fibrin hydrogel is a central biological material in tissue engineering and drug delivery applications. As such, fibrin is typically combined with cells and biomolecules targeted to the regenerated tissue. Previous studies have analyzed the release of different molecules from fibrin hydrogels; however, the effect of embedded cells on the release profile has yet to be quantitatively explored. This study focused on the release of Fluorescein isothiocyanate (FITC)-dextran (FD) 250 kDa from fibrin hydrogels, populated with different concentrations of fibroblast or endothelial cells, during a 48-h observation period. The addition of cells to fibrin gels decreased the overall release by a small percentage (by 7-15% for fibroblasts and 6-8% for endothelial cells) relative to acellular gels. The release profile was shown to be modulated by various cellular activities, including gel degradation and physical obstruction to diffusion. Cell-generated forces and matrix deformation (i.e., densification and fiber alignment) were not found to significantly influence the release profiles. This knowledge is expected to improve fibrin integration in tissue engineering and drug delivery applications by enabling predictions and ways to modulate the release profiles of various biomolecules.

Topics: Animals; Cell Survival; Dextrans; Drug Delivery Systems; Endothelial Cells; Extracellular Matrix; Fibrin; Fluorescein-5-isothiocyanate; Heterocyclic Compounds, 4 or More Rings; Human Umbilical Vein Endothelial Cells; Humans; Hydrogels; Mice; Models, Theoretical; NIH 3T3 Cells; Regeneration; Regenerative Medicine; Tissue Engineering

Osteocytes differentiated from osteoblasts play significant roles as mechanosensors in modulating the bone remodeling process. While the well-aligned osteocyte network along the trabeculae with slender cell processes perpendicular to the trabeculae surface is known to facilitate the sensing of mechanical stimuli by cells and the intracellular communication in the bone matrix, the mechanisms underlying osteocyte network formation remains unclear. Here, we developed a novel in vitro collagen matrix system exerting a uniaxially-fixed mechanical boundary condition on which mouse osteoblast-like MC3T3-E1 cells were subcultured, evoking cellular alignment along the uniaxial boundary condition. Using a myosin II inhibitor, blebbistatin, we showed that the intracellular tension via contraction of actin fibers contributed to the cellular alignment under the influence of isometric matrix condition along the uniaxially-fixed mechanical boundary condition. Furthermore, the cells actively migrated inside the collagen matrix and promoted the expression of osteoblast and osteocyte genes with their orientations aligned along the uniaxially-fixed boundary condition. Collectively, our results suggest that the intracellular tension of osteoblasts under a uniaxially-fixed mechanical boundary condition is one of the factors that determines the osteocyte alignment inside the bone matrix.

Topics: Animals; Biomechanical Phenomena; Cell Culture Techniques; Cell Line; Cell Movement; Collagen; Heterocyclic Compounds, 4 or More Rings; Mice; Models, Biological; Osteoblasts; Osteogenesis

The knowledge of cell mechanics is required to understand cellular processes and functions, such as the movement of cells, and the development of tissue engineering in cancer therapy. Cell mechanical properties depend on a variety of factors, such as cellular environments, and may also rely on external factors, such as the ambient temperature. The impact of temperature on cell mechanics is not clearly understood. To explore the effect of temperature on cell mechanics, we employed magnetic tweezers to apply a force of 1 nN to 4.5 µm superparamagnetic beads. The beads were coated with fibronectin and coupled to human epithelial breast cancer cells, in particular MCF-7 and MDA-MB-231 cells. Cells were measured in a temperature range between 25 and 45 °C. The creep response of both cell types followed a weak power law. At all temperatures, the MDA-MB-231 cells were pronouncedly softer compared to the MCF-7 cells, whereas their fluidity was increased. However, with increasing temperature, the cells became significantly softer and more fluid. Since mechanical properties are manifested in the cell's cytoskeletal structure and the paramagnetic beads are coupled through cell surface receptors linked to cytoskeletal structures, such as actin and myosin filaments as well as microtubules, the cells were probed with pharmacological drugs impacting the actin filament polymerization, such as Latrunculin A, the myosin filaments, such as Blebbistatin, and the microtubules, such as Demecolcine, during the magnetic tweezer measurements in the specific temperature range. Irrespective of pharmacological interventions, the creep response of cells followed a weak power law at all temperatures. Inhibition of the actin polymerization resulted in increased softness in both cell types and decreased fluidity exclusively in MDA-MB-231 cells. Blebbistatin had an effect on the compliance of MDA-MB-231 cells at lower temperatures, which was minor on the compliance MCF-7 cells. Microtubule inhibition affected the fluidity of MCF-7 cells but did not have a significant effect on the compliance of MCF-7 and MDA-MB-231 cells. In summary, with increasing temperature, the cells became significant softer with specific differences between the investigated drugs and cell lines.

Topics: Actins; Biomechanical Phenomena; Breast Neoplasms; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Demecolcine; Female; Fibronectins; Heterocyclic Compounds, 4 or More Rings; Humans; Magnetic Iron Oxide Nanoparticles; MCF-7 Cells; Microtubules; Temperature; Thiazolidines

Simple and consistent chiral HPLC methods for the efficient separation of enantiomeric blebbistatin derivatives, namely parent compound blebbistatin and derivatives 4-nitroblebbistatin, 4-aminoblebbistatin, 4-dimethylaminoblebbistatin, and 4-t-butylblebbistatin were developed using cellulose tris(3,5-dimethylphenylcarbamate) as a stationary phase (Lux cellulose-1 column). Blebbistatin, 4-aminoblebbistatin, and 4-dimethylaminoblebbistatin racemates were well-separated in normal-phase HPLC conditions while 4-nitroblebbistatin and 4-t-butylblebbistatin were effectively separated in both normal- and reversed-phase HPLC conditions. Furthermore, the order of elution of enantiopure compounds was found to be independent of mobile phase compositions and conditions used, and solely depends on the interaction between the enantiomer and the chiral stationary phase. We found that despite the chiral center being present far from the D-ring in the blebbistatin structure, the D-ring substitutions prominently affect the chiral separation. Ex vivo racemization studies of the most popular blebbistatin derivative (S)-(-)-4-aminoblebbistatin in rat blood and brain tissues revealed that the compound does not convert into the inactive enantiomer. This confirms that (S)-(-)-4-aminoblebbistatin is a useful tool compound in cellular and molecular biology studies without the risks of racemization and degradation effects.

Topics: Animals; Chromatography, High Pressure Liquid; Heterocyclic Compounds, 4 or More Rings; Rats; Stereoisomerism; Vertebrates

The oxygen-dependent nature and limited penetration capacity of visible light render the low efficiency of photodynamic therapy in hypoxic and deep-seated tumors. Therefore, the development of oxygen-free photoactivated chemotherapy (PACT) to generate cytotoxic reactive oxygen species by near-IR (NIR) light-cleavable photocages is in high demand. Here, an oxygen-irrelevant PACT strategy based on NIR light-triggered hydroxyl radicals (•OH) generation is developed for free-radical nanotherapy. Blebbistatin-loaded upconversion of mesoporous silica nanoparticles (UCSNs-B) is established to facilitate the high loading efficiency of blebbistatin and implement the efficient transformation of NIR light into blue light for unprecedented direct photorelease of oxygen-independent •OH. Under NIR laser irradiation, UCSNs-B converted NIR light into blue light, thus enabling the photocleavage of blebbistatin to induce the burst of •OH. The •OH burst under NIR laser irradiation further induces cancer cell apoptosis and significant suppression of hypoxic tumors. In addition, the gadolinium ion (Gd

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Free Radicals; Gadolinium; Heterocyclic Compounds, 4 or More Rings; Humans; Infrared Rays; Magnetic Resonance Imaging; Mice; Nanomedicine; Nanoparticles; Neoplasms; Neoplasms, Experimental; Oxygen; Photochemotherapy; Reactive Oxygen Species; Silicon Dioxide

Although quiescent hepatic stellate cells (HSCs) have been suggested to regulate hepatic blood flow, there is no direct evidence that quiescent HSCs display contractile abilities. Here, we developed a new method to quantitatively measure the contraction of single isolated HSCs and evaluated whether endothelin-1 (ET-1) induced contraction of HSCs in a non-activated state. HSCs isolated from mice were seeded on collagen gel containing fluorescent beads. The beads around a single HSC were observed gravitating toward the cell upon contraction. By recording the movement of each bead by fluorescent microscopy, the real-time contraction of HSCs was quantitatively evaluated. ET-1 induced a slow contraction of non-activated HSCs, which was inhibited by the non-muscle myosin II inhibitor blebbistatin, the calmodulin inhibitor W-7, and the ETA receptor antagonist ambrisentan. ET-1-induced contraction was also largely reduced in Ca2+-free conditions, but sustained contraction still remained. The tonic contraction was further diminished by the Rho-kinase inhibitor H-1152. The mRNA expression of P/Q-type voltage-dependent Ca2+ channels (VDCC), as well as STIM and Orai, constituents of store-operated channels (SOCs), was observed in mouse non-activated HSCs. ET-1-induced contraction was not affected by amlodipine, a VDCC blocker, whereas it was partly reduced by Gd3+ and amiloride, non-selective cation channel blockers. However, neither YM-58483 nor SKF-96365, which inhibit SOCs, had any effects on the contraction. These results suggest that ET-1 leads to Ca2+-influx through cation channels other than SOCs and produces myosin II-mediated contraction of non-activated HSCs via ETA receptors, as well as via mechanisms involving Ca2+-calmodulin and Rho kinase.

Topics: Animals; Calcium; Calcium Channels, N-Type; Calmodulin; Cell Physiological Phenomena; Cells, Cultured; Endothelin Receptor Antagonists; Endothelin-1; Hepatic Stellate Cells; Heterocyclic Compounds, 4 or More Rings; Male; Mice; Myosin Type II; Phenylpropionates; Pyridazines; Receptor, Endothelin A; rho-Associated Kinases; RNA, Messenger; Signal Transduction; Sulfonamides

In the fabrication of cardiac tissue, an important factor is continuous measurement of its contraction features. A module that allows for a dynamic system capable of noninvasive and label-free monitoring of the contraction profile under administering chemicals and drugs is highly valuable for understanding accurate tissue mechanobiology. In this research, we have successfully demonstrated the use of surface plasmon resonance (SPR) technology for the first time to characterize the contractility of cardiac cells in response to Blebbistatin and ATP drug exposure in real tme. An optimal flow rate of 10 μL/min was selected for a continuous flow of warm media,and 10 μM drug administration effect was detected with high spatiotemporal sensitivity on contracting cardiomyocytes. Our drug screening has identified the source of the SPR periodic signal to be direct cell contraction rather than action potentials or calcium signaling. Per our results, SPR has high potential in applications in least-interference real-time and label-free tissue characterizations and cellular properties analysis from a functional and structural point of view.

Topics: Animals; Cells, Cultured; Heterocyclic Compounds, 4 or More Rings; Myocytes, Cardiac; Rats; Surface Plasmon Resonance; Time Factors

Arterial thrombosis (AT) causes various ischemia-related diseases, which impose a serious medical burden worldwide. As an inhibitor of myosin II, blebbistatin has an important role in thrombosis development. We investigated the effect of blebbistatin on carotid artery ligation (CAL)-induced carotid AT and its potential underlying mechanism. A model of carotid AT in mice was generated by CAL. Mice were divided into three groups: CAL model, blebbistatin-treated, and sham-operation. After 7 days, blood vessels were harvested from mice in each group. The procoagulant activity of tissue factor (TF) was tested by a chromogenic assay, and thrombus severity assessed by histopathology scores. Expression of non-muscle myosin heavy chain II A (NMMHCIIA), TF, glycogen synthase kinase 3β (GSK3β), and nuclear factor-kappa B (NF-κB) was detected by immunohistochemical and immunofluorescence staining. mRNA expression was measured by quantitative polymerase chain reaction. Blebbistatin (1 mg/kg) inhibited development of carotid AT, reduced infiltration of inflammatory cells, and prevented vascular-tissue damage, relative to the model group. Furthermore, blebbistatin also reduced the procoagulant activity of TF. Immunohistochemical and immunofluorescence data demonstrated that, compared with the model group, blebbistatin intervention reduced expression of NMMHCIIA, TF, GSK3β, p65, and p-p65 in carotid-artery endothelia in the CAL-induced AT model, but it increased levels of p-GSK3β. Blebbistatin could inhibit expression of NMMHCIIA mRNA in the CAL model. Overall, our data demonstrated that blebbistatin could inhibit TF expression and AT development in arterial endothelia (at least in part) via GSK3β/NF-κB signaling.

Topics: Animals; Arteries; Cytoskeletal Proteins; Glycogen Synthase Kinase 3 beta; Heterocyclic Compounds, 4 or More Rings; Male; Mice; Mice, Inbred C57BL; Myosin Type II; NF-kappa B; Signal Transduction; Thrombosis

Fibrosis is a complication of tendon injury where excessive scar tissue accumulates in and around the injured tissue, leading to painful and restricted joint motion. Unfortunately, fibrosis tends to recur after surgery, creating a need for alternative approaches to disrupt scar tissue. We posited a strategy founded on mechanobiological principles that collagen under tension generated by fibroblasts is resistant to degradation by collagenases. In this study, we tested the hypothesis that blebbistatin, a drug that inhibits cellular contractile forces, would increase the susceptibility of scar tissue to collagenase degradation. Decellularized tendon scaffolds (DTS) were treated with bacterial collagenase with or without external or cell-mediated internal tension. External tension producing strains of 2-4% significantly reduced collagen degradation compared with non-tensioned controls. Internal tension exerted by human fibroblasts seeded on DTS significantly reduced the area of the scaffolds compared to acellular controls and inhibited collagen degradation compared to free-floating DTS. Treatment of cell-seeded DTS with 50 mM blebbistatin restored susceptibility to collagenase degradation, which was significantly greater than in untreated controls (p < 0.01). These findings suggest that therapies combining collagenases with drugs that reduce cell force generation should be considered in cases of tendon fibrosis that do not respond to physiotherapy.

Topics: Collagen; Collagenases; Fibroblasts; Fibrosis; Heterocyclic Compounds, 4 or More Rings; Humans; Stress, Mechanical; Tendons; Tissue Scaffolds

Blebbistatin, a potent inhibitor of myosin II, is known to suppress smooth muscle contraction without affecting myosin light chain phosphorylation level. In order to clarify the regulatory mechanisms of blebbistatin on phasic and tonic smooth muscles in detail, we examined the effects of blebbistatin on relaxation process by Ca

Topics: Animals; Calcium; Cecum; Cell Membrane; Cells, Cultured; Escin; Guinea Pigs; Heterocyclic Compounds, 4 or More Rings; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Trachea

Cardiac constructs fabricated using human induced pluripotent stem cells-derived cardiomyocytes (iPSCs-CMs) are useful for evaluating the cardiotoxicity of and cardiac response to new drugs. Previously, we fabricated scaffold-free three-dimensional (3D) tubular cardiac constructs using a bio-3D printer, which can load cardiac spheroids onto a needle array. In this study, we developed a method to measure the contractile force and to evaluate the drug response in cardiac constructs. Specifically, we measured the movement of the needle tip upon contraction of the cardiac constructs on the needle array. The contractile force and beating rate of the cardiac constructs were evaluated by analysing changes in the movement of the needle tip. To evaluate the drug response, contractile properties were measured following treatment with isoproterenol, propranolol, or blebbistatin, in which the movement of the needle tip was increased following isoproterenol treatment, but was decreased following propranolol or blebbistain, treatments. To evaluate cardiotoxicity, contraction and cell viability of the cardiac constructs were measured following doxorubicin treatment. Cell viability was found to decrease with decreasing movement of the needle tip following doxorubicin treatment. Collectively, our results show that this method can aid in evaluating the contractile force of cardiac constructs.

Topics: Cardiotoxicity; Cell Survival; Doxorubicin; Drug Evaluation, Preclinical; Heterocyclic Compounds, 4 or More Rings; Humans; Induced Pluripotent Stem Cells; Isoproterenol; Myocardial Contraction; Myocytes, Cardiac; Printing, Three-Dimensional; Propranolol; Tissue Engineering; Tissue Scaffolds; Toxicity Tests

Previous findings have shown that non-muscle myosin heavy-chain IIA (NMMHC IIA) is involved in autophagy induction triggered by starvation in D. melanogaster; however, its functional contribution to neuronal autophagy remains unclear. The aim of this study is to explore the function of NMMHC IIA in cerebral ischemia-induced neuronal autophagy and the underlying mechanism related to autophagy-related gene 9A (ATG9A) trafficking. Functional assays and molecular mechanism studies were used to investigate the role of NMMHC IIA in cerebral ischemia-induced neuronal autophagy in vivo and in vitro. A middle cerebral artery occlusion (MCAO) model in mice was used to evaluate the therapeutic effect of blebbistatin, a myosin II ATPase inhibitor. Herein, either depletion or knockdown of NMMHC IIA led to increased cell viability in both primary cultured cortical neurons and pheochromocytoma (PC12) cells exposed to oxygen-glucose deprivation/reoxygenation (OGD/R). In addition, NMMHC IIA and autophagic marker LC3B were upregulated by OGD/R, and inhibition of NMMHC IIA significantly reduced OGD-induced neuronal autophagy. Furthermore, NMMHC IIA-induced autophagy is through its interactions with F-actin and ATG9A in response to OGD/R. The NMMHC IIA-actin interaction contributes to ATG9A trafficking and autophagosome formation. Inhibition of the NMMHC IIA-actin interaction using blebbistatin and the F-actin polymerization inhibitor cytochalasin D significantly suppressed ATG9A trafficking and autophagy induction. Furthermore, blebbistatin significantly improved neurological deficits and infarct volume after ischemic attack in mice, accompanied by ATG9A trafficking and autophagy inhibition. These findings demonstrate neuroprotective effects of NMMHC IIA inhibition on regulating ATG9A trafficking-dependent autophagy activation in the context of cerebral ischemia/reperfusion.

Topics: Actins; Animals; Autophagic Cell Death; Autophagy-Related Proteins; Brain Ischemia; Heterocyclic Compounds, 4 or More Rings; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Nonmuscle Myosin Type IIA; PC12 Cells; Rats; Reperfusion Injury; Vesicular Transport Proteins

Subcellular dynamics of non-muscle myosin 2 (NM2) is crucial for a broad-array of cellular functions. To unveil mechanisms of NM2 pharmacological control, we determined how the dynamics of NM2 diffusion is affected by NM2's allosteric inhibitors, i.e. blebbistatin derivatives, as compared to Y-27632 inhibiting ROCK, NM2's upstream regulator. We found that NM2 diffusion is markedly faster in central fibers than in peripheral stress fibers. Y-27632 accelerated NM2 diffusion in both peripheral and central fibers, whereas in peripheral fibers blebbistatin derivatives slightly accelerated NM2 diffusion at low, but markedly slowed it at high inhibitor concentrations. In contrast, rapid NM2 diffusion in central fibers was unaffected by direct NM2 inhibition. Using our optopharmacological tool, Molecular Tattoo, sub-effective concentrations of a photo-crosslinkable blebbistatin derivative were increased to effective levels in a small, irradiated area of peripheral fibers. These findings suggest that direct allosteric inhibition affects the diffusion profile of NM2 in a markedly different manner compared to the disruption of the upstream control of NM2. The pharmacological action of myosin inhibitors is channeled through autonomous molecular processes and might be affected by the load acting on the NM2 proteins.

Topics: Allosteric Regulation; Animals; Cell Line, Tumor; Diffusion; HeLa Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Myosin Type II; Rats

Topics: Adult; Aged; Case-Control Studies; Cell Differentiation; Cell Proliferation; Cells, Cultured; Collagen; Female; Heterocyclic Compounds, 4 or More Rings; Humans; Keloid; Male; Middle Aged; Myofibroblasts; Nonmuscle Myosin Type IIA; RNA Interference; Signal Transduction; Skin; Transforming Growth Factor beta

Living cells and tissues experience various complex modes of forces that are important in physiology and disease. However, how different force modes impact gene expression is elusive. Here we apply local forces of different modes via a magnetic bead bound to the integrins on a cell and quantified cell stiffness, chromatin deformation, and DHFR (dihydrofolate reductase) gene transcription. In-plane stresses result in lower cell stiffness than out-of-plane stresses that lead to bead rolling along the cell long axis (i.e., alignment of actin stress fibers) or at different angles (90° or 45°). However, chromatin stretching and ensuing DHFR gene upregulation by the in-plane mode are similar to those induced by the 45° stress mode. Disrupting stress fibers abolishes differences in cell stiffness, chromatin stretching, and DHFR gene upregulation under different force modes and inhibiting myosin II decreases cell stiffness, chromatin deformation, and gene upregulation. Theoretical modeling using discrete anisotropic stress fibers recapitulates experimental results and reveals underlying mechanisms of force-mode dependence. Our findings suggest that forces impact biological responses of living cells such as gene transcription via previously underappreciated means.

Topics: Animals; Anisotropy; Biomechanical Phenomena; CHO Cells; Chromatin; Cricetulus; Heterocyclic Compounds, 4 or More Rings; Intravital Microscopy; Microscopy, Fluorescence; Myosin Type II; Stress Fibers; Stress, Mechanical; Tetrahydrofolate Dehydrogenase; Transcription, Genetic; Up-Regulation

The spectrin-based membrane skeleton is a major component of the cell cortex. While expressed by all metazoans, its dynamic interactions with the other cortex components, including the plasma membrane or the acto-myosin cytoskeleton, are poorly understood. Here, we investigate how spectrin re-organizes spatially and dynamically under the membrane during changes in cell mechanics. We find spectrin and acto-myosin to be spatially distinct but cooperating during mechanical challenges, such as cell adhesion and contraction, or compression, stretch and osmolarity fluctuations, creating a cohesive cortex supporting the plasma membrane. Actin territories control protrusions and contractile structures while spectrin territories concentrate in retractile zones and low-actin density/inter-contractile regions, acting as a fence that organize membrane trafficking events. We unveil here the existence of a dynamic interplay between acto-myosin and spectrin necessary to support a mesoscale organization of the lipid bilayer into spatially-confined cortical territories during cell mechanoresponse.

Topics: Actins; Actomyosin; Animals; Cell Membrane; Coated Pits, Cell-Membrane; Endocytosis; Fibroblasts; Green Fluorescent Proteins; Heterocyclic Compounds, 4 or More Rings; Lipid Bilayers; Mice; Microscopy, Confocal; NIH 3T3 Cells; Spectrin; Stress, Mechanical

The motor protein myosin drives a wide range of cellular and muscular functions by generating directed movement and force, fueled through adenosine triphosphate (ATP) hydrolysis. Release of the hydrolysis product adenosine diphosphate (ADP) is a fundamental and regulatory process during force production. However, details about the molecular mechanism accompanying ADP release are scarce due to the lack of representative structures. Here we solved a novel blebbistatin-bound myosin conformation with critical structural elements in positions between the myosin pre-power stroke and rigor states. ADP in this structure is repositioned towards the surface by the phosphate-sensing P-loop, and stabilized in a partially unbound conformation via a salt-bridge between Arg131 and Glu187. A 5 Å rotation separates the mechanical converter in this conformation from the rigor position. The crystallized myosin structure thus resembles a conformation towards the end of the two-step power stroke, associated with ADP release. Computationally reconstructing ADP release from myosin by means of molecular dynamics simulations further supported the existence of an equivalent conformation along the power stroke that shows the same major characteristics in the myosin motor domain as the resolved blebbistatin-bound myosin-II·ADP crystal structure, and identified a communication hub centered on Arg232 that mediates chemomechanical energy transduction.

Topics: Actins; Adenosine Diphosphate; Adenosine Triphosphate; Catalytic Domain; Crystallization; Heterocyclic Compounds, 4 or More Rings; Hydrolysis; Molecular Dynamics Simulation; Myosins; Protein Conformation, beta-Strand

Malignant pleural mesothelioma (MPM) has an overall poor prognosis and unsatisfactory treatment options. MPM nodules, protruding into the pleural cavity may have growth and spreading dynamics distinct that of other solid tumors. We demonstrate that multicellular aggregates can develop spontaneously in the majority of tested MPM cell lines when cultured at high cell density. Surprisingly, the nodule-like aggregates do not arise by excessive local cell proliferation, but by myosin II-driven cell contractility. Prominent actin cables, spanning several cells, are abundant both in cultured aggregates and in MPM surgical specimens. We propose a computational model for in vitro MPM nodule development. Such a self-tensioned Maxwell fluid exhibits a pattern-forming instability that was studied by analytical tools and computer simulations. Altogether, our findings may underline a rational for targeting the actomyosin system in MPM.

Topics: Actins; Amides; Animals; Cell Count; Cell Line, Tumor; Heterocyclic Compounds, 4 or More Rings; Humans; Male; Mesothelioma, Malignant; Mice, SCID; Myosins; Pyridines; Stochastic Processes; Time-Lapse Imaging; Xenograft Model Antitumor Assays

Vascular leakage is an important pathophysiological process of critical conditions such as shock and ischemia-reperfusion (I/R)-induced lung injury. Microparticles (MPs), including endothelial cell-derived microparticles (EMPs), platelet-derived microparticles (PMPs) and leukocyte-derived microparticles (LMPs), have been shown to participate in many diseases. Whether and which of these MPs take part in pulmonary vascular leakage and lung injury after I/R and whether these MPs have synergistic effect and the underlying mechanism are not known.. Using hemorrhage/transfusion (Hemo/Trans) and aorta abdominalis occlusion-induced I/R rat models, the role of EMPs, PMPs and LMPs and the mechanisms in pulmonary vascular leakage and lung injury were observed.. The concentrations of EMPs, PMPs and LMPs were significantly increased after I/R. Intravenous administration of EMPs and PMPs but not LMPs induced pulmonary vascular leakage and lung injury. Furthermore, EMPs induced pulmonary sequestration of platelets and promoted more PMPs production, and played a synergistic effect on pulmonary vascular leakage. MiR-1, miR-155 and miR-542 in EMPs, and miR-126 and miR-29 in PMPs, were significantly increased after hypoxia/reoxygenation (H/R). Of which, inhibition of miR-155 in EMPs and miR-126 in PMPs alleviated the detrimental effects of EMPs and PMPs on vascular barrier function and lung injury. Overexpression of miR-155 in EMPs down-regulated the expression of tight junction related proteins such as ZO-1 and claudin-5, while overexpression of miR-126 up-regulated the expression of caveolin-1 (Cav-1), the trans-cellular transportation related protein such as caveolin-1 (Cav-1). Inhibiting EMPs and PMPs production with blebbistatin (BLE) and amitriptyline (AMI) alleviated I/R induced pulmonary vascular leakage and lung injury.. EMPs and PMPs contribute to the pulmonary vascular leakage and lung injury after I/R. EMPs mediate pulmonary sequestration of platelets, producing more PMPs to play synergistic effect. Mechanically, EMPs carrying miR-155 that down-regulates ZO-1 and claudin-5 and PMPs carrying miR-126 that up-regulates Cav-1, synergistically mediate pulmonary vascular leakage and lung injury after I/R. Video Abstract.

Topics: Amitriptyline; Animals; Blood Platelets; Capillary Permeability; Caveolin 1; Cell-Derived Microparticles; Claudin-5; Endothelial Cells; Heterocyclic Compounds, 4 or More Rings; Leukocytes; Lung; Lung Injury; MicroRNAs; Rats, Sprague-Dawley; Reperfusion Injury; Zonula Occludens-1 Protein

The paracellular barrier function of tight junctions (TJs) in epithelial cell sheets is robustly maintained against mechanical fluctuations, by molecular mechanisms that are poorly understood. Vinculin is an adaptor of a mechanosensory complex at the adherens junction. Here, we generated vinculin KO Eph4 epithelial cells and analyzed their confluent cell-sheet properties. We found that vinculin is dispensable for the basic TJ structural integrity and the paracellular barrier function for larger solutes. However, vinculin is indispensable for the paracellular barrier function for ions. In addition, TJs stochastically showed dynamically distorted patterns in vinculin KO cell sheets. These KO phenotypes were rescued by transfecting full-length vinculin and by relaxing the actomyosin tension with blebbistatin, a myosin II ATPase activity inhibitor. Our findings indicate that vinculin resists mechanical fluctuations to maintain the TJ paracellular barrier function for ions in epithelial cell sheets.

Topics: Actomyosin; Cell Line; Epithelial Cells; Gene Knockout Techniques; HEK293 Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Ions; Stochastic Processes; Tight Junctions; Vinculin

In skeletal muscle, the Na/K ATPase (NKA) plays essential roles in processes linked to muscle contraction, fatigue, and energy metabolism; however, very little information exists regarding the regulation of NKA activity. The scarcity of information regarding NKA function in skeletal muscle likely stems from methodological constraints, as NKA contributes minimally to total cellular ATP utilization, and therefore contamination from other ATPases prevents the assessment of NKA activity in muscle homogenates. Here we introduce a method that improves accuracy and feasibility for the determination of NKA activity in small rodent muscle samples (5-10 mg) and in human skeletal muscle.. Skeletal muscle homogenates from mice (n = 6) and humans (n = 3) were used to measure NKA and sarcoplasmic reticulum Ca ATPase (SERCA) activities with the addition of specific ATPase inhibitors to minimize "background noise.". We observed that myosin ATPase activity was the major interfering factor for estimation of NKA activity in skeletal muscle homogenates, as the addition of 25 μM of blebbistatin, a specific myosin ATPase inhibitor, considerably minimized "background noise" (threefold) and enabled the determination of NKA maximal activity with values three times higher than previously reported. The specificity of the assay was demonstrated after the addition of 2 mM ouabain, which completely inhibited NKA. On the other hand, the addition of blebbistatin did not affect the ability to measure SERCA function. The coefficient of variation for NKA and SERCA assays were 6.2% and 4.4%, respectively.. The present study has improved the methodology to determine NKA activity. We further show the feasibility of measuring NKA and SERCA activities from a common muscle homogenate. This methodology is expected to aid in our long-term understanding of how NKA affects skeletal muscle metabolic homeostasis and contractile function in diverse situations.

Topics: Aged; Animals; Energy Metabolism; Excitation Contraction Coupling; Feasibility Studies; Female; Fluorometry; Heterocyclic Compounds, 4 or More Rings; Humans; Male; Mice, Inbred C57BL; Middle Aged; Muscle, Skeletal; Myosins; Ouabain; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sodium-Potassium-Exchanging ATPase

Recent studies have found discordant mechanosensitive outcomes when comparing 2D and 3D, highlighting the need for tools to study mechanotransduction in 3D across a wide spectrum of stiffness. A gelatin methacryloyl (GelMA) hydrogel with a continuous stiffness gradient ranging from 5 to 38 kPa was developed to recapitulate physiological stiffness conditions. Adipose-derived stem cells (ASCs) were encapsulated in this hydrogel, and their morphological characteristics and expression of both mechanosensitive proteins (Lamin A, YAP, and MRTFa) and differentiation markers (PPARγ and RUNX2) were analyzed. Low-stiffness regions (∼8 kPa) permitted increased cellular and nuclear volume and enhanced mechanosensitive protein localization in the nucleus. This trend was reversed in high stiffness regions (∼30 kPa), where decreased cellular and nuclear volumes and reduced mechanosensitive protein nuclear localization were observed. Interestingly, cells in soft regions exhibited enhanced osteogenic RUNX2 expression, while those in stiff regions upregulated the adipogenic regulator PPARγ, suggesting that volume, not substrate stiffness, is sufficient to drive 3D stem cell differentiation. Inhibition of myosin II (Blebbistatin) and ROCK (Y-27632), both key drivers of actomyosin contractility, resulted in reduced cell volume, especially in low-stiffness regions, causing a decorrelation between volume expansion and mechanosensitive protein localization. Constitutively active and inactive forms of the canonical downstream mechanotransduction effector TAZ were stably transfected into ASCs. Activated TAZ resulted in higher cellular volume despite increasing stiffness and a consistent, stiffness-independent translocation of YAP and MRTFa into the nucleus. Thus, volume adaptation as a function of 3D matrix stiffness can control stem cell mechanotransduction and differentiation.

Topics: Actin Cytoskeleton; Actomyosin; Acyltransferases; Adipogenesis; Amides; Cell Cycle Proteins; Cell Differentiation; Cell Encapsulation; Cell Nucleus; Cell Size; Core Binding Factor Alpha 1 Subunit; Gelatin; Heterocyclic Compounds, 4 or More Rings; Humans; Hydrogels; Lamin Type A; Mechanotransduction, Cellular; Mesenchymal Stem Cells; Myosin Type II; Osteogenesis; PPAR gamma; Pyridines; rho-Associated Kinases; Trans-Activators; Transcription Factors

Throughout the body, epithelial tissues contain curved features (e.g. cysts, ducts and crypts) that influence cell behaviors. These structures have varied curvature, with flat structures having zero curvature and structures such as crypts having large curvature. In the ovary, cortical inclusion cysts (CICs) of varying curvatures are found, and fallopian tube epithelial (FTE) cells have been found trapped within these cysts. FTE are the precursor for ovarian cancer, and the CIC niche has been proposed to play a role in ovarian cancer progression. We hypothesized that variations in ovarian CIC curvature that occur during cyst resolution impact the ability of trapped FTE cells to invade into the surrounding stroma. Using a lumen model in collagen gels, we determined that increased curvature resulted in more invasions of mouse FTE cells. To isolate curvature as a system parameter, we developed a novel technique to pattern concave curvatures into collagen gels. When FTE cells were seeded to confluency on curved substrates, increases in curvature increased the number of invading FTE cells and the invasion distance. FTE invasion into collagen substrates with higher curvature depended on matrix metalloproteinases (MMPs), but expression of collagen I degrading Mmps was not different on curved and flat regions. A finite-element model predicted that contractility and cell-cell connections were essential for increased invasion on substrates with higher curvature, while cell-substrate interactions had minimal effect. Experiments supported these predictions, with invasion decreased by blebbistatin, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or N-cadherin-blocking antibody, but with no effect from a focal adhesion kinase inhibitor. Finally, experimental evidence supports that cell invasion on curved substrates occurs in two phases-a cell-cell-dependent initiation phase where individual cells break away from the monolayer and an MMP-dependent phase as cells migrate further into the collagen matrix.

Topics: Animals; Cadherins; Cell Adhesion; Cell Communication; Collagen; Disease Progression; Egtazic Acid; Epithelial Cells; Fallopian Tubes; Female; Finite Element Analysis; Heterocyclic Compounds, 4 or More Rings; Matrix Metalloproteinases; Mice; Microfluidics; Microscopy, Confocal; Ovarian Cysts; Ovarian Neoplasms; Ovary; Phenotype

The interaction of light with biological tissues has been considered for various therapeutic applications. Light-induced neurite growth has the potential to be a clinically useful technique for neuron repair. However, most previous studies used either a large illumination area to accelerate overall neurite growth or employed a light spot to guide a growing neurite. It is not clear if optical stimulation can induce the regrowth of a retracted neurite. In the present work, we used blue light (wavelength: 473 nm) to cause neurite retraction, and we proved that using a red-light (wavelength: 650 nm) spot to illuminate the soma near the junction of the retracted neurite could induce neurite regrowth. As a comparison, we found that green light (wavelength 550 nm) had a 62% probability of inducing neurite regrowth, while red light had a 75% probability of inducing neurite regrowth at the same power level. Furthermore, the neurite regrowth length induced by red light was increased by the pre-treatment with inhibitors of myosin functions. We also observed actin propagation from the soma to the tip of the re-growing neurite following red-light stimulation of the soma. The red light-induced extension and regrowth were abrogated in the calcium-free medium. These results suggest that illumination with a red-light spot on the soma may trigger the regrowth of a neurite after the retraction caused by blue-light illumination.

Topics: Actins; Animals; Calcium; Cell Line, Tumor; Color; Culture Media; Heterocyclic Compounds, 4 or More Rings; Hippocampus; Light; Low-Level Light Therapy; Mice; Myosin Type II; Nerve Regeneration; Neurites; Primary Cell Culture; Rats

The intestinal epithelium is one of the most rapidly self-renewing tissues throughout life in mammals. A small population of stem cells at the base of crypt in the epithelium can continually self-renew and give rise to differentiated epithelial cells. The self-renewal and differentiation of intestinal stem cells are under a tight control during homeostasis, and disruption of this balancing regulation leads to intestinal degeneration or tumorigenesis. Accordingly, exploration of the mechanism underlying the regulation of stem cells is essential for the understanding and treatment of intestinal disorders. As traditional methods using mice models are costly and time-consuming, the recently established ex vivo intestinal organoids model provides an ideal tool to investigate the mechanisms regulating the self-renewal and differentiation of intestinal stem cells. The intestinal organoids recapitulate major characteristics in both structure and function of intestinal epithelium in vivo. Here, we describe a new protocol to generate the intestinal organoids from both crypts and single stem cells with a higher efficiency using the small molecule blebbistatin and provide an approach to assess the self-renewal and differentiation of stem cells in intestinal organoids.

Topics: Animals; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Heterocyclic Compounds, 4 or More Rings; Intestines; Mice; Organoids; Stem Cells; Tissue Engineering

Overactive bladder (OAB) coexists with depression in women. Here, we assessed the effects of a 1-week treatment with blebbistatin, a myosin II inhibitor, on changes in behavior and detrusor overactivity (DO) symptoms induced by a 6-week administration of 13-cis-retinoic acid (13-cis-RA), with the aid of the forced swim test (FST), spontaneous locomotor activity test, and in vivo cystometric investigations in female Wistar rats. 13-cis-RA-induced depressive-like behavior and DO symptoms were associated with increased corticotropin-releasing factor (CRF) level in the plasma, prefrontal cortex (PFC), hippocampus (Hp), Barrington's nucleus (BN), and urinary bladder. Moreover, 13-cis-RA decreased brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) levels in plasma, PFC, Hp, and BN, while it increased BDNF and NGF levels in urinary bladder. Blebbistatin exerted antidepressant-like effect and attenuated changes in the cystometric parameters as well as the central and peripheral levels of CRF, BDNF, and NGF that were induced by 13-cis-RA, while it did not affect urine production, mean, systolic or diastolic blood pressure, or heart rate. The results point to blebbistatin as a potential treatment option for OAB coexisting with depression.

Topics: Animals; Antidepressive Agents; Autonomic Agents; Brain; Brain-Derived Neurotrophic Factor; Corticotropin-Releasing Hormone; Depression; Disease Models, Animal; Female; Heterocyclic Compounds, 4 or More Rings; Isotretinoin; Myosin Type II; Nerve Growth Factor; Random Allocation; Rats, Wistar; Urinary Bladder; Urinary Bladder, Overactive

Proneural transcription factors (TFs) drive highly efficient differentiation of pluripotent stem cells to lineage-specific neurons. However, current strategies mainly rely on genome-integrating viruses. Here, we used synthetic mRNAs coding two proneural TFs (Atoh1 and Ngn2) to differentiate induced pluripotent stem cells (iPSCs) into midbrain dopaminergic (mDA) neurons. mRNAs coding Atoh1 and Ngn2 with defined phosphosite modifications led to higher and more stable protein expression, and induced more efficient neuron conversion, as compared to mRNAs coding wild-type proteins. Using these two modified mRNAs with morphogens, we established a 5-day protocol that can rapidly generate mDA neurons with >90% purity from normal and Parkinson's disease iPSCs. After in vitro maturation, these mRNA-induced mDA (miDA) neurons recapitulate key biochemical and electrophysiological features of primary mDA neurons and can provide high-content neuron cultures for drug discovery. Proteomic analysis of Atoh1-binding proteins identified the nonmuscle myosin II (NM-II) complex as a new binding partner of nuclear Atoh1. The NM-II complex, commonly known as an ATP-dependent molecular motor, binds more strongly to phosphosite-modified Atoh1 than the wild type. Blebbistatin, an NM-II complex antagonist, and bradykinin, an NM-II complex agonist, inhibited and promoted, respectively, the transcriptional activity of Atoh1 and the efficiency of miDA neuron generation. These findings established the first mRNA-driven strategy for efficient iPSC differentiation to mDA neurons. We further identified the NM-II complex as a positive modulator of Atoh1-driven neuron differentiation. The methodology described here will facilitate the development of mRNA-driven differentiation strategies for generating iPSC-derived progenies widely applicable to disease modeling and cell replacement therapy. Stem Cells Translational Medicine 2019;8:112&12.

Topics: Animals; Bradykinin; Cell Differentiation; Cells, Cultured; Dopamine; Dopaminergic Neurons; Heterocyclic Compounds, 4 or More Rings; Humans; Induced Pluripotent Stem Cells; Mice; Parkinson Disease; Pluripotent Stem Cells; Proteomics; RNA, Messenger; Transcription, Genetic

Carcinogenesis occurs in elastin-rich tissues and leads to local inflammation and elastolytic proteinase release. This contributes to bioactive matrix fragment (Matrikine) accumulation like elastin degradation products (EDP) stimulating tumour cell invasive and metastatic properties. We previously demonstrate that EDPs exert protumoural activities through Hsp90 secretion to stabilised extracellular proteinases.. EDP influence on cancer cell blebbing and extracellular vesicle shedding were examined with a videomicroscope coupled with confocal Yokogawa spinning disk, by transmission electron microscopy, scanning electron microscopy and confocal microscopy. The ribosomal protein SA (RPSA) elastin receptor was identified after affinity chromatography by western blotting and cell immunolocalisation. mRNA expression was studied using real-time PCR. SiRNA were used to confirm the essential role of RPSA.. We demonstrate that extracellular matrix degradation products like EDPs induce tumour amoeboid phenotype with cell membrane blebbing and shedding of extracellular vesicle containing Hsp90 and proteinases in the extracellular space. EDPs influence intracellular calcium influx and cytoskeleton reorganisation. Among matrikines, VGVAPG and AGVPGLGVG peptides reproduced EDP effects through RPSA binding.. Our data suggests that matrikines induce cancer cell blebbing and extracellular vesicle release through RPSA binding, favouring dissemination, cell-to-cell communication and growth of cancer cells in metastatic sites.

Topics: Amides; Calcium; Cell Communication; Cell Line, Tumor; Elastin; Extracellular Matrix Proteins; Extracellular Vesicles; Heterocyclic Compounds, 4 or More Rings; HSP90 Heat-Shock Proteins; Humans; Neoplasms; Peptide Fragments; Pyridines; Receptors, Laminin; rho-Associated Kinases; Ribosomal Proteins; Signal Transduction

Tissue internalisation is a key morphogenetic mechanism by which embryonic tissues generate complex internal organs and a number of studies of epithelia have outlined a general view of tissue internalisation. Here we have used quantitative live imaging and mutant analysis to determine whether similar mechanisms are responsible for internalisation in a tissue that apparently does not have a typical epithelial organisation - the zebrafish neural plate. We found that although zebrafish embryos begin neurulation without a conventional epithelium, medially located neural plate cells adopt strategies typical of epithelia in order to constrict their dorsal surface membrane during cell internalisation. Furthermore, we show that Myosin-II activity is a significant driver of this transient cell remodeling which also depends on Cdh2 (N-cadherin). Abrogation of Cdh2 results in defective Myosin-II distribution, mislocalised internalisation events and defective neural plate morphogenesis. Our work suggests Cdh2 coordinates Myosin-II dependent internalisation of the zebrafish neural plate.

Topics: Actins; Animals; Animals, Genetically Modified; Body Patterning; Cadherins; Cell Membrane; Cell Movement; Epithelium; Gene Expression Regulation; Gene Expression Regulation, Developmental; Green Fluorescent Proteins; Heterocyclic Compounds, 4 or More Rings; Microscopy, Confocal; Morphogenesis; Myosin Type II; Myosins; Neural Crest; Neural Plate; Oligonucleotides; Prospective Studies; Zebrafish; Zebrafish Proteins

The aims of the study were to determine the effectiveness of blebbistatin (BLEB) on detrusor overactivity (DO) in an animal model induced by retinyl acetate (RA) and, because of potential urothelial permeability, to evaluate the degenerative impact of BLEB on the urothelium. Three days after RA instillation into the urinary bladder, BLEB was administered into the bladder and immediately after cystometric assessment was performed. Furthermore, Evans Blue extravasation into bladder tissue and urothelium thickness were measured. Sixty female Wistar rats were used and randomly assigned to one of four groups (n = 15 in each group): (1) control, (2) RA, (3) BLEB, and (4) RA + BLEB. RA administration induced changes in cystometric parameters reflecting DO, as previously reported. Treatment with BLEB did not significantly alter cystometric parameters in rats which did not receive RA. Administration of BLEB to rats pretreated with RA reversed changes in cystometric parameters induced by RA in basal pressure, threshold pressure, detrusor overactivity index, amplitude of nonvoiding contractions, frequency of nonvoiding contractions, voided volume, volume threshold, intercontraction interval, bladder compliance, and volume threshold to elicit nonvoiding contractions. There were no significant differences in Evans Blue extravasation into bladder tissue or urothelium thickness between the groups. The current research provides new data on the possible utility of blebbistatin in the pharmacotherapy of DO, which is an important feature of overactive bladder (OAB). Further studies in human patients with DO/OAB are warranted to confirm these preclinical results.

Topics: Administration, Intravesical; Animals; Disease Models, Animal; Diterpenes; Female; Heterocyclic Compounds, 4 or More Rings; Rats, Wistar; Retinyl Esters; Urinary Bladder, Overactive; Urination; Urodynamics; Vitamin A

Cells select from a diverse repertoire of migration strategies. Recent developments in tunable biomaterials have helped identify how extracellular matrix properties influence migration, however, many settings lack the fibrous architecture characteristic of native tissues. To investigate migration in fibrous contexts, we independently varied the alignment and stiffness of synthetic 3D fiber matrices and identified two phenotypically distinct migration modes. In contrast to stiff matrices where cells migrated continuously in a traditional mesenchymal fashion, cells in deformable matrices stretched matrix fibers to store elastic energy; subsequent adhesion failure triggered sudden matrix recoil and rapid cell translocation. Across a variety of cell types, traction force measurements revealed a relationship between cell contractility and the matrix stiffness where this migration mode occurred optimally. Given the prevalence of fibrous tissues, an understanding of how matrix structure and mechanics influences migration could improve strategies to recruit repair cells to wound sites or inhibit cancer metastasis.

Topics: Acrylic Resins; Actomyosin; Amides; Animals; Biocompatible Materials; Cell Line, Tumor; Cell Movement; Dextrans; Elastic Modulus; Extracellular Matrix; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Humans; Intravital Microscopy; Marine Toxins; Materials Testing; Methacrylates; Mice; Microscopy, Confocal; NIH 3T3 Cells; Oxazoles; Pyridines; Time-Lapse Imaging

Damage to alveoli, the gas-exchanging region of the lungs, is a component of many chronic and acute lung diseases. In addition, insufficient generation of alveoli results in bronchopulmonary dysplasia, a disease of prematurity. Therefore visualising the process of alveolar development (alveologenesis) is critical for our understanding of lung homeostasis and for the development of treatments to repair and regenerate lung tissue. Here we show live alveologenesis, using long-term, time-lapse imaging of precision-cut lung slices. We reveal that during this process, epithelial cells are highly mobile and we identify specific cell behaviours that contribute to alveologenesis: cell clustering, hollowing and cell extension. Using the cytoskeleton inhibitors blebbistatin and cytochalasin D, we show that cell migration is a key driver of alveologenesis. This study reveals important novel information about lung biology and provides a new system in which to manipulate alveologenesis genetically and pharmacologically.

Topics: Actomyosin; Animals; Animals, Newborn; Cell Movement; Cytochalasin D; Epithelial Cells; Heterocyclic Compounds, 4 or More Rings; Intravital Microscopy; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Models, Animal; Organogenesis; Pulmonary Alveoli; Time-Lapse Imaging

The main goal of our study was to investigate whether blebbistatin would prevent the cyclophosphamide (CYP)-induced changes in cystometric and inflammatory parameters indicating the development of bladder inflammation and bladder overactivity. As the nature of CYP-induced urotoxicity is inflammatory, we assume that agents presenting an anti-inflammatory potential, such as blebbistatin, are worth special attention.. The experiments were carried out in female Wistar rats. Surgical procedures, cystometric investigations, measurements of bladder edema and urothelium thickness as well as biochemical analyses were performed according to the published literature.. As expected, an acute administration of CYP (200 mg/kg, intraperitoneally) induced changes in the cystometric parameters and the levels of the tested biomarkers (ie, interleukin 1-β, interleukin 6, interleukin 10, tumor necrosis factor-α, nerve growth factor, brain-derived neurotrophic factor, heparin-binding epidermal growth factor-like growth factor, insulin-like growth factor-binding protein 3, C-X-C motif chemokine 10, orosomucoid-1, Tamm-Horsfall protein, hemopexin, and occludin), indicating the development of bladder overactivity and bladder inflammation, respectively. These changes were accompanied by bladder edema and increased urothelium thickness. Intravesical infusion of blebbistatin for 7 days (125 nmol/day) prevented all symptoms of the CYP-induced urotoxicity.. Blebbistatin might be a promising novel agent for the treatment of bladder dysfunctions, like CYP-induced hemorrhage cystitis or bladder overactivity, since it diminished the increased urinary bladder levels of proinflammatory markers and normalized the concentrations of the anti-inflammatory ones. This effect was accompanied by amelioration of bladder edema and permeability, and normalization of both urothelium thickness and values of the cystometric parameters.

Topics: Administration, Intravesical; Animals; Cyclophosphamide; Cystitis; Disease Models, Animal; Female; Heterocyclic Compounds, 4 or More Rings; Rats; Rats, Wistar; Treatment Outcome; Urinary Bladder; Urinary Bladder, Overactive

Breast cancer nuclei have highly irregular shapes, which are diagnostic and prognostic markers of breast cancer progression. The mechanisms by which irregular cancer nuclear shapes develop are not well understood. Here we report the existence of vertical, apical cell protrusions in cultured MDA-MB-231 breast cancer cells. Once formed, these protrusions persist over time scales of hours and are associated with vertically upward nuclear deformations. They are absent in normal mammary epithelial cells (MCF-10A cells). Microtubule disruption enriched these protrusions preferentially in MDA-MB-231 cells compared with MCF-10A cells, whereas inhibition of nonmuscle myosin II (NMMII) abolished this enrichment. Dynamic confocal imaging of the vertical cell and nuclear shape revealed that the apical cell protrusions form first, and in response, the nucleus deforms and/or subsequently gets vertically extruded into the apical protrusion. Overexpression of lamin A/C in MDA-MB-231 cells reduced nuclear deformation in apical protrusions. These data highlight the role of mechanical stresses generated by moving boundaries, as well as abnormal nuclear mechanics in the development of abnormal nuclear shapes in breast cancer cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cytochalasin D; Cytoskeleton; Female; Heterocyclic Compounds, 4 or More Rings; Humans; Nocodazole; Stress, Mechanical; Tubulin Modulators

Three-dimensional matrices often contain highly structured adhesive tracks that require cells to turn corners and bridge non-adhesive areas. Here, we investigate these complex processes using micropatterned cell adhesive frames. Spreading kinetics on these matrices depend strongly on initial adhesive position and are predicted by a cellular Potts model (CPM), which reflects a balance between adhesion and intracellular tension. As cells spread, new stress fibers (SFs) assemble periodically and parallel to the leading edge, with spatial intervals of ∼2.5 μm, temporal intervals of ∼15 min, and characteristic lifetimes of ∼50 min. By incorporating these rules into the CPM, we can successfully predict SF network architecture. Moreover, we observe broadly similar behavior when we culture cells on arrays of discrete collagen fibers. Our findings show that ECM geometry and initial cell position strongly determine cell spreading and that cells encode a memory of their spreading history through SF network organization.

Topics: Actin Cytoskeleton; Cell Adhesion; Cell Line, Tumor; Cell Movement; Collagen; Computer Simulation; Extracellular Matrix; Half-Life; Heterocyclic Compounds, 4 or More Rings; Humans; Kinetics; Models, Biological; Pseudopodia; Stress Fibers; Time Factors

Mechanosensing of the mechanical microenvironment by cells regulates cell phenotype and function. The nucleus is critical in mechanosensing, as it transmits external forces from the cellular microenvironment to the nuclear envelope housing chromatin. This study aims to elucidate how mechanical confinement affects nuclear deformation within several cell types, and to determine the role of cytoskeletal elements in controlling nuclear deformation. Human cancer cells (MDA-MB-231), human mesenchymal stem cells (MSCs), and mouse fibroblasts (L929) were seeded within polydimethylsiloxane (PDMS) microfluidic devices containing microchannels of varying cross-sectional areas, and nuclear morphology and volume were quantified via image processing of fluorescent cell nuclei. We found that the nuclear major axis length remained fairly constant with increasing confinement in MSCs and MDA-MB-231 cells, but increased with increasing confinement in L929 cells. Nuclear volume of L929 cells and MSCs decreased in the most confining channels. However, L929 nuclei were much more isotropic in unconfined channels than MSC nuclei. When microtubule polymerization or myosin II contractility was inhibited, nuclear deformation was altered only in MSCs in wide channels. This work informs our understanding of nuclear mechanics in physiologically relevant spaces, and suggests diverging roles of the cytoskeleton in regulating nuclear deformation in different cell types.

Topics: Animals; Cell Line; Cell Nucleus; Cell Nucleus Shape; Female; Heterocyclic Compounds, 4 or More Rings; Humans; Mesenchymal Stem Cells; Mice; Microtubules; Myosin Type II; Nocodazole; Polymerization; Young Adult

Tissue regeneration at an injured site depends on proliferation, migration, and differentiation of resident stem or progenitor cells, but solid tissues are often sufficiently dense and constricting that nuclei are highly stressed by migration. In this study, constricted migration of myoblastic cell types and mesenchymal stem cells (MSCs) increases nuclear rupture, increases DNA damage, and modulates differentiation. Fewer myoblasts fuse into regenerating muscle in vivo after constricted migration in vitro, and myodifferentiation in vitro is likewise suppressed. Myosin II inhibition rescues rupture and DNA damage, implicating nuclear forces, while mitosis and the cell cycle are suppressed by constricted migration, consistent with a checkpoint. Although perturbed proliferation fails to explain defective differentiation, nuclear rupture mislocalizes differentiation-relevant MyoD and KU80 (a DNA repair factor), with nuclear entry of the DNA-binding factor cGAS. Human MSCs exhibit similar damage, but osteogenesis

Topics: Animals; Cell Count; Cell Cycle; Cell Differentiation; Cell Movement; Cell Nucleus; Cells, Cultured; DNA Damage; Heterocyclic Compounds, 4 or More Rings; Humans; Mesenchymal Stem Cells; Mice; Muscles; Myoblasts; MyoD Protein; Myosin Type II; Osteogenesis; Regeneration

mTORC1 is regarded as an important key regulator of protein synthesis and hypertrophy following mechanical stimuli in skeletal muscle. However, as excitation and tension development is tightly coupled in most experimental models, very little and largely indirect evidence exist for such a mechanosensitive pathway. Here, we sought to examine whether activation of mTORC1 signalling is dependent on tension per se in rat skeletal muscle.. To examine the mechanosensitivity of mTORC1, rat EDL muscles were exposed to either excitation-induced eccentric contractions (ECC), passive stretching (PAS) with identical peak tension (T. PAS and ECC with equal T. The current study directly links tension per se to activation of mTORC1 signalling, which is independent of an active EC-coupling sequence. Moreover, activation of mTORC1 signalling displays a positive dose-response relationship with peak tension.

Topics: Animals; Biomechanical Phenomena; Endoplasmic Reticulum; Gene Expression Regulation; Heterocyclic Compounds, 4 or More Rings; Mechanistic Target of Rapamycin Complex 1; Muscle Contraction; Muscle, Skeletal; Myosins; Rats; Signal Transduction; Sulfonamides; Toluene

Beating cardiomyocytes undergo fast morphodynamics during the contraction-relaxation cycle. However, imaging these morphodynamics with a high spatial and temporal resolution is difficult, owing to a lack of suitable techniques. Here, we combine scanning ion conductance microscopy (SICM) with a microelectrode array (MEA) to image the three-dimensional (3D) topography of cardiomyocytes during a contraction-relaxation cycle with 1 μm spatial and 1 ms time resolution. We record the vertical motion of cardiomyocytes at many locations across a cell by SICM and synchronize these data using the simultaneously recorded action potential by the MEA as a time reference. This allows us to reconstruct the time-resolved 3D morphology of cardiomyocytes during a full contraction-relaxation cycle with a raw data rate of 200 μs/frame and to generate spatially resolved images of contractile parameters (maximum displacement, time delay, asymmetry factor). We use the MEA-SICM setup to visualize the effect of blebbistatin, a myosin II inhibitor, on the morphodynamics of contractions. Further, we find an upper limit of 0.02% for cell volume changes during an action potential. The results show that MEA-SICM provides an ultrafast imaging platform for investigating the functional interplay of cardiomyocyte electrophysiology and mechanics.

Topics: Animals; Cell Line; Cell Movement; Electrophysiological Phenomena; Heterocyclic Compounds, 4 or More Rings; Microelectrodes; Microscopy; Myocytes, Cardiac

Mechanical homeostasis describes how cells sense physical cues from the microenvironment and concomitantly remodel both the cytoskeleton and the surrounding extracellular matrix (ECM). Such feedback is thought to be essential to healthy development and maintenance of tissue. However, the nature of the dynamic coupling between microscale cell and ECM mechanics remains poorly understood. Here we investigate how and whether cells remodel their cortex and basement membrane to adapt to their microenvironment. We measured both intracellular and extracellular viscoelasticity, generating a full factorial dataset on 5 cell lines in 2 ECMs subjected to 4 cytoskeletal drug treatments at 2 time points. Nonmalignant breast epithelial cells show a similar viscoelasticity to that measured for the local ECM when cultured in 3D laminin-rich ECM. In contrast, the malignant counterpart is stiffer than the local environment. We confirmed that other mammary cancer cells embedded in tissue-mimetic hydrogels are nearly 4-fold stiffer than the surrounding ECM. Perturbation of actomyosin did not yield uniform responses but instead depended on the cell type and chemistry of the hydrogel. The observed viscoelasticity of both ECM and cells were well described by power laws in a frequency range that governs single filament cytoskeletal dynamics. Remarkably, the intracellular and extracellular power law parameters for the entire dataset collectively fall onto 2 parallel master curves described by just 2 parameters. Our work shows that tumor cells are mechanically plastic to adapt to many environments and reveals dynamical scaling behavior in the microscale mechanical responses of both cells and ECM.

Topics: Actomyosin; Amides; Cell Culture Techniques; Cell Movement; Cytoskeleton; Extracellular Matrix; Heterocyclic Compounds, 4 or More Rings; Humans; Hydrogels; Laminin; Marine Toxins; MCF-7 Cells; Mechanotransduction, Cellular; Oxazoles; Pyridines; Rheology; Viscosity

Corneal endothelial cell (CEC) dysfunction causes corneal edema that may lead to blindness. In addition to corneal transplantation, simple descemetorhexis has been proposed to treat centrally located disease with adequate peripheral cell reserve, but promoting the centripetal migration of CECs is pivotal to this strategy. Here, we show that targeting non-muscle myosin II (NMII) activity by Y27632, a ROCK inhibitor, or blebbistatin, a selective NMII inhibitor, promotes directional migration of CECs and accelerates in vitro wound healing. The lamellipodial protrusion persistence is increased, and actin retrograde flow is decreased after NMII inhibition. Counteracting lamellipodial protrusion by actin-related protein 2/3 (ARP2/3) inhibitor abolishes this migration-promoting effect. Although both Y27632 and blebbistatin accelerate wound healing, cell junctional integrity and barrier function are better preserved after blebbistatin treatment, leading to more rapid corneal deturgescence in rabbit corneal endothelial wounding model. Our findings indicate that NMII is a promising therapeutic target in the treatment of CEC dysfunction. KEY MESSAGES: NMII inhibition promotes directional migration and wound healing of CECs in vitro. Lamellipodial protrusion persistence is increased after NMII inhibition. Selective NMII inhibitor preserves junctional integrity better than ROCK inhibitor. Selective NMII inhibitor accelerates corneal deturgescence after wounding in vivo.

Topics: Actin-Related Protein 2-3 Complex; Actins; Amides; Animals; Cattle; Cell Movement; Cells, Cultured; Endothelium, Corneal; Heterocyclic Compounds, 4 or More Rings; Myosin Type II; Pyridines; Rabbits; rho-Associated Kinases; Wound Healing

Topics: Administration, Intravesical; Animals; Cyclophosphamide; Female; Heterocyclic Compounds, 4 or More Rings; Rats; Rats, Wistar; Urinary Bladder; Urodynamics

We identify a novel endothelial membrane behaviour in transgenic zebrafish. Cerebral blood vessels extrude large transient spherical structures that persist for an average of 23 min before regressing into the parent vessel. We term these structures "kugeln", after the German for sphere. Kugeln are only observed arising from the cerebral vessels and are present as late as 28 days post fertilization. Kugeln do not communicate with the vessel lumen and can form in the absence of blood flow. They contain little or no cytoplasm, but the majority are highly positive for nitric oxide reactivity. Kugeln do not interact with brain lymphatic endothelial cells (BLECs) and can form in their absence, nor do they perform a scavenging role or interact with macrophages. Inhibition of actin polymerization, Myosin II, or Notch signalling reduces kugel formation, while inhibition of VEGF or Wnt dysregulation (either inhibition or activation) increases kugel formation. Kugeln represent a novel Notch-dependent NO-containing endothelial organelle restricted to the cerebral vessels, of currently unknown function.

Topics: Actins; Animals; Animals, Genetically Modified; Blood Vessels; Brain; Bridged Bicyclo Compounds, Heterocyclic; Cerebrovascular Circulation; Embryo, Nonmammalian; Endothelial Cells; Gene Expression Regulation, Developmental; Heterocyclic Compounds, 4 or More Rings; Myosin Type II; Neovascularization, Physiologic; Nitric Oxide; Organelles; Polymerization; Receptors, Notch; Signal Transduction; Thiazolidines; Vascular Endothelial Growth Factor A; Wnt Proteins; Zebrafish; Zebrafish Proteins

Omecamtiv mecarbil and blebbistatin perturb the regulatory state of the thick filament in heart muscle. Omecamtiv mecarbil increases contractility at low levels of activation by stabilizing the ON state of the thick filament. Omecamtiv mecarbil decreases contractility at high levels of activation by disrupting the acto-myosin ATPase cycle. Blebbistatin reduces contractility by stabilizing the thick filament OFF state and inhibiting acto-myosin ATPase. Thick filament regulation is a promising target for novel therapeutics in heart disease.. Contraction of heart muscle is triggered by a transient rise in intracellular free calcium concentration linked to a change in the structure of the actin-containing thin filaments that allows the head or motor domains of myosin from the thick filaments to bind to them and induce filament sliding. It is becoming increasingly clear that cardiac contractility is also regulated through structural changes in the thick filaments, although the molecular mechanisms underlying thick filament regulation are still relatively poorly understood. Here we investigated those mechanisms using small molecules - omecamtiv mecarbil (OM) and blebbistatin (BS) - that bind specifically to myosin and respectively activate or inhibit contractility in demembranated cardiac muscle cells. We measured isometric force and ATP utilization at different calcium and small-molecule concentrations in parallel with in situ structural changes determined using fluorescent probes on the myosin regulatory light chain in the thick filaments and on troponin C in the thin filaments. The results show that BS inhibits contractility and actin-myosin ATPase by stabilizing the OFF state of the thick filament in which myosin head domains are more parallel to the filament axis. In contrast, OM stabilizes the ON state of the thick filament, but inhibits contractility at high intracellular calcium concentration by disrupting the actin-myosin ATPase pathway. The effects of BS and OM on the calcium sensitivity of isometric force and filament structural changes suggest that the co-operativity of calcium activation in physiological conditions is due to positive coupling between the regulatory states of the thin and thick filaments.

Topics: Actin Cytoskeleton; Animals; Calcium; Cardiac Myosins; Heterocyclic Compounds, 4 or More Rings; Male; Myocardial Contraction; Myocardium; Myocytes, Cardiac; Rats; Rats, Wistar; Signal Transduction; Urea

Myofibroblasts orchestrate wound healing processes, and if they remain activated, they drive disease progression such as fibrosis and cancer. Besides growth factor signaling, the local extracellular matrix (ECM) and its mechanical properties are central regulators of these processes. It remains unknown whether transforming growth factor-β (TGF-β) and tensile forces work synergistically in up-regulating the transition of fibroblasts into myofibroblasts and whether myofibroblasts undergo apoptosis or become deactivated by other means once tissue homeostasis is reached. We used three-dimensional microtissues grown in vitro from fibroblasts in macroscopically engineered clefts for several weeks and found that fibroblasts transitioned into myofibroblasts at the highly tensed growth front as the microtissue progressively closed the cleft, in analogy to closing a wound site. Proliferation was up-regulated at the growth front, and new highly stretched fibronectin fibers were deposited, as revealed by fibronectin fluorescence resonance energy transfer probes. As the tissue was growing, the ECM underneath matured into a collagen-rich tissue containing mostly fibroblasts instead of myofibroblasts, and the fibronectin fibers were under reduced tension. This correlated with a progressive rounding of cells from the growth front inward, with decreased α-smooth muscle actin expression, YAP nuclear translocation, and cell proliferation. Together, this suggests that the myofibroblast phenotype is stabilized at the growth front by tensile forces, even in the absence of endogenously supplemented TGF-β, and reverts into a quiescent fibroblast phenotype already 10 μm behind the growth front, thus giving rise to a myofibroblast-to-fibroblast transition. This is the hallmark of reaching prohealing homeostasis.

Topics: Actins; Cell Differentiation; Cell Nucleus; Cell Proliferation; Cells, Cultured; Cytoskeleton; Dermis; Extracellular Matrix; Fibronectins; Heterocyclic Compounds, 4 or More Rings; Humans; Myofibroblasts; Tensile Strength; Tissue Engineering; Transforming Growth Factor beta

The mechanosensitivity of neurons in the central nervous system (CNS) is an interesting issue as regards understanding neuronal development and designing compliant materials as neural interfaces between neurons and external devices for treating CNS injuries and disorders. Although neurite initiation from a cell body is known to be the first step towards forming a functional nervous network during development or regeneration, less is known about how the mechanical properties of the extracellular microenvironment affect neuritogenesis. Here, we investigated the filamentous actin (F-actin) cytoskeletal structures of neurons, which are a key factor in neuritogenesis, on gel substrates with a stiffness-controlled substrate, to reveal the relationship between substrate stiffness and neuritogenesis. We found that neuritogenesis was significantly suppressed on a gel substrate with an elastic modulus higher than the stiffness of in vivo brain. Fluorescent images of the F-actin cytoskeletal structures showed that the F-actin organization depended on the substrate stiffness. Circumferential actin meshworks and arcs were formed at the edge of the cell body on the stiff gel substrates unlike with soft substrates. The suppression of F-actin cytoskeleton formation improved neuritogenesis. The results indicate that the organization of neuronal F-actin cytoskeletons is strongly regulated by the mechanical properties of the surrounding environment, and the mechanically-induced F-actin cytoskeletons regulate neuritogenesis.

Topics: Animals; Cells, Cultured; Cytochalasin D; Heterocyclic Compounds, 4 or More Rings; Hippocampus; Neurogenesis; Neurons; Rats; Rats, Wistar

Focal adhesions (FAs) are subcellular regions at the micrometer scale that link the cell to the surrounding microenvironment and control vital cell functions. However, the spatial architecture of FAs remains unclear at the nanometer scale. We used two-color and three-color super-resolution stimulated emission depletion microscopy to determine the spatial distributions and co-localization of endogenous FA components in fibroblasts. Our data indicate that adhesion proteins inside, but not outside, FAs are organized into nanometer size units of multi-protein assemblies. The loss of contractile force reduced the nanoscale co-localization between different types of proteins, while it increased this co-localization between markers of the same type. This suggests that actomyosin-dependent force exerts a nonrandom, specific, control of the localization of adhesion proteins within cell-matrix adhesions. These observations are consistent with the possibility that proteins in cell-matrix adhesions are assembled in nanoscale particles, and that force regulates the localization of the proteins therein in a protein-specific manner. This detailed knowledge of how the organization of FA components at the nanometer scale is linked to the capacity of the cells to generate contractile forces expands our understanding of cell adhesion in health and disease.

Topics: 3T3 Cells; Actomyosin; Animals; Cell Adhesion Molecules; Cell Line; Contractile Proteins; Extracellular Matrix Proteins; Fibroblasts; Focal Adhesions; Heterocyclic Compounds, 4 or More Rings; Humans; Mice; Microscopy, Fluorescence; Multiprotein Complexes; Stress, Mechanical

The biconcave disk shape and deformability of mammalian RBCs rely on the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network of RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by superresolution fluorescence microscopy. MgATP disrupts NMIIA association with the membrane skeleton, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical properties. In addition, the phosphorylation of the RBC NMIIA heavy and light chains in vivo indicates active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disk cell shape. As structures similar to the RBC membrane skeleton exist in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin-F-actin networks.

Topics: Actins; Adenosine Triphosphate; Cell Shape; Erythrocyte Membrane; Heterocyclic Compounds, 4 or More Rings; Humans; Nonmuscle Myosin Type IIA

Physiologically, cells experience and respond to a variety of mechanical stimuli such as rigidity and topography of the extracellular matrix. However, little is known about the effects of substrate curvature on cell behavior. We developed a novel, to our knowledge, method to fabricate cell culture substrates with semicylindrical grooves of negative curvatures (radius of curvature, R

Topics: Actins; Animals; Biomechanical Phenomena; Cell Adhesion; Cell Shape; Dogs; Epithelial Cells; Heterocyclic Compounds, 4 or More Rings; Madin Darby Canine Kidney Cells; Mechanical Phenomena; Mice; Models, Biological; NIH 3T3 Cells

Neural progenitor cells (NPCs), which are apicobasally elongated and densely packed in the developing brain, systematically move their nuclei/somata in a cell cycle-dependent manner, called interkinetic nuclear migration (IKNM): apical during G2 and basal during G1. Although intracellular molecular mechanisms of individual IKNM have been explored, how heterogeneous IKNMs are collectively coordinated is unknown. Our quantitative cell-biological and in silico analyses revealed that tissue elasticity mechanically assists an initial step of basalward IKNM. When the soma of an M-phase progenitor cell rounds up using actomyosin within the subapical space, a microzone within 10 μm from the surface, which is compressed and elastic because of the apical surface's contractility, laterally pushes the densely neighboring processes of non-M-phase cells. The pressed processes then recoil centripetally and basally to propel the nuclei/somata of the progenitor's daughter cells. Thus, indirect neighbor-assisted transfer of mechanical energy from mother to daughter helps efficient brain development.

Topics: Actomyosin; Animals; Biomechanical Phenomena; Cell Cycle; Cell Nucleus; Cell Nucleus Division; Cell Proliferation; Cerebral Cortex; Elasticity; Embryo, Mammalian; Energy Transfer; Heterocyclic Compounds, 4 or More Rings; Mice; Mice, Inbred ICR; Movement; Neural Stem Cells; Neuroepithelial Cells; Time-Lapse Imaging

Human antigen R (HuR) is a RNA-binding protein, which binds to the AU-rich element (ARE) in the 3'-untranslated region (3'-UTR) of certain mRNA and is involved in the export and stabilization of ARE-mRNA. HuR constitutively relocates to the cytoplasm in many cancer cells, however the mechanism of intracellular HuR trafficking is poorly understood. To address this question, we examined the functional role of the cytoskeleton in HuR relocalization. We tested the effect of actin depolymerizing macrolide latrunculin A or myosin II ATPase activity inhibitor blebbistatin for HuR relocalization induced by the vasoactive hormone Angiotensin II in cancer and control normal cells. Western blot and confocal imaging data revealed that both inhibitors attenuated the cytoplasmic HuR in normal cells but no such alteration was observed in cancer cells. Concomitant with changes in intracellular HuR localization, both inhibitors markedly decreased the accumulation and half-lives of HuR target ARE-mRNAs in normal cells, whereas no change was observed in cancer cells. Furthermore, co-immunoprecipitation experiments with HuR proteins revealed clear physical interaction with ß-actin only in normal cells. The current study is the first to verify that cancer cells can implicate a microfilament independent HuR transport. We hypothesized that when cytoskeleton structure is impaired, cancer cells can acquire an alternative HuR trafficking strategy.

Topics: 3' Untranslated Regions; Actins; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cytoplasm; Cytoskeleton; ELAV-Like Protein 1; HeLa Cells; Hep G2 Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Myosins; Neoplasms; Protein Binding; Protein Transport; RNA Stability; RNA, Messenger; Thiazolidines

(S)-Blebbistatin is a micromolar myosin II ATPase inhibitor that is extensively used in research. In search of analogs with improved potency, we have synthesized for the first time C-ring modified analogs. We introduced hydroxymethyl or allyloxymethyl functionalities in search of additional favorable interactions and a more optimal filling of the binding pocket. Unfortunately, the resulting compounds did not significantly inhibit the ATPase activity of rabbit skeletal-muscle myosin II. This and earlier reports suggest that rational design of potent myosin II inhibitors based on the architecture of the blebbistatin binding pocket is an ineffective strategy.

Topics: Animals; Binding Sites; Drug Design; Enzyme Assays; Enzyme Inhibitors; Heterocyclic Compounds, 4 or More Rings; Rabbits; Skeletal Muscle Myosins; Stereoisomerism

How mechanical stress applied to the actin network modifies actin turnover has attracted considerable attention. Actomyosin exerts the major force on the actin network, which has been implicated in actin stability regulation. However, direct monitoring of immediate changes in F-actin stability on alteration of actomyosin contraction has not been achieved. Here we reexamine myosin regulation of actin stability by using single-molecule speckle analysis of actin. To avoid possible errors attributable to actin-binding probes, we employed DyLight-labeled actin that distributes identical to F-actin in lamellipodia. We performed time-resolved analysis of the effect of blebbistatin on actin turnover. Blebbistatin enhanced actin disassembly in lamellipodia of fish keratocytes and lamellar of Xenopus XTC cells at an early stage of the inhibition, indicating that actomyosin contraction stabilizes cellular F-actin. In addition, our data show a previously unrecognized relationship between the actin network-driving force and the actin turnover rates in lamellipodia. These findings point to the power of direct viewing of molecular behavior in elucidating force regulation of actin filament turnover.

Topics: Actin Cytoskeleton; Actins; Animals; Cell Movement; Goldfish; Half-Life; Heterocyclic Compounds, 4 or More Rings; Keratinocytes; Myosins; Pseudopodia; Single Molecule Imaging; Time-Lapse Imaging

Topics: Amides; Cell Line; Cell Movement; Heterocyclic Compounds, 4 or More Rings; Humans; Keratinocytes; Pyridines; rho GTP-Binding Proteins; rho-Associated Kinases; Signal Transduction; Wound Healing

Essentials Endothelial activation initiates multiple processes, including hemostasis and inflammation. The molecules that contribute to these processes are co-stored in secretory granules. How can the cells control release of granule content to allow differentiated responses? Selected agonists recruit an exocytosis-linked actin ring to boost release of a subset of cargo.. Background Endothelial cells harbor specialized storage organelles, Weibel-Palade bodies (WPBs). Exocytosis of WPB content into the vascular lumen initiates primary hemostasis, mediated by von Willebrand factor (VWF), and inflammation, mediated by several proteins including P-selectin. During full fusion, secretion of this large hemostatic protein and smaller pro-inflammatory proteins are thought to be inextricably linked. Objective To determine if secretagogue-dependent differential release of WPB cargo occurs, and whether this is mediated by the formation of an actomyosin ring during exocytosis. Methods We used VWF string analysis, leukocyte rolling assays, ELISA, spinning disk confocal microscopy, high-throughput confocal microscopy and inhibitor and siRNA treatments to demonstrate the existence of cellular machinery that allows differential release of WPB cargo proteins. Results Inhibition of the actomyosin ring differentially effects two processes regulated by WPB exocytosis; it perturbs VWF string formation but has no effect on leukocyte rolling. The efficiency of ring recruitment correlates with VWF release; the ratio of release of VWF to small cargoes decreases when ring recruitment is inhibited. The recruitment of the actin ring is time dependent (fusion events occurring directly after stimulation are less likely to initiate hemostasis than later events) and is activated by protein kinase C (PKC) isoforms. Conclusions Secretagogues differentially recruit the actomyosin ring, thus demonstrating one mechanism by which the prothrombotic effect of endothelial activation can be modulated. This potentially limits thrombosis whilst permitting a normal inflammatory response. These results have implications for the assessment of WPB fusion, cargo-content release and the treatment of patients with von Willebrand disease.

Topics: 1-Methyl-3-isobutylxanthine; Actomyosin; Cytochalasins; Endothelial Cells; Epinephrine; Exocytosis; Hemostasis; Heterocyclic Compounds, 4 or More Rings; Histamine; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Leukocyte Rolling; P-Selectin; Protein Conformation; RNA Interference; RNA, Small Interfering; Tetradecanoylphorbol Acetate; von Willebrand Factor; Weibel-Palade Bodies

Crawling migration plays an essential role in a variety of biological phenomena, including development, wound healing, and immune system function. Keratocytes are wound-healing cells in fish skin. Expansion of the leading edge of keratocytes and retraction of the rear are respectively induced by actin polymerization and contraction of stress fibers in the same way as for other cell types. Interestingly, stress fibers in keratocytes align almost perpendicular to the migration-direction. It seems that in order to efficiently retract the rear, it is better that the stress fibers align parallel to it. From the unique alignment of stress fibers in keratocytes, we speculated that the stress fibers may play a role for migration other than the retraction. Here, we reveal that the stress fibers are stereoscopically arranged so as to surround the cytoplasm in the cell body; we directly show, in sequential three-dimensional recordings, their rolling motion during migration. Removal of the stress fibers decreased migration velocity and induced the collapse of the left-right balance of crawling migration. The rotation of these stress fibers plays the role of a "wheel" in crawling migration of keratocytes.

Topics: Animals; Cell Movement; Cells, Cultured; Cichlids; Fishes; Heterocyclic Compounds, 4 or More Rings; Keratinocytes; Stress Fibers; Wound Healing

Cyclic interactions between myosin II motors and actin filaments driven by ATP turnover underlie muscle contraction and have key roles in the motility of nonmuscle cells. A remaining enigma in the understanding of this interaction is the relationship between the force-generating structural change and the release of the ATP-hydrolysis product, inorganic phosphate (Pi), from the active site of myosin. Here, we use the small molecular compound blebbistatin to probe otherwise hidden states and transitions in this process. Different hypotheses for the Pi release mechanism are tested by interpreting experimental results from in vitro motility assays and isolated muscle fibers in terms of mechanokinetic actomyosin models. The data fit with ideas that actomyosin force generation is preceded by Pi release, which in turn is preceded by two serial transitions after/coincident with cross-bridge attachment. Blebbistatin changes the rate limitation of the cycle from the first to the second of these transitions, uncovering functional roles of an otherwise short-lived pre-power stroke state that has been implicated by structural data.

Topics: Actins; Animals; Biomechanical Phenomena; Heterocyclic Compounds, 4 or More Rings; Mechanical Phenomena; Models, Molecular; Myosins; Protein Conformation; Rabbits

Using pharmacologic and genetic approaches targeting actin or the actin-driving molecular motor, nonmuscle myosin II (NMII), we previously discovered an immediate, retrieval-independent, and long-lasting disruption of methamphetamine- (METH-) and amphetamine-associated memories. A single intrabasolateral amygdala complex infusion or systemic administration of the NMII inhibitor Blebbistatin (Blebb) is sufficient to produce this disruption, which is selective, having no retrieval-independent effect on memories for fear, food reward, cocaine, or morphine. However, it was unclear if Blebb treatment would disrupt memories of other stimulants and amphetamine class drugs, such as nicotine (NIC) or mephedrone (MEPH; bath salts). Moreover, many individuals abuse multiple drugs, but it was unknown if Blebb could disrupt polydrug memories, or if the inclusion of another substance would render Blebb no longer able to disrupt METH-associated memories. Therefore, the present study had two primary goals: (1) to determine the ability of Blebb to disrupt NIC- or MEPH-associated memories, and (2) to determine the ability of METH to modify other unconditioned stimulus (US) associations' susceptibility to Blebb. To this end, using the conditional place preference model, mice were conditioned to NIC and MEPH alone or METH in combination with NIC, morphine, or foot shock. We report that, unlike METH, there was no retrieval-independent effect of Blebb on NIC- or MEPH-associated memories. However, similar to cocaine, reconsolidation of the memory for both drugs was disrupted. Further, when combined with METH administration, NIC- and morphine-, but not fear-, associated memories were rendered susceptible to disruption by Blebb. Given the high rate of polydrug use and the resurgence of METH use, these results have important implications for the treatment of substance use disorder.

Topics: Animals; Central Nervous System Stimulants; Conditioning, Classical; Heterocyclic Compounds, 4 or More Rings; Male; Memory Consolidation; Mental Recall; Methamphetamine; Mice; Mice, Inbred C57BL; Nicotine; Nonmuscle Myosin Type IIA; Nonmuscle Myosin Type IIB; Peripheral Nervous System Agents

Mitochondrial fission is the essential mechanisms of myocardial ischemia/reperfusion (MI/R)-induced cardiomyocytes apoptosis. Myosin II plays a key role in fission due to the recruitment and actomyosin constriction at the fission site in U2OS cells. However, the role of myosin IIA-actin interaction in regulating MI/R-induced cardiomyocytes mitochondrial fission and apoptosis remains to be fully elucidated.. When cardiomyocytes are exposed to simulated I/R injury, the myosin IIA protein translocated from the juxtamembrane to the cytoplasm, interacted with actin filaments, formed stress fibers and generated contractile forces. Treatment with the myosin II inhibitor blebbistatin attenuated the myosin IIA-actin complex induced actomyosin contractility and prevented cardiomyocytes apoptosis as reflected by inhibition of cleaved caspase-3 expression, normalization of Bcl-2/Bax levels and decreased apoptotic cells. Meanwhile, blebbistatin inhibited the activation of PINK1/Parkin pathway and ameliorated mitochondrial fission as evidenced by improvement of mitochondrial morphology, inhibition of Drp1 phosphorylation at Ser616 and translocation. Furthermore, CRISPR/Cas9 knockout of myosin IIA blocked I/R-induced apoptosis, suppressed PINK1/Parkin pathway and reduced mitochondrial fission. Importantly, blebbistatin attenuated myocardial apoptosis, inhibited myosin IIA-actin interaction and PINK1/Parkin pathway, suppressed myocardial ultrastructure abnormalities and mitochondrial fission in a mouse MI/R injury model.. Inhibition of actomyosin contractility induced by myosin IIA-actin interaction could impede myocardial apoptosis and MI/R injury via PINK1/Parkin pathway and mitochondrial fission modulation both in vitro and in vivo, which may be applicable for the development of therapies for cardiovascular diseases.

Topics: Animals; Apoptosis; Cells, Cultured; Gene Knockout Techniques; Heterocyclic Compounds, 4 or More Rings; Male; Mice; Mice, Inbred ICR; Mitochondrial Dynamics; Myocytes, Cardiac; Nonmuscle Myosin Type IIA; Protein Kinases; Rats; Reperfusion Injury; Signal Transduction; Ubiquitin-Protein Ligases

Topics: Adenoviridae; Animals; Biosensing Techniques; Cyclic AMP-Dependent Protein Kinases; Fluorescence Resonance Energy Transfer; Heterocyclic Compounds, 4 or More Rings; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myocytes, Cardiac; Primary Cell Culture; Rabbits; Rats, Zucker; Signal Transduction

Shear forces between cells occur during global changes in multicellular organization during morphogenesis and tissue growth, yet how cells sense shear forces and propagate a response across a tissue is unknown. We found that applying exogenous shear at the midline of an epithelium induced a local, short-term deformation near the shear plane, and a long-term collective oscillatory movement across the epithelium that spread from the shear-plane and gradually dampened. Inhibiting actomyosin contraction or E-cadherin

Topics: Actins; Actomyosin; Animals; Cadherins; Cell Adhesion; Cell Count; Cell Movement; Depsipeptides; Dogs; Epithelial Cells; Epithelium; Heterocyclic Compounds, 4 or More Rings; Madin Darby Canine Kidney Cells; Rheology; Stress, Mechanical

Cytoplasmic flows are an ubiquitous feature of biological systems, in particular in large cells, such as oocytes and eggs in early animal development. Here we show that cytoplasmic flows in starfish oocytes, which can be imaged well with transmission light microscopy, are fully determined by the cortical dynamics during surface contraction waves. We first show that the dynamics of the oocyte surface is highly symmetric around the animal-vegetal axis. We then mathematically solve the Stokes equation for flows inside a deforming sphere using the measured surface displacements as boundary conditions. Our theoretical predictions agree very well with the intracellular flows quantified by particle image velocimetry, proving that during this stage the starfish cytoplasm behaves as a simple Newtonian fluid on the micrometer scale. We calculate the pressure field inside the oocyte and find that its gradient is too small as to explain polar body extrusion, in contrast to earlier suggestions. Myosin II inhibition by blebbistatin confirms this conclusion, because it diminishes cell shape changes and hydrodynamic flow, but does not abolish polar body formation.

Topics: Actins; Algorithms; Animals; Cytoplasm; Heterocyclic Compounds, 4 or More Rings; Imaging, Three-Dimensional; Models, Theoretical; Myosin Type II; Normal Distribution; Oocytes; Polar Bodies; Rotation; Seawater; Starfish; Surface Properties

As cells with aberrant force-generating phenotypes can directly lead to disease, cellular force-generation mechanisms are high-value targets for new therapies. Here, we show that single-cell force sensors embedded in elastomers enable single-cell force measurements with ~100-fold improvement in throughput than was previously possible. The microtechnology is scalable and seamlessly integrates with the multi-well plate format, enabling highly parallelized time-course studies. In this regard, we show that airway smooth muscle cells isolated from fatally asthmatic patients have innately greater and faster force-generation capacity in response to stimulation than healthy control cells. By simultaneously tracing agonist-induced calcium flux and contractility in the same cell, we show that the calcium level is ultimately a poor quantitative predictor of cellular force generation. Finally, by quantifying phagocytic forces in thousands of individual human macrophages, we show that force initiation is a digital response (rather than a proportional one) to the proper immunogen. By combining mechanobiology at the single-cell level with high-throughput capabilities, this microtechnology can support drug-discovery efforts for clinical conditions associated with aberrant cellular force generation.

Topics: Asthma; Cell Differentiation; Cells, Cultured; Elastomers; Fluorescent Dyes; Formoterol Fumarate; Heterocyclic Compounds, 4 or More Rings; Humans; Macrophages; Mesenchymal Stem Cells; Microscopy, Fluorescence; Myocardial Contraction; Myocytes, Smooth Muscle; Phagocytosis; Single-Cell Analysis

In neurons, axons possess a molecularly defined and highly organised proximal region - the axon initial segment (AIS) - that is a key regulator of both electrical excitability and cellular polarity. Despite existing as a large, dense structure with specialised cytoskeletal architecture, the AIS is surprisingly plastic, with sustained alterations in neuronal activity bringing about significant alterations to its position, length or molecular composition. However, although the upstream activity-dependent signalling pathways that lead to such plasticity have begun to be elucidated, the downstream mechanisms that produce structural changes at the AIS are completely unknown. Here, we use dissociated cultures of rat hippocampus to show that two forms of AIS plasticity in dentate granule cells - long-term relocation, and more rapid shortening - are completely blocked by treatment with blebbistatin, a potent and selective myosin II ATPase inhibitor. These data establish a link between myosin II and AIS function, and suggest that myosin II's primary role at the structure may be to effect activity-dependent morphological alterations.

Topics: Animals; Axon Initial Segment; Calcineurin; Cells, Cultured; Central Nervous System Agents; Dentate Gyrus; Endocytosis; Heterocyclic Compounds, 4 or More Rings; Myosin Type II; Neuronal Plasticity; Rats, Wistar

(S)-Blebbistatin is a widely used research tool to study myosin II, an important regulator of many motility based diseases. Its potency is too low to be of clinical relevance, but identification of analogs with enhanced potency could deliver leads for targeted pharmacotherapeutics. This, however, requires a profound insight into the structure-activity relationship of the (S)-blebbistatin scaffold. Therefore, new D-ring modified (S)-blebbistatin derivatives were prepared to extend the existing small library of analogs. These molecules were obtained via an improved synthesis pathway and their myosin II inhibitory properties were evaluated in vitro. Finally, all new and known D-ring modified (S)-blebbistatin analogs were compared and the most potent ones underwent a screening of their physicochemical properties.

Topics: Caco-2 Cells; Cell Membrane Permeability; Dose-Response Relationship, Drug; Enzyme Inhibitors; Heterocyclic Compounds, 4 or More Rings; Humans; Molecular Structure; Myosin Type II; Stereoisomerism; Structure-Activity Relationship

Myosin II is an interesting target for therapeutic intervention, as it is involved in a large number of motility-based diseases. (S)-Blebbistatin is a known micromolar inhibitor of this protein. A new series of (S)-blebbistatin derivatives with a modified A-ring was synthesized and the myosin II inhibitory properties were evaluated in vitro. In this way, we gained insight into the influence of structural modifications in this part of the scaffold on myosin II inhibitory potency. Our results indicate there are few possibilities for potency enhancement via ring A modification of the blebbistatin scaffold.

Topics: Dictyostelium; Dose-Response Relationship, Drug; Enzyme Inhibitors; Heterocyclic Compounds, 4 or More Rings; Molecular Structure; Myosin Type II; Structure-Activity Relationship

The strain-induced reorientation response of cyclically stretched cells has been well characterized in uniform strain fields. In the present study, we comprehensively analyse the behaviour of human fibroblasts subjected to a highly non-uniform strain field within a polymethylsiloxane microdevice. Our results indicate that the strain gradient amplitude and direction regulate cell reorientation through a coordinated gradient avoidance response. We provide critical evidence that strain gradient is a key physical cue that can guide cell organization. Specifically, our work suggests that cells are able to pinpoint the location under the cell of multiple physical cues and integrate this information (strain and strain gradient amplitudes and directions), resulting in a coordinated response. To gain insight into the underlying mechanosensing processes, we studied focal adhesion reorganization and the effect of modulating myosin-II contractility. The extracted focal adhesion orientation distributions are similar to those obtained for the cell bodies, and their density is increased by the presence of stretching forces. Moreover, it was found that the myosin-II activity promoter calyculin-A has little effect on the cellular response, while the inhibitor blebbistatin suppresses cell and focal adhesion alignment and reduces focal adhesion density. These results confirm that similar internal structures involved in sensing and responding to strain direction and amplitude are also key players in strain gradient mechanosensing and avoidance.

Topics: Anisotropy; Bioengineering; Biomechanical Phenomena; Cell Polarity; Cellular Microenvironment; Equipment Design; Fibroblasts; Focal Adhesions; Heterocyclic Compounds, 4 or More Rings; Humans; Marine Toxins; Mechanotransduction, Cellular; Myosin Type II; Oxazoles; Stress, Mechanical

Adiponectin is a protein secreted by white adipocytes that plays an important role in insulin action, energy homeostasis and the development of atherosclerosis. The intracellular localization and trafficking of GLUT4 and leptin in adipocytes has been well studied, but little is known regarding the intracellular trafficking of adiponectin. Recent studies have demonstrated that constitutive adiponectin secretion is dependent on PIP2 levels and the integrity of cortical F-actin. Non-muscle myosin II is an actin-based motor that is associated with membrane vesicles and participates in vesicular trafficking in mammalian cells. Therefore, we investigated the role of myosin II in the trafficking and secretion of adiponectin in 3T3-L1 adipocytes. Confocal microscopy revealed that myosin IIA and IIB were dispersed throughout the cytoplasm of the adipocyte. Both myosin isoforms were localized in the Golgi/TGN region as evidenced by colocalization with the cis-Golgi marker, p115 and the trans-Golgi marker, γ-adaptin. Inhibition of myosin II activity by blebbistatin or actin depolymerization by latrunculin B dispersed myosin IIA and IIB towards the periphery while significantly inhibiting adiponectin secretion. Therefore, the constitutive trafficking and secretion of adiponectin in 3T3-L1 adipocytes occurs by an actin-dependent mechanism that involves the actin-based motors, myosin IIA and IIB.

Topics: 3T3-L1 Cells; Adipocytes; Adiponectin; Animals; Biological Transport; Bridged Bicyclo Compounds, Heterocyclic; Cells, Cultured; Dose-Response Relationship, Drug; Heterocyclic Compounds, 4 or More Rings; Mice; Nonmuscle Myosin Type IIA; Nonmuscle Myosin Type IIB; Structure-Activity Relationship; Thiazolidines

Myosin-1 (Myo1) represents a mechanical link between the membrane and actin-cytoskeleton in animal cells. We have studied the effect of Myo1 inhibitor PClP in 1-8 cell Zebrafish embryos. Our results indicate a unique involvement of Myo1 in early development of Zebrafish embryos. Inhibition of Myo1 (by PClP) and Myo2 (by Blebbistatin) lead to arrest in cell division. While Myo1 isoforms appears to be important for both the formation and the maintenance of cleavage furrows, Myo2 is required only for the formation of furrows. We found that the blastodisc of the embryo, which contains a thick actin cortex (~13 μm), is loaded with cortical Myo1. Myo1 appears to be crucial for maintaining the blastodisc morphology and the actin cortex thickness. In addition to cell division and furrow formation, inhibition of Myo1 has a drastic effect on the dynamics and distribution of lipid droplets (LDs) in the blastodisc near the cleavage furrow. All these results above are effects of Myo1 inhibition exclusively; Myo2 inhibition by blebbistatin does not show such phenotypes. Therefore, our results demonstrate a potential role for Myo1 in the maintenance and formation of furrow, blastodisc morphology, cell-division and LD organization within the blastodisc during early embryogenesis.

Topics: Actin Cytoskeleton; Actins; Animals; Blastomeres; Blotting, Western; Cell Division; Cell Membrane; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Heterocyclic Compounds, 4 or More Rings; Hydrocarbons, Chlorinated; Lipid Droplets; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Myosin Heavy Chains; Pyrroles; Reverse Transcriptase Polymerase Chain Reaction; Zebrafish; Zebrafish Proteins

Blebbistatin is a potent and specific inhibitor of the motor functions of class II myosins, including striated muscle myosin and nonmuscle myosin-2 (NM2). However, the blebbistatin inhibition of NM2c has not been assessed and remains controversial with respect to its efficacy with smooth muscle myosin (SmM), which is highly homologous to NM2. To clarify these issues, we analyzed the effects of blebbistatin on the motor activities of recombinant SmM and three NM2s (NM2a, -2b, and -2c). We found that blebbistatin potently inhibits the actin-activated ATPase activities of SmM and NM2s with following IC

Topics: Amino Acid Substitution; Animals; Avian Proteins; Chickens; Heterocyclic Compounds, 4 or More Rings; Humans; Mice; Mutation, Missense; Nonmuscle Myosin Type IIB; Smooth Muscle Myosins

External chest impacts (commotio cordis) can cause mechanically induced premature ventricular excitation (PVE. Subcontusional mechanical stimuli were applied to isolated rabbit hearts during optical voltage mapping, combined with pharmacological block of ATP-inactivated potassium or stretch-activated cation-nonselective channels. We demonstrate that local mechanical stimulation reliably triggers PVE. Local mechanical tissue deformation determines PVE

Topics: Animals; Commotio Cordis; Electrocardiography; Female; Heart Conduction System; Heterocyclic Compounds, 4 or More Rings; Ion Channels; Mechanotransduction, Cellular; Potassium Channel Blockers; Pressoreceptors; Rabbits; Tachycardia, Ventricular; Wounds, Nonpenetrating

SM22α, also named transgelin, is an actin filament-associated protein in smooth muscle and fibroblasts. Three decades after its discovery, the biological function of SM22α remains under investigation. Here we report a novel finding that the expression and degradation of SM22α/transgelin are regulated by mechanical tension. Following a mass spectrometry identification of SM22α degradation in isolated and tension-unloaded mouse aorta, we developed specific monoclonal antibodies to study the regulation of SM22α in human fetal lung myofibroblast line MRC-5 and primary cultures of neonatal mouse skin fibroblasts. The level of SM22α is positively related to the mechanical tension in the cytoskeleton produced by the myosin II motor in response to the stiffness of the culture matrix. Quantitative reverse transcription polymerase chain reaction demonstrated that the expression of SM22α is regulated at the transcriptional level. This mechanical regulation resembles that of calponin 2, another actin filament-associated protein. Immunofluorescent staining co-localized SM22α with F-actin, myosin, and calponin 2 in mouse skin fibroblasts. The close phylogenetic relationship between SM22α and the calponin family supports that SM22α is a calponin-like regulatory protein. The level of SM22α is decreased in skin fibroblasts isolated from calponin 2 knockout mice, suggesting interrelated regulation and function of the two proteins. On the other hand, SM22α expression was maximized at a matrix stiffness higher than that for calponin 2 in the same cell type, indicating differentiated regulation and tension responsiveness. The novel mechanoregulation of SM22α/transgelin lays the groundwork for understanding its cellular functions.

Topics: Animals; Biomarkers; Calcium-Binding Proteins; Calpain; Calponins; Cell Line; Cells, Cultured; Cytoskeleton; Enzyme Inhibitors; Extracellular Matrix; Gene Expression Regulation; Heterocyclic Compounds, 4 or More Rings; Humans; Keratinocytes; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Microfilament Proteins; Muscle Proteins; Myofibroblasts; Myosin Type II; Organ Specificity; Pliability; Protein Transport

Engineered 3D cardiac tissue constructs (ECTCs) can replicate complex cardiac physiology under normal and pathological conditions. Currently, most measurements of ECTC contractility are either made isometrically, with fixed length and without control of the applied force, or auxotonically against a variable force, with the length changing during the contraction. The "I-Wire" platform addresses the unmet need to control the force applied to ECTCs while interrogating their passive and active mechanical and electrical characteristics. A six-well plate with inserted PDMS casting molds containing neonatal rat cardiomyocytes cultured with fibrin for 13-15days is mounted on the motorized mechanical stage of an inverted microscope equipped with a fast sCMOS camera. A calibrated flexible probe provides strain load of the ECTC via lateral displacement, and the microscope detects the deflections of both the probe and the ECTC. The ECTCs exhibited longitudinally aligned cardiomyocytes with well-developed sarcomeric structure, recapitulated the Frank-Starling force-tension relationship, and demonstrated expected transmembrane action potentials, electrical and mechanical restitutions, and responses to both β-adrenergic stimulation and blebbistatin. The I-Wire platform enables creation and mechanical and electrical characterization of ECTCs, and hence can be valuable in the study of cardiac diseases, drug screening, drug development, and the qualification of cells for tissue-engineered regenerative medicine.. There is a growing interest in creating engineered heart tissue constructs for basic cardiac research, applied research in cardiac pharmacology, and repair of damaged hearts. We address an unmet need to characterize fully the performance of these tissues with our simple "I-Wire" assay that allows application of controlled forces to three-dimensional cardiac fiber constructs and measurement of both the electrical and mechanical properties of the construct. The advantage of I-Wire over other approaches is that the constructs being measured are truly three-dimensional, rather than a single layer of cells grown within a microfluidic device. We anticipate that the I-Wire will be extremely useful for the evaluation of myocardial constructs created using cardiomyocytes derived from human induced pluripotent stem cells.

Topics: Action Potentials; Animals; Cells, Cultured; Elasticity; Heart; Heterocyclic Compounds, 4 or More Rings; Isoproterenol; Lab-On-A-Chip Devices; Myocardial Contraction; Phenotype; Rats, Sprague-Dawley; Tissue Engineering; Tissue Scaffolds

Cortical actin is a thin layer of filamentous (F-)actin that lies beneath the plasma membrane, and its role in pathophysiology remains unclear. We investigated the subcellular localization of cortical actin by the histopathological and experimental studies of lung adenocarcinomas.. The subcellular localization of cortical actin was studied in surgically resected lung adenocarcinomas tissues and in 3-dimensionally cultured lung adenocarcinoma A549 cells.. In normal type II alveolar cells and the bronchiolar epithelium, cortical actin was localized to the apical-side cytoplasm. In invasive adenocarcinoma cells, cortical actin was frequently localized to the matrix side. The degree of cortical actin localized to the matrix side was associated with the loss of basement membrane and a poor prognosis. In A549 cell spheroids cultured in a type I collagen and basement membrane extract Matrigel™ mixed gel, cortical F-actin was localized to the matrix side with phosphorylated myosin light chain. Super-resolution and electron microscopy results suggest that compact wrinkling of the plasma membrane by myosin-mediated F-actin contraction is an explanation for cortical actin accumulation at the matrix side. The myosin II inhibitor blebbistatin suppressed the 3-dimensional collective migration of A549 cells induced by constitutively active Cdc42 and MT1-MMP.. Cortical actin accumulation at the matrix-side cytoplasm of cancer cells occurs in invasive lung adenocarcinomas and it possibly participates in the migration of cancer cells through myosin-mediated contraction.

Topics: A549 Cells; Actins; Adenocarcinoma; Adenocarcinoma of Lung; Cell Membrane; Cell Movement; Cytoplasm; Heterocyclic Compounds, 4 or More Rings; Humans; Immunohistochemistry; Lung Neoplasms; Myosins; Neoplasm Invasiveness; Neoplasm Metastasis; Prognosis

Hepatocellular carcinoma (HCC) was usually coupled with increased stiffness of the extracellular matrix (ECM) and elevated level of transforming growth factor-β1 (TGF-β1). However, the mechanism by which substrate rigidity modulated TGF-β1 signaling transduction remained unknown. This paper investigated the molecular mechanism of how matrix stiffness regulating TGF-β1 signaling in HCC cells. By means of stiffness tunable collagen I-coated polyacrylamide (PA) gels, we found that the expressions of β1 integrin, p-FAK

Topics: Amides; Carcinoma, Hepatocellular; Cell Line, Tumor; Cytoskeleton; Extracellular Matrix; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Integrin beta1; Liver Neoplasms; Oligopeptides; Protein Binding; Pyridines; rho GTP-Binding Proteins; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta1; Up-Regulation

Mechanical properties of cells are known to be influenced by the actin cytoskeleton. In this article, the action of drugs that interact with the actin cortex is investigated by tether extraction and rheology experiments using optical tweezers. The influences of Blebbistatin, Cytochalasin D and Jasplakinolide on the cell mechanical properties are evaluated. The results, in contradiction to current views for Jasplakinolide, show that all three drugs and treatments destabilize the actin cytoskeleton, decreasing the cell membrane tension. The cell membrane bending modulus increased when the actin cytoskeleton was disorganized by Cytochalasin D. This effect was not observed for Blebbistatin and Jasplakinolide. All drugs decreased by two-fold the cell viscoelastic moduli, but only Cytochalasin D was able to alter the actin network into a more fluid-like structure. The results can be interpreted as the interplay between the actin network and the distribution of myosins as actin cross-linkers in the cytoskeleton. This information may contribute to a better understanding of how the membrane and cytoskeleton are involved in cell mechanical properties, underlining the role that each one plays in these properties.

Topics: Actin Cytoskeleton; Animals; Biomechanical Phenomena; Cell Membrane; Cytochalasin D; Depsipeptides; Elasticity; Heterocyclic Compounds, 4 or More Rings; Humans; Mice; Myosins; NIH 3T3 Cells; Optical Tweezers; Rheology; Viscosity

Memories associated with drug use can trigger strong motivation for the drug, which increases relapse vulnerability in substance use disorder (SUD). Currently there are no treatments for relapse to abuse of psychostimulants, such as methamphetamine (METH). We previously reported that storage of memories associated with METH, but not those for fear or food reward, and the concomitant spine density increase are disrupted in a retrieval-independent manner by depolymerizing actin in the basolateral amygdala complex (BLC) of adult male rats and mice. Similar results are achieved in males through intra-BLC or systemic inhibition of nonmuscle myosin II (NMII), a molecular motor that directly drives actin polymerization. Given the substantial differences in physiology between genders, we sought to determine if this immediate and selective disruption of METH-associated memory extends to adult females. A single intra-BLC infusion of the NMII inhibitor Blebbistatin (Blebb) produced a long-lasting disruption of context-induced drug seeking for at least 30days in female rats that mirrored our prior results in males. Furthermore, a single systemic injection of Blebb prior to testing disrupted METH-associated memory and the concomitant increase in BLC spine density in females. Importantly, as in males, the same manipulation had no effect on an auditory fear memory or associated BLC spine density. In addition, we established that the NMII-based disruption of METH-associated memory extends to both male and female adolescents. These findings provide further support that small molecular inhibitors of NMII have strong therapeutic potential for the prevention of relapse to METH abuse triggered by associative memories.

Topics: Animals; Central Nervous System Stimulants; Dendritic Spines; Drug-Seeking Behavior; Female; Heterocyclic Compounds, 4 or More Rings; Memory; Methamphetamine; Nonmuscle Myosin Type IIA; Nonmuscle Myosin Type IIB; Rats; Reward; Self Administration

Depolymerizing actin in the amygdala through nonmuscle myosin II inhibition (NMIIi) produces a selective, lasting, and retrieval-independent disruption of the storage of methamphetamine-associated memories. Here we report a similar disruption of memories associated with amphetamine, but not cocaine or morphine, by NMIIi. Reconsolidation appeared to be disrupted with cocaine. Unlike in the amygdala, methamphetamine-associated memory storage was not disrupted by NMIIi in the hippocampus, nucleus accumbens, or orbitofrontal cortex. NMIIi in the hippocampus did appear to disrupt reconsolidation. Identification of the unique mechanisms responsible for NMII-mediated, amygdala-dependent disruption of memory storage associated with the amphetamine class may enable induction of retrieval-independent vulnerability to other pathological memories.

Topics: Analysis of Variance; Anesthetics, Local; Animals; Brain; Central Nervous System Stimulants; Cocaine; Conditioning, Operant; Dendritic Spines; Green Fluorescent Proteins; Heterocyclic Compounds, 4 or More Rings; Memory Disorders; Mental Recall; Methamphetamine; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microinjections; Morphine Derivatives; Myosin Type II

The endothelial cytoskeleton is a barrier for leukocyte transendothelial migration (TEM). Mononuclear and polymorphonuclear leukocytes generate gaps of similar micron-scale size when squeezing through inflamed endothelial barriers in vitro and in vivo. To elucidate how leukocytes squeeze through these barriers, we co-tracked the endothelial actin filaments and leukocyte nuclei in real time. Nuclear squeezing involved either preexistent or de novo-generated lobes inserted into the leukocyte lamellipodia. Leukocyte nuclei reversibly bent the endothelial actin stress fibers. Surprisingly, formation of both paracellular gaps and transcellular pores by squeezing leukocytes did not require Rho kinase or myosin II-mediated endothelial contractility. Electron-microscopic analysis suggested that nuclear squeezing displaced without condensing the endothelial actin filaments. Blocking endothelial actin turnover abolished leukocyte nuclear squeezing, whereas increasing actin filament density did not. We propose that leukocyte nuclei must disassemble the thin endothelial actin filaments interlaced between endothelial stress fibers in order to complete TEM.

Topics: Actin Cytoskeleton; Actins; Amides; Antigens, CD; Cadherins; Cells, Cultured; Endothelial Cells; Endothelium, Vascular; Heterocyclic Compounds, 4 or More Rings; Humans; Interleukin-1beta; Leukocytes; Muscle Contraction; Myosin Type II; Neutrophils; Pyridines; rho-Associated Kinases; T-Lymphocytes; Time-Lapse Imaging; Transendothelial and Transepithelial Migration

In search of myosin II inhibitors with superior research tool properties, a chemical optimization campaign of the blebbistatin scaffold was conducted in this paper. (S)-Blebbistatin is the best known small-molecule inhibitor of myosin II ATPase activity. Unfortunately, as a research tool this compound has several deficiencies: it is photolabile and (photo)toxic, has low water solubility, and its (fluorescent) precipitates interfere in (fluorescence) readouts. In view of obtaining tool compounds with improved properties, both enantiomers of a series of D-ring modified polar analogs were prepared. We identified (S)-3'-hydroxyblebbistatin (S)-2 and (S)-3'-aminoblebbistatin (S)-3 as two myosin II inhibitors with a 30-fold higher water solubility than (S)-blebbistatin. These molecules furthermore do not cause interference in (fluorescence) readouts. (S)-2 and (S)-3 thus are superior alternatives to (S)-blebbistatin as research tools to study myosin II.

Topics: Dose-Response Relationship, Drug; Drug Discovery; Enzyme Inhibitors; Heterocyclic Compounds, 4 or More Rings; Humans; Molecular Structure; Myosin Type II; Structure-Activity Relationship; Tumor Cells, Cultured

Memories associated with drug use increase vulnerability to relapse in substance use disorder (SUD), and there are no pharmacotherapies for the prevention of relapse. Previously, we reported a promising finding that storage of memories associated with methamphetamine (METH), but not memories for fear or food reward, is vulnerable to disruption by actin depolymerization in the basolateral amygdala complex (BLC). However, actin is not a viable therapeutic target because of its numerous functions throughout the body. Here we report the discovery of a viable therapeutic target, nonmuscle myosin IIB (NMIIB), a molecular motor that supports memory by directly driving synaptic actin polymerization. A single intra-BLC treatment with Blebbistatin (Blebb), a small-molecule inhibitor of class II myosin isoforms, including NMIIB, produced a long-lasting disruption of context-induced drug seeking (at least 30 days). Further, postconsolidation genetic knockdown of Myh10, the heavy chain of the most highly expressed NMII in the BLC, was sufficient to produce METH-associated memory loss. Blebb was found to be highly brain penetrant. A single systemic injection of the compound selectively disrupted the storage of METH-associated memory and reversed the accompanying increase in BLC spine density. This effect was specific to METH-associated memory, as it had no effect on an auditory fear memory. The effect was also independent of retrieval, as METH-associated memory was disrupted 24 h after a single systemic injection of Blebb delivered in the home cage. Together, these results argue for the further development of small-molecule inhibitors of NMII as potential therapeutics for the prevention of SUD relapse triggered by drug associations.

Topics: Amphetamine-Related Disorders; Amygdala; Animals; Central Nervous System Agents; Conditioning, Psychological; Disease Models, Animal; Drug-Seeking Behavior; Exploratory Behavior; Fear; Gene Knockdown Techniques; Heterocyclic Compounds, 4 or More Rings; Male; Memory; Methamphetamine; Mice; Motor Activity; Nonmuscle Myosin Type IIB; Rats; Secondary Prevention; Self Administration; Spatial Behavior

Local anesthetics are administered intraarticularly for pain control in orthopedic clinics and surgeries. Although previous studies have shown that local anesthetics can be toxic to chondrocytes, the underlying cellular mechanisms remain unclear. The present study investigates acute cellular responses associated with lidocaine-induced toxicity to articular chondrocytes. Rabbit articular chondrocytes were exposed to lidocaine and their morphological changes were monitored with live cell microscopy. The viability of chondrocytes was evaluated using a fluorescence based LIVE/DEAD assay. Acute treatment of chondrocytes with lidocaine (3-30 mM) induced spherical protrusions on the cell surface (so called "membrane blebbing") in a time- and concentration-dependent manner. The concentration-response relationship for the lidocaine effect was shifted leftward by elevating extracellular pH, as expected for the non-ionized lidocaine being involved in the bleb formation. ROCK (Rho-kinase) inhibitors Y-27632 and fasudil completely prevented the lidocaine-induced membrane blebbing, suggesting that ROCK activation is required for bleb formation. Caspase-3 levels were unchanged by 10 mM lidocaine (p = 0.325) and a caspase inhibitor z-VAD-fmk did not affect the lidocaine-induced blebbing (p = 0.964). GTP-RhoA levels were significantly increased (p < 0.001), but Rho inhibitor-1 failed to suppress the membrane blebbing (p = 0.875). Lidocaine (30 mM) reduced the cell viability of isolated chondrocytes (p < 0.001) and in situ chondrocytes (p < 0.001). The chondrotoxicity was attenuated by pretreatment of cells with ROCK inhibitors or a myosin-II inhibitor blebbistatin (p < 0.001). These findings suggest that lidocaine induces ROCK-dependent membrane blebbing and thereby produces a cytotoxic effect on chondrocytes. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:754-762, 2016.

Topics: Anesthetics, Local; Animals; Cartilage, Articular; Caspase 3; Cell Membrane; Chondrocytes; Heterocyclic Compounds, 4 or More Rings; Lidocaine; Male; Rabbits; rho-Associated Kinases

The question of how cells sense substrate mechanical cues has gained increasing attention among biologists. By introducing contour-based data analysis to single-cell force spectroscopy, we identified a loading-rate threshold for the integrin α2β1-DGEA bond beyond which a dramatic increase in bond lifetime was observed. On the basis of mechanical cues (elasticity or topography), the effective spring constant of substrates k is mapped to the loading rate r under actomyosin pulling speed v, which, in turn, affects the lifetime of the integrin-ligand bond. Additionally, downregulating v with a low-dose blebbistatin treatment promotes the neuronal lineage specification of mesenchymal stem cells on osteogenic stiff substrates. Thus, sensing of the loading rate is central to how cells sense mechanical cues that affect cell-extracellular matrix interactions and stem cell differentiation.

Topics: Actomyosin; Biomechanical Phenomena; Cell Differentiation; Collagen Type I; Core Binding Factor Alpha 1 Subunit; Elasticity; Extracellular Matrix; Gene Expression Regulation; Hardness; Heterocyclic Compounds, 4 or More Rings; Humans; Integrin alpha2beta1; Mechanotransduction, Cellular; Mesenchymal Stem Cells; Nestin; Neurofilament Proteins; Neurons; Oligopeptides; Primary Cell Culture; Single-Cell Analysis

The phosphorylation of the 20-kD myosin light chain (MLC) and actin filament formation play a key role in endothelial barrier disruption. MLC is either mono- or di-phosphorylated (pMLC and ppMLC) at T18 or S19. The present study investigated whether there are any distinct roles of pMLC and ppMLC in barrier disruption induced by thrombin. Thrombin induced a modest bi-phasic increase in pMLC and a robust mono-phasic increase in ppMLC. pMLC localized in the perinuclear cytoplasm during the initial phase, while ppMLC localized in the cell periphery, where actin bundles were formed. Later, the actin bundles were rearranged into stress fibers, where pMLC co-localized. Rho-kinase inhibitors inhibited thrombin-induced barrier disruption and peripheral localization of ppMLC and actin bundles. The double, but not single, mutation of phosphorylation sites abolished the formation of peripheral actin bundles and the barrier disruption, indicating that mono-phosphorylation of MLC at either T18 or S19 is functionally sufficient for barrier disruption. Namely, the peripheral localization, but not the degree of phosphorylation, is suggested to be essential for the functional effect of ppMLC. These results suggest that MLC phosphorylation and actin bundle formation in cell periphery are initial events during barrier disruption.

Topics: Actins; Animals; Aorta; Calcium; Capillary Permeability; Electric Impedance; Endothelial Cells; Endothelium, Vascular; Gene Expression Regulation; Heterocyclic Compounds, 4 or More Rings; Mutation; Myosin Light Chains; Myosins; Phosphorylation; Primary Cell Culture; Protein Kinase Inhibitors; Receptor, PAR-1; rho-Associated Kinases; Serine; Swine; Threonine; Thrombin

Mesenchymal precursors (MPs) present some advantageous features, such as differentiation and migration, which make them promising candidates for cell therapy. A better understanding of MP migration characteristics would aid the development of cell delivery protocols. Traditionally, cell migration is thought to occur only through the formation of lamellipodia. More recently, contractility-driven bleb formation has emerged as an alternative mechanism of motility. Here we report that MPs derived from different tissues present spontaneously dynamic cytoplasmic projections in sub-confluent culture, which appear as a combination of lamellipodia with blebs in the leading edge. Upon initial seeding, however, only bleb structures could be observed. Immunofluorescence revealed the presence of pERM, RhoA and F-actin during the blebbing process. Results from migration assays in the presence of blebbistatin, a myosin II inhibitor, showed that bleb formation correlated with migratory capacity, suggesting a functional role for blebs in migration. Bleb formation might be a useful mechanism to improve cell migration in cellular therapy protocols.

Topics: Actin Cytoskeleton; Actins; Animals; Cell Differentiation; Cell Membrane; Cell Movement; Heterocyclic Compounds, 4 or More Rings; Mesenchymal Stem Cells; Mice; Pseudopodia; rhoA GTP-Binding Protein

Myofibrils are the main protein structures that generate force in the beating heart. Myofibril disassembly is related to many physiological and pathological processes. This study investigated, in a cultured rat adult cardiomyocyte model, the effect of force imbalance on myofibril disassembly. The imbalance of forces that were exerted on Z-discs was induced by the synergistic effect of broken intercalated discs and actin-myosin interaction. Cardiomyocytes with well-preserved intercalated discs were isolated from adult rat ventricles. The ultrastructure of cardiomyocyte was observed using a customized two-photon excitation fluorescence and second harmonic generation imaging system. The contraction of cardiomyocytes was recorded with a high-speed CCD camera, and the movement of cellular components was analyzed using a contractile imaging assay technique. The cardiomyocyte dynamic remodeling process was recorded using a time-lapse imaging system. The role of actin-myosin interaction in myofibril disassembly was investigated by incubating cardiomyocytes with blebbistatin (25 μM). Results demonstrated that the hierarchical disassembly process of myofibrils was initiated from cardiomyocyte free ends where intercalated discs had broken, during which the desmin network near the free cell ends was destroyed to release single myofibrils. Analysis of force (based on a schematic model of cardiomyocytes connected at intercalated discs) suggests that breaking of intercalated discs caused force imbalance on both sides of the Z-discs adjacent to the cell ends due to actin-myosin interaction. The damaged intercalated discs and actin-myosin interaction induced force imbalance on both sides of the Z-discs, which played an important role in the hierarchical disassembly of myofibrils. © 2016 Wiley Periodicals, Inc.

Topics: Actins; Animals; Heart Ventricles; Heterocyclic Compounds, 4 or More Rings; Models, Biological; Myofibrils; Myosins; Rats; Rats, Sprague-Dawley

Collective cell migrations are essential in several physiological processes and are driven by both chemical and mechanical cues. The roles of substrate stiffness and confinement on collective migrations have been investigated in recent years, however few studies have addressed how geometric shapes influence collective cell migrations. Here, we address the hypothesis that the relative position of a cell within the confinement influences its motility. Monolayers of two types of epithelial cells--MCF7, a breast epithelial cancer cell line, and MDCK, a control epithelial cell line--were confined within circular, square, and cross-shaped stencils and their migration velocities were quantified upon release of the constraint using particle image velocimetry. The choice of stencil geometry allowed us to investigate individual cell motility within convex, straight and concave boundaries. Cells located in sharp, convex boundaries migrated at slower rates than those in concave or straight edges in both cell types. The overall cluster migration occurred in three phases: an initial linear increase with time, followed by a plateau region and a subsequent decrease in cluster speeds. An acto-myosin contractile ring, present in the MDCK but absent in MCF7 monolayer, was a prominent feature in the emergence of leader cells from the MDCK clusters which occurred every ~125 μm from the vertex of the cross. Further, coordinated cell movements displayed vorticity patterns in MDCK which were absent in MCF7 clusters. We also used cytoskeletal inhibitors to show the importance of acto-myosin bounding cables in collective migrations through translation of local movements to create long range coordinated movements and the creation of leader cells within ensembles. To our knowledge, this is the first demonstration of how bounding shapes influence long-term migratory behaviours of epithelial cell monolayers. These results are important for tissue engineering and may also enhance our understanding of cell movements during developmental patterning and cancer metastasis.

Topics: Actomyosin; Animals; Cadherins; Cell Adhesion; Cell Culture Techniques; Cell Movement; Cytoskeleton; Dogs; Epithelial Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Madin Darby Canine Kidney Cells; MCF-7 Cells; Microscopy, Fluorescence; Stress, Mechanical

Gingival junctional epithelial cell apoptosis caused by periodontopathic bacteria exacerbates periodontitis. This pathological apoptosis is involved in the activation of transforming growth factor β (TGF-β). However, the molecular mechanisms by which microbes induce the activation of TGF-β remain unclear. We previously reported that Aggregatibacter actinomycetemcomitans (Aa) activated TGF-β receptor (TGF-βR)/smad2 signalling to induce epithelial cell apoptosis, even though Aa cannot bind to TGF-βR. Additionally, outer membrane protein 29 kDa (Omp29), a member of the Aa Omps family, can induce actin rearrangements via focal adhesion kinase (FAK) signalling, which also plays a role in the activation of TGF-β by cooperating with integrin. Accordingly, we hypothesized that Omp29-induced actin rearrangements via FAK activity would enhance the activation of TGF-β, leading to gingival epithelial cell apoptosis in vitro. By using human gingival epithelial cell line OBA9, we found that Omp29 activated TGF-βR/smad2 signalling and decreased active TGF-β protein levels in the extracellular matrix (ECM) of cell culture, suggesting the transactivation of TGF-βR. Inhibition of actin rearrangements by cytochalasin D or blebbistatin and knockdown of FAK or integrinβ1 expression by siRNA transfection attenuated TGF-βR/smad2 signalling activity and reduction of TGF-β levels in the ECM caused by Omp29. Furthermore, Omp29 bound to fibronectin (Fn) to induce its aggregation on integrinβ1, which is associated with TGF-β signalling activity. All the chemical inhibitors and siRNAs tested blocked Omp29-induced OBA9 cells apoptosis. These results suggest that Omp29 binds to Fn in order to facilitate Fn/integrinβ1/FAK signalling-dependent TGF-β release from the ECM, thereby inducing gingival epithelial cell apoptosis via TGF-βR/smad2 pathway.

Topics: Aggregatibacter actinomycetemcomitans; Apoptosis; Bacterial Outer Membrane Proteins; Cell Line, Transformed; Cytochalasin D; Epithelial Cells; Fibronectins; Focal Adhesion Kinase 1; Gene Expression Regulation; Gingiva; Heterocyclic Compounds, 4 or More Rings; Host-Pathogen Interactions; Humans; Integrin beta1; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Recombinant Proteins; RNA, Small Interfering; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta

Crawling cells can generate polarity for migration in response to forces applied from the substratum. Such reaction varies according to cell type: there are both fast- and slow-crawling cells. In response to periodic stretching of the elastic substratum, the intracellular stress fibers in slow-crawling cells, such as fibroblasts, rearrange themselves perpendicular to the direction of stretching, with the result that the shape of the cells extends in that direction; whereas fast-crawling cells, such as neutrophil-like differentiated HL-60 cells and Dictyostelium cells, which have no stress fibers, migrate perpendicular to the stretching direction. Fish epidermal keratocytes are another type of fast-crawling cell. However, they have stress fibers in the cell body, which gives them a typical slow-crawling cell structure. In response to periodic stretching of the elastic substratum, intact keratocytes rearrange their stress fibers perpendicular to the direction of stretching in the same way as fibroblasts and migrate parallel to the stretching direction, while blebbistatin-treated stress fiber-less keratocytes migrate perpendicular to the stretching direction, in the same way as seen in HL-60 cells and Dictyostelium cells. Our results indicate that keratocytes have a hybrid mechanosensing system that comprises elements of both fast- and slow-crawling cells, to generate the polarity needed for migration.

Topics: Animals; Cell Movement; Cell Polarity; Depsipeptides; Elasticity; Fibroblasts; Goldfish; Heterocyclic Compounds, 4 or More Rings; Mechanotransduction, Cellular; Stress Fibers; Stress, Mechanical

Cells interact mechanically with their environment, exerting mechanical forces that probe the extracellular matrix (ECM). The mechanical properties of the ECM determine cell behavior and control cell differentiation both in 2D and 3D environments. Gelatin (Gel) is a soft hydrogel into which cells can be embedded. This study shows significant 3D Gel shrinking due to the high traction cellular forces exerted by the cells on the matrix, which prevents cell differentiation. To modulate this process, Gel with hyaluronic acid (HA) has been combined in an injectable crosslinked hydrogel with controlled Gel-HA ratio. HA increases matrix stiffness. The addition of small amounts of HA leads to a significant reduction in hydrogel shrinking after cell encapsulation (C2C12 myoblasts). We show that hydrogel stiffness counterbalanced traction forces of cells and this was decisive in promoting cell differentiation and myotube formation of C2C12 encapsulated in the hybrid hydrogels.

Topics: Animals; Biomechanical Phenomena; Cell Differentiation; Cell Line; Compressive Strength; Cross-Linking Reagents; Elastic Modulus; Gelatin; Heterocyclic Compounds, 4 or More Rings; Hyaluronic Acid; Hydrogels; Kinetics; Mice; Muscle Development; Myoblasts; Polymers; Stress, Mechanical; Sus scrofa; Water

Blebbistatin is a commonly used molecular tool for the specific inhibition of various myosin II isoforms both in vitro and in vivo. Despite its popularity, the use of blebbistatin is hindered by its poor water-solubility (below 10 micromolar in aqueous buffer) and blue-light sensitivity, resulting in the photoconversion of the molecule, causing severe cellular phototoxicity in addition to its cytotoxicity. Furthermore, blebbistatin forms insoluble aggregates in water-based media above 10 micromolar with extremely high fluorescence and also high adherence to different types of surfaces, which biases its experimental usage. Here, we report a highly soluble (440 micromolar in aqueous buffer), non-fluorescent and photostable C15 amino-substituted derivative of blebbistatin, called para-aminoblebbistatin. Importantly, it is neither photo- nor cytotoxic, as demonstrated on HeLa cells and zebrafish embryos. Additionally, para-aminoblebbistatin bears similar myosin II inhibitory properties to blebbistatin or para-nitroblebbistatin (not to be confused with the C7 substituted nitroblebbistatin), tested on rabbit skeletal muscle myosin S1 and on M2 and HeLa cells. Due to its drastically improved solubility and photochemical feature, as well as lack of photo- or cytotoxicity, para-aminoblebbistatin may become a feasible replacement for blebbistatin, especially at applications when high concentrations of the inhibitor or blue light irradiation is required.

Topics: Animals; Cell Survival; Chemical Precipitation; Dermatitis, Phototoxic; Drug Stability; Fluorescence; HeLa Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Myosin Type II; Rabbits; Solubility; Survival Analysis; Zebrafish

Coordinated motions of close-packed multicellular systems typically generate cooperative packs, swirls, and clusters. These cooperative motions are driven by active cellular forces, but the physical nature of these forces and how they generate collective cellular motion remain poorly understood. Here, we study forces and motions in a confined epithelial monolayer and make two experimental observations: 1) the direction of local cellular motion deviates systematically from the direction of the local traction exerted by each cell upon its substrate; and 2) oscillating waves of cellular motion arise spontaneously. Based on these observations, we propose a theory that connects forces and motions using two internal state variables, one of which generates an effective cellular polarization, and the other, through contractile forces, an effective cellular inertia. In agreement with theoretical predictions, drugs that inhibit contractility reduce both the cellular effective elastic modulus and the frequency of oscillations. Together, theory and experiment provide evidence suggesting that collective cellular motion is driven by at least two internal variables that serve to sustain waves and to polarize local cellular traction in a direction that deviates systematically from local cellular velocity.

Topics: Acrylic Resins; Animals; Butadienes; Cell Adhesion; Cell Count; Cell Culture Techniques; Cell Movement; Cell Polarity; Cell Size; Collagen Type I; Dogs; Elastic Modulus; Enzyme Inhibitors; Epidermal Growth Factor; Green Fluorescent Proteins; Heterocyclic Compounds, 4 or More Rings; Madin Darby Canine Kidney Cells; Microscopy, Fluorescence; Models, Biological; Nitriles; Periodicity; Surface Properties

Tumor metastasis is one of the main causes of mortality among patients with malignant tumors. Previous studies concerning tumor metastasis have merely focused on the cancer cells in the tumor. However, an increasing number of studies show that the tumor microenvironment plays a vital role in the progression of cancer, particularly in tumor metastasis. Since fibroblasts and adipocytes are two of the most representative mesenchymal cells in the tumor microenvironment, we established a hypoxia-induced cancer-associated fibroblast (CAF) model and a chemically induced adipocyte model to reveal the effect of the microenvironment on cancer development. In these models, the conditioned medium from the tumor microenvironment was found to significantly promote the migration of human lung cancer cell line 95D and regulate the expression of non-muscle myosin IIA (NMIIA), which is consistent with results in the published literature. Then, we confirmed the hypothesis that the tumor microenvironment can regulate NMIIA in cancer cells and facilitate migration by using the non-muscle myosin II inhibitor, blebbistatin. Thus, this is the first report that the tumor microenvironment can promote cancer cell migration by regulating the expression of NMIIA. Our present data also indicated that DT-13, the saponin monomer 13 of dwarf lilyturf tuber, inhibited cancer cell migration in the tumor microenvironment model. Further results showed that DT-13 exhibited anti-migratory effects by inhibiting the c-raf/ERK1/2 signaling pathway. Consequently, our research confirmed that DT-13 significantly inhibited 95D cell migration in vitro, indicating the potential anti-metastatic effect of DT-13 on lung cancer and the scientific basis for drug development.

Topics: Cell Line, Tumor; Cell Movement; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Humans; Hypoxia; Lung Neoplasms; MAP Kinase Signaling System; Molecular Motor Proteins; Myosin Heavy Chains; Proto-Oncogene Proteins c-raf; Saponins; Signal Transduction; Tumor Microenvironment

Cells organize actin filaments into higher-order structures by regulating the composition, distribution and concentration of actin crosslinkers. Palladin is an actin crosslinker found in the lamellar actin network and stress fibers, which are critical for mechanosensing of the environment. Palladin also serves as a molecular scaffold for α-actinin, another key actin crosslinker. By virtue of its close interactions with actomyosin structures in the cell, palladin may play an important role in cell mechanics. However, the role of palladin in cellular force generation and mechanosensing has not been studied. Here, we investigate the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. Traction force microscopy revealed that tumor-associated fibroblasts generate larger forces on substrates of increased stiffness. Contrary to expectations, knocking down palladin increased the forces generated by cells and inhibited their ability to sense substrate stiffness for very stiff gels. This was accompanied by significant differences in actin organization, adhesion dynamics and altered myosin organization in palladin knock-down cells. Our results suggest that actin crosslinkers such as palladin and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis.

Topics: Actins; Cancer-Associated Fibroblasts; Cell Adhesion; Cells, Cultured; Cytoskeletal Proteins; Focal Adhesions; Heterocyclic Compounds, 4 or More Rings; Humans; Mechanotransduction, Cellular; Phosphoproteins

Variance analysis of postsynaptic current amplitudes suggests the presence of distinct docking sites (also called release sites) where vesicles pause before exocytosis. Docked vesicles participate in the readily releasable pool (RRP), but the relation between docking site number and RRP size remains unclear. It is also unclear whether all vesicles of the RRP are equally release competent, and what cellular mechanisms underlie RRP renewal. We address here these questions at single glutamatergic synapses, counting released vesicles using deconvolution. We find a remarkably low variance of cumulative vesicle counts during action potential trains. This, combined with Monte Carlo simulations, indicates that vesicles transit through two successive states before exocytosis, so that the RRP is up to 2-fold higher than the docking site number. The transition to the second state has a very rapid rate constant, and is specifically inhibited by latrunculin B and blebbistatin, suggesting the involvement of actin and myosin.

Topics: Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Cerebellum; Egtazic Acid; Excitatory Postsynaptic Potentials; Exocytosis; Heterocyclic Compounds, 4 or More Rings; Membrane Fusion; Models, Neurological; Monte Carlo Method; Myosins; Presynaptic Terminals; Rats; Synaptic Vesicles; Thiazolidines

The chemokine receptor cysteine (C)-X-C receptor (CXCR4) is a G-protein-coupled receptor that exerts a vital role in distant metastasis of renal cell carcinoma (RCC). Emerging evidence demonstrates that CXCR4 as the cytomembrane receptor translocated into the nucleus to facilitate cell migration and, therefore, determine the prognosis of several types of malignancies. However, the biological mechanism of nuclear location of CXCR4 remains unclear. In the present study, we confirmed the significant implications of the putative nuclear localization sequence (NLS) '146RPRK149̓ on CXCR4 subcellular localization and metastatic potential by point-mutation assay in RCC cell lines. Importantly, mass spectrum followed by immunoprecipitation identified non-muscle myosin heavy chain-IIA (NMMHC-IIA) as the CXCR4-interacting protein. Furthermore, pharmaceutical inhibition of NMMHC-IIA by blebbistatin dampened the nuclear translocation of CXCR4 as well as the metastatic capacity of RCC cells. In conclusion, the present study may drive the comprehensive progress toward elucidating the mechanism responsible for CXCR4 nuclear function and metastasis in tumors.

Topics: Carcinoma, Renal Cell; Cell Line, Tumor; Cell Movement; Cell Nucleus; Heterocyclic Compounds, 4 or More Rings; Humans; Molecular Motor Proteins; Myosin Heavy Chains; Neoplasm Invasiveness; Neoplasm Metastasis; Point Mutation; Receptors, CXCR4; Signal Transduction

Repeated rounds of nuclear envelope (NE) rupture and repair have been observed in laminopathy and cancer cells and result in intermittent loss of nucleus compartmentalization. Currently, the causes of NE rupture are unclear. Here, we show that NE rupture in cancer cells relies on the assembly of contractile actin bundles that interact with the nucleus via the linker of nucleoskeleton and cytoskeleton (LINC) complex. We found that the loss of actin bundles or the LINC complex did not rescue nuclear lamina defects, a previously identified determinant of nuclear membrane stability, but did decrease the number and size of chromatin hernias. Finally, NE rupture inhibition could be rescued in cells treated with actin-depolymerizing drugs by mechanically constraining nucleus height. These data suggest a model of NE rupture where weak membrane areas, caused by defects in lamina organization, rupture because of an increase in intranuclear pressure from actin-based nucleus confinement.

Topics: Actins; Cell Line, Tumor; Chromatin; Cytochalasin D; Heterocyclic Compounds, 4 or More Rings; Humans; Multiprotein Complexes; Nuclear Envelope; Stress, Mechanical; Time-Lapse Imaging

Tissue fusion, whereby two or more spheroids coalesce, is a process that is fundamental to biofabrication. We have designed a quantitative, high-throughput platform to investigate the fusion of multicellular spheroids using agarose micro-molds. Spheroids of primary human chondrocytes (HCH) or human breast cancer cells (MCF-7) were self-assembled for 24 h and then brought together to form an array comprised of two spheroids (one doublet) per well. To quantify spheroid fusogenicity, we developed two assays: (1) an initial tack assay, defined as the minimum amount of time for two spheroids to form a mechanically stable tissue complex or doublet, and (2) a fusion assay, in which we defined and tracked key morphological parameters of the doublets as a function of time using wide-field fluorescence microscopy over a 24 h time-lapse. The initial tack of spheroid fusion was measured by inverting the micro-molds and centrifuging doublets at various time points to assess their connectedness. We found that the initial tack between two spheroids forms rapidly, with the majority of doublets remaining intact after centrifugation following just 30 min of fusion. Over the course of 24 h of fusion, several morphological changes occurred, which were quantified using a custom image analysis pipeline. End-to-end doublet lengths decreased over time, doublet widths decreased for chondrocytes and increased for MCF-7, contact lengths increased over time, and chondrocyte doublets exhibited higher intersphere angles at the end of fusion. We also assessed fusion by measuring the fluorescence intensity at the plane of fusion, which increased over time for both cell types. Interestingly, we observed that doublets moved and rotated in the micro-wells during fusion and this rotation was inhibited by ROCK inhibitor Y-27632 and myosin II inhibitor blebbistatin. Understanding and optimizing tissue fusion is essential for creating larger tissues, organs, or other structures using individual microtissues as building parts.

Topics: Amides; Cell Fusion; Cell Shape; Cells, Cultured; Chondrocytes; Heterocyclic Compounds, 4 or More Rings; Humans; Kinetics; MCF-7 Cells; Microscopy, Fluorescence; Pyridines; Spheroids, Cellular; Time-Lapse Imaging; Tissue Scaffolds

Integrins coupled with other proteins form protein complexes named focal adhesions (FA) which are considered as the primary sites for cellular forces transduction during cell stable adhesion. Cell traction forces transmitted by FAs and integrin tensions inside FAs have been extensively studied. However, it remains unknown whether integrins outside FAs can transmit tension, and if so, what is the tension range. We previously developed a tension sensor named tension gauge tether (TGT). To calibrate integrin tensions outside FAs, here we applied multiplex TGT (mTGT) to simultaneously monitor integrin tensions at separate levels. mTGT unambiguously revealed that integrins outside FAs also transmit tension after FA formation. These tensions are mainly located in the range of 43 ~ 54 pN which is lower than integrin tensions inside FAs. Integrin tensions both inside and outside FAs substantially contribute to bulk cellular forces and they respond independently to actin and myosin II inhibition, serum deprivation and microtubule inhibition, indicating their different tension sources and independent dynamics. Our work identified integrin tensions outside FAs and calibrated the tension range for the first time. We also demonstrated that mTGT is a valuable tool to monitor integrin tension profile in a broad detection range of 10 ~ 60 pN.

Topics: Animals; Base Sequence; Cell Adhesion; CHO Cells; Cricetinae; Cricetulus; Cytochalasin D; Focal Adhesions; Heterocyclic Compounds, 4 or More Rings; Integrins; Microtubules

The impact of the RNA-binding protein HuR for the post-transcriptional deregulation of tumor-relevant genes is well established. Despite of elevations in HuR expression levels, an increase in cytoplasmic HuR abundance in many cases correlates with a high grade of malignancy. Here, we demonstrated that administration of the actin-depolymerizing macrolide latrunculin A, or blebbistatin, an inhibitor of myosin II ATPase activity, caused a dose- and time-dependent reduction in the high cytoplasmic HuR content of HepG2 and Huh7 hepatocellular carcinoma (HCC) cells. Subcellular fractionation revealed that in addition, both inhibitors strongly attenuated cytoskeletal and membrane-bound HuR abundance and conversely increased the HuR amount in nuclear cell fractions. Concomitant with changes in intracellular HuR localization, both cytoskeletal inhibitors markedly decreased the half-lives of cyclooxygenase-2 (COX-2), cyclin A and cyclin D1 encoding mRNAs resulting in a significant reduction in their expression levels in HepG2 cells. Importantly, a similar reduction in the expression of these HuR targets was achieved by a RNA interference (RNAi)-mediated knockdown of either HuR or nonmuscle myoin IIA. Using polysomal fractionation, we further demonstrate that the decrease in cytoplasmic HuR by latrunculin A or blebbistatin is accompanied by a marked change in the allocation of HuR and its mRNA cargo from polysomes to ribonucleoprotein (RNP) particles. Functionally, the basal migration and prostaglandin E2 synthesis are similarly impaired in inhibitor-treated and stable HuR-knockdown HepG2 cells. Our data demonstrate that interfering with the actomyosin-dependent HuR trafficking may comprise a valid therapeutic option for antagonizing pathologic posttranscriptional gene expression by HuR and furthermore emphasize the potential benefit of HuR inhibitory strategies for treatment of HCC.

Topics: Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Hepatocellular; Cyclin A; Cyclin D; Cyclooxygenase 2; Cytoskeleton; Dinoprostone; ELAV Proteins; Hep G2 Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Liver Neoplasms; Nonmuscle Myosin Type IIA; Polyribosomes; Protein Transport; Ribonucleoproteins; RNA, Messenger; Thiazolidines

The cytokinetic furrow is organized by the RhoA GTPase, which recruits actin and myosin II to the furrow and drives contractility. Here, we show that the RhoA GTPase-activting protein (GAP) p190RhoGAP-A (also known as ARHGAP35) has a role in cytokinesis and is involved in regulating levels of RhoA-GTP and contractility. Cells depleted of p190RhoGAP-A accumulate high levels of RhoA-GTP and markers of high RhoA activity in the furrow, resulting in failure of the cytokinetic furrow to progress to abscission. The loss of p190RhoGAP-A can be rescued by a low dose of the myosin II inhibitor blebbistatin, suggesting that cells fail cytokinesis because they have too much myosin activity. p190RhoGAP-A binds the cytokinetic organizer anillin, and mutants of p190RhoGAP-A that are unable to bind anillin or unable to inactivate RhoA fail to rescue cytokinesis defects in p190RhoGAP-A-depleted cells. Taken together, these data demonstrate that a complex of p190RhoGAP-A and anillin modulates RhoA-GTP levels in the cytokinetic furrow to ensure progression of cytokinesis.

Topics: Cytokinesis; Guanine Nucleotide Exchange Factors; HeLa Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Microfilament Proteins; Mutation; Protein Binding; Repressor Proteins; rhoA GTP-Binding Protein

Nonmuscle myosin II (NM II) is the name given to the multi-subunit protein product of three genes encoding different nonmuscle myosin heavy chains including NM II-A, NM II-B, and NM II-C. Blebbistatin is a small molecule that has been shown to be a relatively specific inhibitor of NM II. Blocking the function of NM II by blebbistatin induces zebrafish embryo cardia bifida at a dose-dependent manner. In situ hybridization analysis with ventricular marker ventricular myosin heavy chain (vmhc) and atrial marker atrial myosin heavy chain (amhc) showed each of the heart contained both distinct atria and ventricle. However, the cardia bifida embryos had highly variable distance between two separate ventricles. We also provided evidence that time window from 12 to 20 h post fertilization (hpf) is necessary and sufficient for cardia bifida formation caused by blebbistatin treatment. Expression of spinster homolog 2 (spns2) was decreased in blebbistatin-treated embryos, suggesting the cardia bifida phenotype caused by NM II inhibition was relevant to precardiac mesoderm migration defects. Through in situ hybridization analysis, we showed that foxa1 was expressed in endoderm of blebbistatin-treated embryos at 24-hpf stage, suggesting the endoderm formation is normal in cardia bifida embryos caused by blebbistatin treatment. In addition, we demonstrated that blebbistatin treatment resulted in morphology alteration of zebrafish cardiomyocytes in vivo and neonatal mouse cardiomyocytes in vitro.

Topics: Animals; Animals, Newborn; Carrier Proteins; Cell Shape; Dose-Response Relationship, Drug; Embryo, Nonmammalian; Fertilization; Heart Defects, Congenital; Heterocyclic Compounds, 4 or More Rings; Membrane Proteins; Mice; Myocytes, Cardiac; Myosin Type II; Zebrafish; Zebrafish Proteins

Mitochondrial membrane potential (ΔΨm) depolarization has been implicated in the loss of excitability (asystole) during global ischemia, which is relevant for the success of defibrillation and resuscitation after cardiac arrest. However, the relationship between ΔΨm depolarization and asystole during no-flow ischemia remains unknown. We applied spatial Fourier analysis to confocally recorded fluorescence emitted by ΔΨm-sensitive dye tetramethylrhodamine methyl ester. The time of ischemic ΔΨm depolarization (tmito_depol) was defined as the time of 50% decrease in the magnitude of spectral peaks reflecting ΔΨm. The time of asystole (tasys) was determined as the time when spontaneous and induced ventricular activity ceased to exist. Interventions included tachypacing (150 ms), myosin II ATPase inhibitor blebbistatin (heart immobilizer), and the combination of blebbistatin and the inhibitor of glycolysis iodoacetate. In the absence of blebbistatin, confocal images were obtained during brief perfusion with hyperkalemic solution and after the contraction failed between 7 and 15 min of ischemia. In control, tmito_depol and tasys were 24.4 ± 6.0 and 26.0 ± 5.0 min, respectively. Tachypacing did not significantly affect either parameter. Blebbistatin dramatically delayed tmito_depol and tasys (51.4 ± 8.6 and 45.7 ± 5.3 min, respectively; both P < 0.0001 vs. control). Iodoacetate combined with blebbistatin accelerated both events (tmito_depol, 12.7 ± 1.8 min; and tasys, 6.5 ± 1.1 min; both P < 0.03 vs. control). In all groups pooled together, tasys was strongly correlated with tmito_depol (R(2) = 0.845; P < 0.0001). These data may indicate a causal relationship between ΔΨm depolarization and asystole or a similar dependence of the two events on energy depletion during ischemia. Our results urge caution against the use of blebbistatin in studies addressing pathophysiology of myocardial ischemia.

Topics: Adenosine Triphosphate; Animals; Female; Heterocyclic Compounds, 4 or More Rings; Male; Membrane Potential, Mitochondrial; Mitochondria, Heart; Myocardial Reperfusion Injury; Rabbits; Systole

Differentiating neurons process the mechanical stimulus by exerting the protrusive forces through lamellipodia and filopodia. We used optical tweezers, video imaging and immunocytochemistry to analyze the role of non-muscle myosin-II on the protrusive force exerted by lamellipodia and filopodia from developing growth cones (GCs) of isolated Dorsal Root Ganglia (DRG) neurons. When the activity of myosin-II was inhibited by 30 μM Blebbistatin protrusion/retraction cycles of lamellipodia slowed down and during retraction lamellipodia could not lift up axially as in control condition. Inhibition of actin polymerization with 25 nM Cytochalasin-D and of microtubule polymerization with 500 nM Nocodazole slowed down the protrusion/retraction cycles, but only Cytochalasin-D decreased lamellipodia axial motion. The force exerted by lamellipodia treated with Blebbistatin decreased by 50%, but, surprisingly, the force exerted by filopodia increased by 20-50%. The concomitant disruption of microtubules caused by Nocodazole abolished the increase of the force exerted by filopodia treated with Blebbistatin. These results suggest that; i- Myosin-II controls the force exerted by lamellipodia and filopodia; ii- contractions of the actomyosin complex formed by filaments of actin and myosin have an active role in ruffle formation; iii- myosin-II is an essential component of the structural stability of GCs architecture.

Topics: Animals; Cytochalasin D; Ganglia, Spinal; Heterocyclic Compounds, 4 or More Rings; Microscopy, Fluorescence; Myosin Heavy Chains; Myosin Type II; Neurons; Nocodazole; Optical Tweezers; Pseudopodia; Rats; Rats, Wistar; Tubulin

Anagrelide represents a treatment option for essential thrombocythemia patients. It lowers platelet counts through inhibition of megakaryocyte maturation and polyploidization, although the basis for this effect remains unclear. Based on its rapid onset of action, we assessed whether, besides blocking megakaryopoiesis, anagrelide represses proplatelet formation (PPF) and aimed to clarify the underlying mechanisms.. Exposure of cord blood-derived megakaryocytes to anagrelide during late stages of culture led to a dose- and time-dependent inhibition of PPF and reduced proplatelet complexity, which were independent of the anagrelide-induced effect on megakaryocyte maturation. Whereas anagrelide was shown to phosphorylate cAMP-substrate VASP, two pharmacologic inhibitors of the cAMP pathway were completely unable to revert anagrelide-induced repression in megakaryopoiesis and PPF, suggesting these effects are unrelated to its ability to inhibit phosphodiesterase (PDE) 3. The reduction in thrombopoiesis was not the result of down-regulation of transcription factors which coordinate PPF, while the myosin pathway was identified as a candidate target, as anagrelide was shown to phosphorylate the myosin light chain and the PPF phenotype was partially rescued after inhibition of myosin activity with blebbistatin.. The platelet-lowering effect of anagrelide results from impaired megakaryocyte maturation and reduced PPF, both of which are deregulated in essential thrombocythemia. These effects seem unrelated to PDE3 inhibition, which is responsible for anagrelide's cardiovascular side-effects and antiplatelet activity. Further work in this field may lead to the potential development of drugs to treat thrombocytosis in myeloproliferative disorders with an improved pharmacologic profile.

Topics: Blood Platelets; Case-Control Studies; Cell Adhesion Molecules; Cells, Cultured; Cyclic AMP; Dose-Response Relationship, Drug; Fetal Blood; Hematopoietic Stem Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Megakaryocytes; Microfilament Proteins; Myosins; Phosphodiesterase 3 Inhibitors; Phosphoproteins; Phosphorylation; Platelet Aggregation Inhibitors; Quinazolines; Signal Transduction; Thrombocythemia, Essential; Thrombopoiesis; Time Factors; Transcription Factors

Activity-dependent bulk endocytosis allows neurons to internalize large portions of the plasma membrane in response to stimulation. However, whether this critical type of compensatory endocytosis is unique to neurons or also occurs in other excitable cells is currently unknown. Here we used fluorescent 70 kDa dextran to demonstrate that secretagogue-induced bulk endocytosis also occurs in bovine chromaffin cells. The relatively large size of the bulk endosomes found in this model allowed us to investigate how the neck of the budding endosomes constricts to allow efficient recruitment of the fission machinery. Using time-lapse imaging of Lifeact-GFP-transfected chromaffin cells in combination with fluorescent 70 kDa dextran, we detected acto-myosin II rings surrounding dextran-positive budding endosomes. Importantly, these rings were transient and contracted before disappearing, suggesting that they might be involved in restricting the size of the budding endosome neck. Based on the complete recovery of dextran fluorescence after photobleaching, we demonstrated that the actin ring-associated budding endosomes were still connected with the extracellular fluid. In contrast, no such recovery was observed following the constriction and disappearance of the actin rings, suggesting that these structures were pinched-off endosomes. Finally, we showed that the rings were initiated by a circular array of phosphatidylinositol(4,5)bisphosphate microdomains, and that their constriction was sensitive to both myosin II and dynamin inhibition. The acto-myosin II rings therefore play a key role in constricting the neck of budding bulk endosomes before dynamin-dependent fission from the plasma membrane of neurosecretory cells.

Topics: Actins; Adrenal Glands; Animals; Biological Transport; Cattle; Cell Membrane; Cells, Cultured; Chromaffin Cells; Dextrans; Dynamins; Endocytosis; Endosomes; Glycosylphosphatidylinositols; Heterocyclic Compounds, 4 or More Rings; Hydrazones; Luminescent Proteins; Myosin Type II; Naphthols; Nicotine; Nicotinic Agonists; Rhodamines; Time Factors; Transfection

Nonmuscle myosin-2 is the primary enzyme complex powering contractility of the F-actin cytoskeleton in the model organism Drosophila. Despite myosin's essential function in fly development and homeostasis, its kinetic features remain elusive. The purpose of this in vitro study is a detailed steady-state and presteady-state kinetic characterization of the Drosophila nonmuscle myosin-2 motor domain. Kinetic features are a slow steady-state ATPase activity, high affinities for F-actin and ADP, and a low duty ratio. Comparative analysis of the overall enzymatic signatures across the nonmuscle myosin-2 complement from model organisms indicates that the Drosophila protein resembles nonmuscle myosin-2s from metazoa rather than protozoa, though modulatory aspects of myosin motor function are distinct. Drosophila nonmuscle myosin-2 is uniquely insensitive toward blebbistatin, a commonly used myosin-2 inhibitor. An in silico modeling approach together with kinetic studies indicate that the nonconsensus amino acid Met466 in the Drosophila nonmuscle myosin-2 active-site loop switch-2 acts as blebbistatin desensitizer. Introduction of the M466I mutation sensitized the protein for blebbistatin, resulting in a half-maximal inhibitory concentration of 36.3 ± 4.1 µM. Together, these data show that Drosophila nonmuscle myosin-2 is a bona fide molecular motor and establish an important link between switch-2 and blebbistatin sensitivity.

Topics: Actins; Adenosine Diphosphate; Adenosine Triphosphate; Amino Acid Sequence; Animals; Binding Sites; Drosophila melanogaster; Drosophila Proteins; Heterocyclic Compounds, 4 or More Rings; Kinetics; Models, Molecular; Molecular Sequence Data; Myosin Type II; Protein Conformation; Recombinant Proteins; Sequence Homology, Amino Acid

Recently we observed that endothelial cells cultured in tightly confluent monolayers display frequent local lamellipodia, and that thrombin, an agent that increases endothelial permeability, reduces lamellipodia protrusions. This led us to test the hypothesis that local lamellipodia contribute to endothelial barrier function. Movements of subcellular structures containing GFP-actin or VE-cadherin-GFP expressed in endothelial cells were recorded using time-lapse microscopy. Transendothelial electrical resistance (TER) served as an index of endothelial barrier function. Changes in both lamellipodia dynamics and TER were assessed during baseline and after cells were treated with either the barrier-disrupting agent thrombin, or the barrier-stabilizing agent sphingosine-1-phosphate (S1P). The myosin II inhibitor blebbistatin was used to selectively block lamellipodia formation, and was used to test their role in the barrier function of endothelial cell monolayers and isolated, perfused rat mesenteric venules. Myosin light chain (MLC) phosphorylation was assessed by immunofluorescence microscopy. Rac1 and RhoA activation were evaluated using G-LISA assays. The role of Rac1 was tested with the specific inhibitor NSC23766 or by expressing wild-type or dominant negative GFP-Rac1. The results show that thrombin rapidly decreased both TER and the lamellipodia protrusion frequency. S1P rapidly increased TER in association with increased protrusion frequency. Blebbistatin nearly abolished local lamellipodia protrusions while cortical actin fibers and stress fibers remained intact. Blebbistatin also significantly decreased TER of cultured endothelial cells and increased permeability of isolated rat mesenteric venules. Both thrombin and S1P increased MLC phosphorylation and activation of RhoA. However, thrombin and S1P had differential impacts on Rac1, correlating with the changes in TER and lamellipodia protrusion frequency. Overexpression of Rac1 elevated, while NSC23766 and dominant negative Rac1 reduced barrier function and lamellipodia activity. Combined, these data suggest that local lamellipodia, driven by myosin II and Rac1, are important for dynamic changes in endothelial barrier integrity.

Topics: Actins; Aminoquinolines; Animals; Antigens, CD; Cadherins; Capillary Permeability; Cell Membrane Permeability; Cells, Cultured; Green Fluorescent Proteins; Heterocyclic Compounds, 4 or More Rings; Human Umbilical Vein Endothelial Cells; Humans; Lysophospholipids; Male; Mesentery; Microscopy, Confocal; Microscopy, Fluorescence; Myosin Light Chains; Phosphorylation; Pseudopodia; Pyrimidines; rac1 GTP-Binding Protein; Rats, Sprague-Dawley; rhoA GTP-Binding Protein; Sphingosine; Thrombin; Time-Lapse Imaging; Venules

Pluripotent human embryonic stem cells (hESCs) have the capability of differentiating into different lineages based on specific environmental cues. We had previously shown that hESCs can be primed to differentiate into either neurons or glial cells, depending on the arrangement, geometry and size of their substrate topography. In particular, anisotropically patterned substrates like gratings were found to favour the differentiation of hESCs into neurons rather than glial cells. In this study, our aim is to elucidate the underlying mechanisms of topography-induced differentiation of hESCs towards neuronal lineages. We show that high actomyosin contractility induced by a nano-grating topography is crucial for neuronal maturation. Treatment of cells with the myosin II inhibitor (blebbistatin) and myosin light chain kinase inhibitor (ML-7) greatly reduces the expression level of microtubule-associated protein 2 (MAP2). On the other hand, our qPCR array results showed that PAX5, BRN3A and NEUROD1 were highly expressed in hESCs grown on nano-grating substrates as compared to unpatterned substrates, suggesting the possible involvement of these genes in topography-mediated neuronal differentiation of hESCs. Interestingly, YAP was localized to the cytoplasm of differentiating hESCs. Taken together, our study has provided new insights in understanding the mechanotransduction of topographical cues during neuronal differentiation of hESCs.

Topics: Actomyosin; Azepines; Cell Differentiation; Cell Line; Cell Lineage; Cytoskeleton; Embryonic Stem Cells; Gene Expression Profiling; Gene Expression Regulation; Heterocyclic Compounds, 4 or More Rings; Humans; Image Processing, Computer-Assisted; Microscopy, Fluorescence; Microtubule-Associated Proteins; Myosin Light Chains; Myosin Type II; Naphthalenes; Neuroglia; Neurons; Polymerase Chain Reaction; rho-Associated Kinases; Stress, Mechanical; Up-Regulation

Actin and myosin within the crystalline lens maintain the structural integrity of lens fiber cells and form a hexagonal lattice cradling the posterior surface of the lens. The actomyosin network was pharmacologically disrupted to examine the effects on lenticular biomechanics and optical quality.. One lens of 7-day-old White Leghorn chickens was treated with 10 µM of a disruptor and the other with 0.01% dimethyl sulfoxide (vehicle). Actin, myosin, and myosin light chain kinase (MLCK) disruptors were used. The stiffness and the optical quality of the control and treated lenses were measured. Western blotting and confocal imaging were used to confirm that treatment led to a disruption of the actomyosin network. The times for the lenses to recover stiffness to match the control values were also measured.. Disruptor-treated lenses were significantly less stiff than their controls (p≤0.0274 for all disruptors). The disruptors led to changes in the relative protein amounts as well as the distributions of proteins within the lattice. However, the disruptors did not affect the clarity of the lenses (p≥0.4696 for all disruptors), nor did they affect spherical aberration (p = 0.02245). The effects of all three disruptors were reversible, with lenses recovering from treatment with actin, myosin, and MLCK disruptors after 4 h, 1 h, and 8 min, respectively.. Cytoskeletal protein disruptors led to a decreased stiffness of the lens, and the effects were reversible. Optical quality was mostly unaffected, but the long-term consequences remain unclear. Our results raise the possibility that the mechanical properties of the avian lens may be actively regulated in vivo via adjustments to the actomyosin lattice.

Topics: Actins; Actomyosin; Animals; Animals, Newborn; Avian Proteins; Azepines; Bridged Bicyclo Compounds, Heterocyclic; Chickens; Compressive Strength; Hardness; Heterocyclic Compounds, 4 or More Rings; Lens, Crystalline; Myosin-Light-Chain Kinase; Myosins; Naphthalenes; Thiazolidines; Tissue Culture Techniques

In neurosecretory cells, secretory vesicles (SVs) undergo Ca(2+)-dependent fusion with the plasma membrane to release neurotransmitters. How SVs cross the dense mesh of the cortical actin network to reach the plasma membrane remains unclear. Here we reveal that, in bovine chromaffin cells, SVs embedded in the cortical actin network undergo a highly synchronized transition towards the plasma membrane and Munc18-1-dependent docking in response to secretagogues. This movement coincides with a translocation of the cortical actin network in the same direction. Both effects are abolished by the knockdown or the pharmacological inhibition of myosin II, suggesting changes in actomyosin-generated forces across the cell cortex. Indeed, we report a reduction in cortical actin network tension elicited on secretagogue stimulation that is sensitive to myosin II inhibition. We reveal that the cortical actin network acts as a 'casting net' that undergoes activity-dependent relaxation, thereby driving tethered SVs towards the plasma membrane where they undergo Munc18-1-dependent docking.

Topics: Actins; Animals; Cattle; Chromaffin Cells; Heterocyclic Compounds, 4 or More Rings; Munc18 Proteins; Myosin Type II; Neurosecretion; PC12 Cells; Rats; Secretory Vesicles

Tendon injuries are common and heal poorly. Strategies to regenerate or replace injured tendons are challenged by an incomplete understanding of normal tendon development. Our previous study showed that embryonic tendon elastic modulus increases as a function of developmental stage. Inhibition of enzymatic collagen crosslink formation abrogated increases in tendon elastic modulus at late developmental stages, but did not affect increases in elastic modulus of early stage embryonic tendons. Here, we aimed to identify potential contributors to the mechanical properties of these early stage embryonic tendons. We characterized tendon progenitor cells in early stage embryonic tendons, and the influence of actin cytoskeleton disruption on tissue elastic modulus. Cells were closely packed in embryonic tendons, and did not change in density during early development. We observed an organized network of actin filaments that seemed contiguous between adjacent cells. The actin filaments exhibited a crimp pattern with a period and amplitude that matched the crimp of collagen fibers at each developmental stage. Chemical disruption of the actin cytoskeleton decreased tendon tissue elastic modulus, measured by atomic force microscopy. Our results demonstrate that early developmental stage embryonic tendons possess a well organized actin cytoskeleton network that contributes significantly to tendon tissue mechanical properties.

Topics: Actin Cytoskeleton; Alginates; Animals; Chick Embryo; Elastic Modulus; Glucuronic Acid; Heterocyclic Compounds, 4 or More Rings; Hexuronic Acids; Stem Cells; Tendons

To examine the role of protein kinase C (PKC) and non-muscle myosin in regulation of wall tension in the hypertrophied urinary bladder.. A partial urinary outflow obstruction was induced in the mouse. Tissue strips from sham operated controls and obstructed bladders were examined in vitro with quantitative gel electrophoresis, immunohistochemistry, and in vitro force recordings.. Outlet obstruction (14-18 days) induced a significant growth of the bladder, 73 ± 6.13 mg compared to 19 ± 1 13 mg in sham operated controls. The hypertrophying bladder tissue had increased expression of non-muscle myosin B (SMemb) mainly localized to serosa and suburothelium. Direct activation of PKC with PDBu did not alter force in the control urinary bladder. In contrast, PDBu initiated a prominent and sustained contraction which had an increased sensitivity to the myosin type II inhibitor blebbistatin.. PKC activates a significant contractile response in the wall of the hypertrophying urinary bladder, possibly supported by non-muscle myosin. This contractile component is not contributing to the physiological response to muscarinic stimulation, but might be separately regulated by other, yet unknown, mechanisms.

Topics: Animals; Biomechanical Phenomena; Disease Models, Animal; Female; Heterocyclic Compounds, 4 or More Rings; Hypertrophy; In Vitro Techniques; Mice; Mice, Inbred C57BL; Muscle Contraction; Muscle, Smooth; Nonmuscle Myosin Type IIB; Phorbol 12,13-Dibutyrate; Protein Kinase C; Urinary Bladder; Urinary Bladder Neck Obstruction

Acute osmotic fluctuations in the brain occur during a number of clinical conditions and can result in a variety of adverse neurological symptoms. Osmotic perturbation can cause changes in the volumes of intra- and extracellular fluid and, due to the rigidity of the skull, can alter intracranial pressure thus making it difficult to analyze purely osmotic effects in vivo. The present study aims to determine the effects of changes in osmolarity on SH-SY5Y human neuroblastoma cells in vitro, and the role of the actin-myosin network in regulating this response. Cells were exposed to hyper- or hypoosmotic media and morphological and cytoskeletal responses were recorded. Hyperosmotic shock resulted in a drop in cell body volume and planar area, a persisting shape deformation, and increases in cellular translocation. Hypoosmotic shock did not significantly alter planar area, but caused a transient increase in cell body volume and an increase in cellular translocation via the development of small protrusions rich in actin. Disruption of the actin-myosin network with latrunculin and blebbistatin resulted in changes to volume and shape regulation, and a decrease in cellular translocation. In both osmotic perturbations, no apparent disruptions to cytoskeletal integrity were observed by light microscopy. Overall, because osmotically induced changes persisted even after volume regulation occurred, it is possible that osmotic stress may play a larger role in neurological dysfunction than currently believed.

Topics: Actins; Cell Line, Tumor; Cell Shape; Cytoskeleton; Heterocyclic Compounds, 4 or More Rings; Humans; Myosins; Neurons; Osmotic Pressure

Primitive streak formation in the chick embryo involves large-scale highly coordinated flows of more than 100,000 cells in the epiblast. These large-scale tissue flows and deformations can be correlated with specific anisotropic cell behaviours in the forming mesendoderm through a combination of light-sheet microscopy and computational analysis. Relevant behaviours include apical contraction, elongation along the apical-basal axis followed by ingression, and asynchronous directional cell intercalation of small groups of mesendoderm cells. Cell intercalation is associated with sequential, directional contraction of apical junctions, the onset, localization and direction of which correlate strongly with the appearance of active myosin II cables in aligned apical junctions in neighbouring cells. Use of class specific myosin inhibitors and gene-specific knockdown shows that apical contraction and intercalation are myosin II dependent and also reveal critical roles for myosin I and myosin V family members in the assembly of junctional myosin II cables.

Topics: Animals; Animals, Genetically Modified; Cell Line; Cell Movement; Cell Proliferation; Cell Shape; Chick Embryo; Chickens; Gastrulation; HEK293 Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Hydrocarbons, Chlorinated; Myosin Type I; Myosin Type II; Myosin Type V; Phosphorylation; Primitive Streak; Pyrroles; RNA Interference; RNA, Small Interfering

The Ciona notochord displays planar cell polarity (PCP), with anterior localization of Prickle (Pk) and Strabismus (Stbm). We report that a myosin is polarized anteriorly in these cells and strongly colocalizes with Stbm. Disruption of the actin/myosin machinery with cytochalasin or blebbistatin disrupts polarization of Pk and Stbm, but not of myosin complexes, suggesting a PCP-independent aspect of myosin localization. Wash out of cytochalasin restored Pk polarization, but not if done in the presence of blebbistatin, suggesting an active role for myosin in core PCP protein localization. On the other hand, in the pk mutant line, aimless, myosin polarization is disrupted in approximately one third of the cells, indicating a reciprocal action of core PCP signaling on myosin localization. Our results indicate a complex relationship between the actomyosin cytoskeleton and core PCP components in which myosin is not simply a downstream target of PCP signaling, but also required for PCP protein localization.

Topics: Actin Cytoskeleton; Animals; Bacterial Proteins; Cell Nucleus; Cell Polarity; Ciona intestinalis; Cytochalasin B; Embryo, Nonmammalian; Fetal Proteins; Gene Expression; Gene Expression Regulation, Developmental; Heterocyclic Compounds, 4 or More Rings; Luminescent Proteins; Myosins; Notochord; Protein Isoforms; ras Guanine Nucleotide Exchange Factors; Recombinant Fusion Proteins; Signal Transduction; T-Box Domain Proteins

Non-muscle myosin heavy chain IIA (NMMHC IIA) has been shown to be involved in thrombus formation and inflammatory microparticle release in endothelial cells. However, the role of NMMHC IIA in regulating the expression of tissue factor (TF) and deep venous thrombosis remains to be elucidated. In the present study, endothelial cells were stimulated with tumour necrosis factor-α (TNF-α) to induce TF expression. Pretreatment with the NMMHC II inhibitor blebbistatin suppressed the mRNA and protein expressions as well as the procoagulant activity of TF in a dose-dependent manner. Blebbistatin enhanced Akt and GSK3β phosphorylation and inhibited NF-κB p65 nuclear translocation and IκBα degradation. These observations were similar to the effect of CHIR99021, a GSK3β inhibitor. TF downregulation by blebbistatin was antagonised by the PI3K inhibitor, wortmannin. Furthermore, siRNA knockdown of NMMHC IIA, but not IIB or IIC, inhibited TF expression, activated Akt/GSK3β and suppressed NF-κB signalling pathways, whereas the overexpression of NMMHC IIA increased TF expression. The binding of NMMHC IIA and TNF receptor 2 mediated signal internalisation in TNF-α-stimulated endothelial cells. Importantly, blebbistatin decreased endothelium NMMHC IIA and TF expression, deactivated GSK3β by inducing its phosphorylation, suppressed p65 nuclear translocation, and inhibited thrombus formation in a mouse deep venous thrombosis model.Our findings provide solid evidence that inhibition of NMMHC II, most likely NMMHC IIA, impedes TF expression and venous thrombosis via Akt/GSK3β-NF-κB signalling pathways in the endothelium both in vitro and in vivo. NMMHC IIA might be a potential novel target for the treatment of thrombotic disorders.

Topics: Active Transport, Cell Nucleus; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelial Cells; Fibrinolytic Agents; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HEK293 Cells; Heterocyclic Compounds, 4 or More Rings; Humans; I-kappa B Proteins; Mice, Inbred C57BL; Molecular Motor Proteins; Myosin Heavy Chains; NF-kappa B; Nonmuscle Myosin Type IIA; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Receptors, Tumor Necrosis Factor, Type II; RNA Interference; Signal Transduction; Thromboplastin; Transcription Factor RelA; Transfection; Tumor Necrosis Factor-alpha; Venous Thrombosis

Defective protein kinase C (PKC) signaling has been suggested to contribute to abnormal vascular contraction in disease conditions including hypertension and diabetes. Our previous work on agonist and pressure-induced cerebral vasoconstriction implicated PKC as a major contributor to force production in a myosin light chain (LC20) phosphorylation-independent manner. Here, we used phorbol dibutyrate to selectively induce a PKC-dependent constriction in rat middle cerebral arteries and delineate the relative contribution of different contractile mechanisms involved. Specifically, we employed an ultra-sensitive 3-step western blotting approach to detect changes in the content of phosphoproteins that regulate myosin light chain phosphatase (MLCP) activity, thin filament activation, and actin cytoskeleton reorganization. Data indicate that PKC activation evoked a greater constriction at a similar level of LC20 phosphorylation achieved by 5-HT. PDBu-evoked constriction persisted in the presence of Gö6976, a selective inhibitor of Ca(2+)-dependent PKC, and in the absence of extracellular Ca(2+). Biochemical evidence indicates that either + or - extracellular Ca(2+), PDBu (i) inhibits MLCP activity via the phosphorylation of myosin targeting subunit of myosin phosphatase (MYPT1) and C-kinase potentiated protein phosphatase-1 inhibitor (CPI-17), (ii) increases the phosphorylation of paxillin and heat shock protein 27 (HSP27), and reduces G-actin content, and (iii) does not change the phospho-content of the thin filament proteins, calponin and caldesmon. PDBu-induced constriction was more sensitive to disruption of actin cytoskeleton compared to inhibition of cross-bridge cycling. In conclusion, this study provided evidence for the pivotal contribution of cytoskeletal actin polymerization in force generation following PKC activation in cerebral resistance arteries.

Topics: Actin Cytoskeleton; Animals; Brain; Bridged Bicyclo Compounds, Heterocyclic; Enzyme Activation; Heterocyclic Compounds, 4 or More Rings; Male; Middle Cerebral Artery; Myosin Light Chains; Phorbol 12,13-Dibutyrate; Phosphorylation; Polymerization; Protein Kinase C; Rats, Sprague-Dawley; Serotonin; Thiazolidines; Vascular Resistance; Vasoconstriction

Lgr5+ stem cells are crucial to gut epithelium homeostasis, and therapies targeting these cells hold promise for treatment of gastrointestinal diseases. Here we report that the non-muscle-myosin-II (NMII) heavy chain Myh9 accumulates at epithelial injury sites in mice distal colon treated with dextran sulphate sodium (DSS). Gut-epithelium-specific Myh9 monoallelic deletion alleviates DSS-induced colonic crypt damage and acute colitis. Consistently, the NMII inhibitor blebbistatin can improve the survival of Lgr5+ stem cells and the growth of Lgr5 organoids. Mechanistically, inhibition of NMII by blebbistatin or Myh9 monoallelic deletion activates Akt through Rac1 and PAK1, which is essential for the survival and pluripotency of Lgr5+ cells. These results establish a critical role of the Myh9-Rac1-PAK1-Akt pathway in the maintenance of Lgr5+ stem cells. As blebbistatin can mitigate DSS-induced colitis and preserve Lgr5+ colonic stem cells in vivo, our findings provide a potential therapeutic intervention of gastrointestinal epithelium injury and degenerative diseases.

Topics: Alleles; Aminoquinolines; Animals; Cell Survival; Colitis; Epithelium; Gene Expression Regulation; Green Fluorescent Proteins; Heterocyclic Compounds, 4 or More Rings; Intestinal Mucosa; Male; Mice; Myosin Heavy Chains; Myosin Type II; Neuropeptides; NIH 3T3 Cells; Nonmuscle Myosin Type IIA; p21-Activated Kinases; Proto-Oncogene Proteins c-akt; Pyrimidines; rac1 GTP-Binding Protein; Receptors, G-Protein-Coupled; Signal Transduction; Stem Cells

Cellular protrusions are highly dynamic structures involved in fundamental processes, including cell migration and invasion. For a cell to migrate, its leading edge must form protrusions, and then adhere or retract. The spatial and temporal coordination of protrusions and retraction is yet to be fully understood. The study of protrusion dynamics mainly relies on live-microscopy often coupled to fluorescent labeling. Here we report the use of an alternative, label-free, quantitative and rapid assay to analyze protrusion dynamics in a cell population based on the real-time recording of cell activity by means of electronic sensors. Cells are seeded on a plate covered with electrodes and their shape changes map into measured impedance variations. Upon growth factor stimulation the impedance increases due to protrusive activity and decreases following retraction. Compared to microscopy-based methods, impedance measurements are suitable to high-throughput studies on different cell lines, growth factors and chemical compounds. We present data indicating that this assay lends itself to dissect the biochemical signaling pathways controlling adhesive protrusions. Indeed, we show that the protrusion phase is sustained by actin polymerization, directly driven by growth factor stimulation. Contraction instead mainly relies on myosin action, pointing at a pivotal role of myosin in lamellipodia retraction.

Topics: Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cell Movement; Cell Surface Extensions; Cetuximab; Electric Impedance; Epidermal Growth Factor; HEK293 Cells; HeLa Cells; Hepatocyte Growth Factor; Heterocyclic Compounds, 4 or More Rings; Humans; Myosins; Pseudopodia; Thiazolidines; Vascular Endothelial Growth Factor A

The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.

Topics: Actin Cytoskeleton; Animals; Cell Line, Tumor; Cell Movement; Heterocyclic Compounds, 4 or More Rings; Microscopy, Atomic Force; Myosin Type II; Protein Isoforms; Rats; RNA, Small Interfering; Tropomyosin

Enveloped viruses enter host cells through membrane fusion and the cells in turn alter their shape to accommodate components of the virus. However, the role of nonmuscle myosin II of the actomyosin complex of host cells in membrane fusion is yet to be understood. Herein, we show that both (-) blebbistatin, a specific inhibitor of nonmuscle myosin II (NMII) and small interfering RNA markedly augment fusion of Sendai virus (SeV), with chinese hamster ovary cells and human hepatocarcinoma cells. Inhibition of RLC phosphorylation using inhibitors against ROCK, but not PKC and MRCK, or overexpression of phospho-dead mutant of RLC enhances membrane fusion. SeV infection increases cellular stiffness and myosin light chain phosphorylation at two hour post infection. Taken together, the present investigation strongly indicates that Rho-ROCK-NMII contractility signaling pathway may provide a physical barrier to host cells against viral fusion.

Topics: Amino Acid Sequence; Animals; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Heterocyclic Compounds, 4 or More Rings; Humans; Microscopy, Atomic Force; Microscopy, Fluorescence; Mutagenesis; Myosin Light Chains; Nonmuscle Myosin Type IIA; Nonmuscle Myosin Type IIB; Phosphorylation; rho-Associated Kinases; RNA Interference; RNA, Small Interfering; Sendai virus; Sequence Alignment; Virus Internalization; Virus Release

There is substantial evidence that cells produce a diverse response to changes in ECM stiffness depending on their identity. Our aim was to understand how stiffness impacts neuronal differentiation of embryonic stem cells (ESC's), and how this varies at three specific stages of the differentiation process. In this investigation, three effects of stiffness on cells were considered; attachment, expansion and phenotypic changes during differentiation. Stiffness was varied from 2 kPa to 18 kPa to finally 35 kPa. Attachment was found to decrease with increasing stiffness for both ESC's (with a 95% decrease on 35 kPa compared to 2 kPa) and neural precursors (with a 83% decrease on 35 kPa). The attachment of immature neurons was unaffected by stiffness. Expansion was independent of stiffness for all cell types, implying that the proliferation of cells during this differentiation process was independent of Young's modulus. Stiffness had no effect upon phenotypic changes during differentiation for mESC's and neural precursors. 2 kPa increased the proportion of cells that differentiated from immature into mature neurons. Taken together our findings imply that the impact of Young's modulus on attachment diminishes as neuronal cells become more mature. Conversely, the impact of Young's modulus on changes in phenotype increased as cells became more mature.

Topics: Animals; Biomarkers; Cell Adhesion; Cell Differentiation; Elastic Modulus; Gelatin; Glutaral; Heterocyclic Compounds, 4 or More Rings; Mice; Microtubule-Associated Proteins; Microtubules; Mouse Embryonic Stem Cells; Neurons; Nocodazole; Phenotype; Tubulin

Embryonic morphogenesis takes place via a series of dramatic collective cell movements. The mechanisms that coordinate these intricate structural transformations across an entire organism are not well understood. In this study, we used gentle mechanical deformation of developing zebrafish embryos to probe the role of physical forces in generating long-range intercellular coordination during epiboly, the process in which the blastoderm spreads over the yolk cell. Geometric distortion of the embryo resulted in nonuniform blastoderm migration and realignment of the anterior-posterior (AP) axis, as defined by the locations at which the head and tail form, toward the new long axis of the embryo and away from the initial animal-vegetal axis defined by the starting location of the blastoderm. We found that local alterations in the rate of blastoderm migration correlated with the local geometry of the embryo. Chemical disruption of the contractile ring of actin and myosin immediately vegetal to the blastoderm margin via Ca(2+) reduction or treatment with blebbistatin restored uniform migration and eliminated AP axis reorientation in mechanically deformed embryos; it also resulted in cellular disorganization at the blastoderm margin. Our results support a model in which tension generated by the contractile actomyosin ring coordinates epiboly on both the organismal and cellular scales. Our observations likewise suggest that the AP axis is distinct from the initial animal-vegetal axis in zebrafish.

Topics: Actins; Animals; Calcium; Cations, Divalent; Cell Movement; Computer Simulation; Extracellular Space; Heterocyclic Compounds, 4 or More Rings; Microscopy, Confocal; Models, Biological; Myosins; Physical Stimulation; Zebrafish

Skeletal condensation occurs when specified mesenchyme cells self-organize over several days to form a distinctive cartilage template. Here, we determine how and when specified mesenchyme cells integrate mechanical and molecular information from their environment, forming cartilage condensations in the pharyngeal arches of chick embryos. By disrupting cytoskeletal reorganization, we demonstrate that dynamic cell shape changes drive condensation and modulate the response of the condensing cells to Fibroblast Growth Factor (FGF), Bone Morphogenetic Protein (BMP) and Transforming Growth Factor beta (TGF-β) signaling pathways. Rho Kinase (ROCK)-driven actomyosin contractions and Myosin II-generated differential cell cortex tension regulate these cell shape changes. Disruption of the condensation process inhibits the differentiation of the mesenchyme cells into chondrocytes, demonstrating that condensation regulates the fate of the mesenchyme cells. We also find that dorsal and ventral condensations undergo distinct cell shape changes. BMP signaling is instructive for dorsal condensation-specific cell shape changes. Moreover, condensations exhibit ventral characteristics in the absence of BMP signaling, suggesting that in the pharyngeal arches ventral morphology is the ground pattern. Overall, this study characterizes the interplay between cytoskeletal dynamics and molecular signaling in a self-organizing system during tissue morphogenesis.

Topics: Actins; Animals; Bone Morphogenetic Proteins; Cell Shape; Cells, Cultured; Chick Embryo; Chondrocytes; Chondrogenesis; Cytochalasin D; Cytoskeleton; Fibroblast Growth Factors; Heterocyclic Compounds, 4 or More Rings; Pyrazoles; Pyrimidines; Pyrroles; Signal Transduction; Transforming Growth Factor beta

The nucleus has a smooth, regular appearance in normal cells, and its shape is greatly altered in human pathologies. Yet, how the cell establishes nuclear shape is not well understood. We imaged the dynamics of nuclear shaping in NIH3T3 fibroblasts. Nuclei translated toward the substratum and began flattening during the early stages of cell spreading. Initially, nuclear height and width correlated with the degree of cell spreading, but over time, reached steady-state values even as the cell continued to spread. Actomyosin activity, actomyosin bundles, microtubules, and intermediate filaments, as well as the LINC complex, were all dispensable for nuclear flattening as long as the cell could spread. Inhibition of actin polymerization as well as myosin light chain kinase with the drug ML7 limited both the initial spreading of cells and flattening of nuclei, and for well-spread cells, inhibition of myosin-II ATPase with the drug blebbistatin decreased cell spreading with associated nuclear rounding. Together, these results show that cell spreading is necessary and sufficient to drive nuclear flattening under a wide range of conditions, including in the presence or absence of myosin activity. To explain this observation, we propose a computational model for nuclear and cell mechanics that shows how frictional transmission of stress from the moving cell boundaries to the nuclear surface shapes the nucleus during early cell spreading. Our results point to a surprisingly simple mechanical system in cells for establishing nuclear shapes.

Topics: 3T3 Cells; Actins; Actomyosin; Animals; Azepines; Cell Movement; Cell Nucleus; Cell Nucleus Shape; Enzyme Inhibitors; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Mice; Microtubules; Models, Biological; Myosin-Light-Chain Kinase; Myosins; Naphthalenes

In isolated thick filaments from many types of muscle, the two head domains of each myosin molecule are folded back against the filament backbone in a conformation called the interacting heads motif (IHM) in which actin interaction is inhibited. This conformation is present in resting skeletal muscle, but it is not known how exit from the IHM state is achieved during muscle activation. Here, we investigated this by measuring the in situ conformation of the light chain domain of the myosin heads in relaxed demembranated fibers from rabbit psoas muscle using fluorescence polarization from bifunctional rhodamine probes at four sites on the C-terminal lobe of the myosin regulatory light chain (RLC). The order parameter 〈P2〉 describing probe orientation with respect to the filament axis had a roughly sigmoidal dependence on temperature in relaxing conditions, with a half-maximal change at ∼19°C. Either lattice compression by 5% dextran T500 or addition of 25 μM blebbistatin decreased the transition temperature to ∼14°C. Maximum entropy analysis revealed three preferred orientations of the myosin RLC region at 25°C and above, two with its long axis roughly parallel to the filament axis and one roughly perpendicular. The parallel orientations are similar to those of the so-called blocked and free heads in the IHM and are stabilized by either lattice compression or blebbistatin. In relaxed skeletal muscle at near-physiological temperature and myofilament lattice spacing, the majority of the myosin heads have their light chain domains in IHM-like conformations, with a minority in a distinct conformation with their RLC regions roughly perpendicular to the filament axis. None of these three orientation populations were present during active contraction. These results are consistent with a regulatory transition of the thick filament in skeletal muscle associated with a conformational equilibrium of the myosin heads.

Topics: Animals; Dextrans; Heterocyclic Compounds, 4 or More Rings; Isometric Contraction; Muscle Fibers, Skeletal; Muscle Relaxation; Myosins; Neuromuscular Agents; Rabbits; Rest; Rigor Mortis; Transition Temperature

Platelets are central to the process of hemostasis, rapidly aggregating at sites of blood vessel injury and acting as coagulation nidus sites. On interaction with the subendothelial matrix, platelets are transformed into balloonlike structures as part of the hemostatic response. It remains unclear, however, how and why platelets generate these structures. We set out to determine the physiological relevance and cellular and molecular mechanisms underlying platelet membrane ballooning.. Using 4-dimensional live-cell imaging and electron microscopy, we show that human platelets adherent to collagen are transformed into phosphatidylserine-exposing balloonlike structures with expansive macro/microvesiculate contact surfaces, by a process that we termed procoagulant spreading. We reveal that ballooning is mechanistically and structurally distinct from membrane blebbing and involves disruption to the platelet microtubule cytoskeleton and inflation through fluid entry. Unlike blebbing, procoagulant ballooning is irreversible and a consequence of Na(+), Cl(-), and water entry. Furthermore, membrane ballooning correlated with microparticle generation. Inhibition of Na(+), Cl(-), or water entry impaired ballooning, procoagulant spreading, and microparticle generation, and it also diminished local thrombin generation. Human Scott syndrome platelets, which lack expression of Ano-6, also showed a marked reduction in membrane ballooning, consistent with a role for chloride entry in the process. Finally, the blockade of water entry by acetazolamide attenuated ballooning in vitro and markedly suppressed thrombus formation in vivo in a mouse model of thrombosis.. Ballooning and procoagulant spreading of platelets are driven by fluid entry into the cells, and are important for the amplification of localized coagulation in thrombosis.

Topics: Acetazolamide; Actomyosin; Amides; Animals; Anoctamins; Blood Coagulation Disorders; Blood Platelets; Carotid Artery Thrombosis; Cell Adhesion; Cell Membrane; Cell Shape; Cell Size; Cell-Derived Microparticles; Chlorides; Collagen; Cytochalasin D; Heterocyclic Compounds, 4 or More Rings; Humans; Mice; Microtubules; Phospholipid Transfer Proteins; Pyridines; Sodium; Thrombin; Thrombosis; Water

While β-adrenergic receptor (β-AR) stimulation ensures adequate cardiac output during stress, it can also trigger life-threatening cardiac arrhythmias. We have previously shown that proarrhythmic Ca(2+) waves during β-AR stimulation temporally coincide with augmentation of reactive oxygen species (ROS) production. In this study, we tested the hypothesis that increased energy demand during β-AR stimulation plays an important role in mitochondrial ROS production and Ca(2+)-wave generation in rabbit ventricular myocytes. We found that β-AR stimulation with isoproterenol (0.1 μM) decreased the mitochondrial redox potential and the ratio of reduced to oxidated glutathione. As a result, β-AR stimulation increased mitochondrial ROS production. These metabolic changes induced by isoproterenol were associated with increased sarcoplasmic reticulum (SR) Ca(2+) leak and frequent diastolic Ca(2+) waves. Inhibition of cell contraction with the myosin ATPase inhibitor blebbistatin attenuated oxidative stress as well as spontaneous SR Ca(2+) release events during β-AR stimulation. Furthermore, we found that oxidative stress induced by β-AR stimulation caused the formation of disulfide bonds between two ryanodine receptor (RyR) subunits, referred to as intersubunit cross-linking. Preventing RyR cross-linking with N-ethylmaleimide decreased the propensity of Ca(2+) waves induced by β-AR stimulation. These data suggest that increased energy demand during sustained β-AR stimulation weakens mitochondrial antioxidant defense, causing ROS release into the cytosol. By inducing RyR intersubunit cross-linking, ROS can increase SR Ca(2+) leak to the critical level that can trigger proarrhythmic Ca(2+) waves.

Topics: Adenosine Triphosphatases; Adrenergic beta-Agonists; Animals; Calcium; Calcium Signaling; Cell Culture Techniques; Cytosol; Enzyme Inhibitors; Ethylmaleimide; Heterocyclic Compounds, 4 or More Rings; Isoproterenol; Mitochondria; Myocytes, Cardiac; Oxidative Stress; Rabbits; Reactive Oxygen Species; Receptors, Adrenergic, beta; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum

Blebbistatin reversibly disrupted both stolon tip pulsations and gastrovascular flow in the colonial hydroid Podocoryna carnea. Epithelial longitudinal muscles of polyps were unaffected by blebbistatin, as polyps contracted when challenged with a pulse of KCl. Latrunculin B, which sequesters G actin preventing F actin assembly, caused stolons to retract, exposing focal adhesions where the tip epithelial cells adhere to the substratum. These results are consistent with earlier suggestions that non-muscle myosin II provides the motive force for stolon tip pulsations and further suggest that tip oscillations are functionally coupled to hydrorhizal axial muscle contraction.

Topics: Animals; Heterocyclic Compounds, 4 or More Rings; Hydrozoa; Muscle Contraction; Myosin Type II; Time-Lapse Imaging

Large quantities of human pluripotent stem cells (hPSCs) needed for therapeutic applications can be grown in scalable suspended microcarrier cultures. These microcarriers are coated with animal or human extracellular matrix (ECM) proteins to promote cell growth and maintain pluripotency. However, the coating is costly for large-scale cultures and it presents safety risks. This study demonstrates that hPSCs can be propagated on noncoated positively charged cellulose microcarriers in a serum-free medium containing the ROCK inhibitor, (Y27632) or myosin inhibitor, Blebbistatin. In the presence of these two inhibitors, myosin phosphatase 1 and myosin light chain 2 were dephosphorylated suggesting that reduced myosin contractility is responsible for hPSC survival and growth on ECM coating-free microcarriers. Cells propagated on the noncoated microcarriers for 12 passages maintained their pluripotency and karyotype stability. Scalability was demonstrated by achieving a cell concentration of 2.3×10⁶ cells/mL with 11.5-fold expansion (HES-3) in a 100-mL spinner flask. The differentiation capability of these cells toward three primary lineages is demonstrated via in vitro embryoid bodies and in vivo teratoma formations. Moreover, the directed differentiation to polysialylated neuronal cell adhesion molecule-positive (PSA-NCAM+) neural progenitors produced high cell concentrations (9.1±1.2×10⁶ cells/mL) with a cell yield of 412±77 neural progenitor cells per seeded HES-3 and a PSA-NCAM expression level of 91±1.1%. This defined serum- and coating-free scalable microcarrier culturing system is a safer and less expensive method for generating large amounts of hPSCs for cell therapies.

Topics: Biomarkers; Cell Culture Techniques; Cell Proliferation; Cells, Cultured; Coated Materials, Biocompatible; Embryonic Stem Cells; Extracellular Matrix; Heterocyclic Compounds, 4 or More Rings; Humans; Microspheres; Myosin Type II; Phosphorylation; Pluripotent Stem Cells; Protein Kinase Inhibitors; rho-Associated Kinases; Signal Transduction

Memories associated with drugs of abuse, such as methamphetamine (METH), increase relapse vulnerability to substance use disorder. There is a growing consensus that memory is supported by structural and functional plasticity driven by F-actin polymerization in postsynaptic dendritic spines at excitatory synapses. However, the mechanisms responsible for the long-term maintenance of memories, after consolidation has occurred, are largely unknown.. Conditioned place preference (n = 112) and context-induced reinstatement of self-administration (n = 19) were used to assess the role of F-actin polymerization and myosin II, a molecular motor that drives memory-promoting dendritic spine actin polymerization, in the maintenance of METH-associated memories and related structural plasticity.. Memories formed through association with METH but not associations with foot shock or food reward were disrupted by a highly-specific actin cycling inhibitor when infused into the amygdala during the postconsolidation maintenance phase. This selective effect of depolymerization on METH-associated memory was immediate, persistent, and did not depend upon retrieval or strength of the association. Inhibition of non-muscle myosin II also resulted in a disruption of METH-associated memory.. Thus, drug-associated memories seem to be actively maintained by a unique form of cycling F-actin driven by myosin II. This finding provides a potential therapeutic approach for the selective treatment of unwanted memories associated with psychiatric disorders that is both selective and does not rely on retrieval of the memory. The results further suggest that memory maintenance depends upon the preservation of polymerized actin.

Topics: Actins; Amygdala; Animals; Bridged Bicyclo Compounds, Heterocyclic; Conditioning, Psychological; Dendritic Spines; Electric Stimulation; Extinction, Psychological; Food; Heterocyclic Compounds, 4 or More Rings; Male; Memory; Mental Recall; Methamphetamine; Mice; Microinjections; Myosin Type II; Neuronal Plasticity; Polymerization; Rats; Reward; Self Administration; Thiazolidines

Mutations in the dysferlin gene resulting in dysferlin-deficiency lead to limb-girdle muscular dystrophy 2B and Myoshi myopathy in humans. Dysferlin has been proposed as a critical regulator of vesicle-mediated membrane resealing in muscle fibers, and localizes to muscle fiber wounds following sarcolemma damage. Studies in fibroblasts and urchin eggs suggest that trafficking and fusion of intracellular vesicles with the plasma membrane during resealing requires the intracellular cytoskeleton. However, the contribution of dysferlin-containing vesicles to resealing in muscle and the role of the cytoskeleton in regulating dysferlin-containing vesicle biology is unclear. Here, we use live-cell imaging to examine the behavior of dysferlin-containing vesicles following cellular wounding in muscle cells and examine the role of microtubules and kinesin in dysferlin-containing vesicle behavior following wounding. Our data indicate that dysferlin-containing vesicles move along microtubules via the kinesin motor KIF5B in muscle cells. Membrane wounding induces dysferlin-containing vesicle-vesicle fusion and the formation of extremely large cytoplasmic vesicles, and this response depends on both microtubules and functional KIF5B. In non-muscle cell types, lysosomes are critical mediators of membrane resealing, and our data indicate that dysferlin-containing vesicles are capable of fusing with lysosomes following wounding which may contribute to formation of large wound sealing vesicles in muscle cells. Overall, our data provide mechanistic evidence that microtubule-based transport of dysferlin-containing vesicles may be critical for resealing, and highlight a critical role for dysferlin-containing vesicle-vesicle and vesicle-organelle fusion in response to wounding in muscle cells.

Topics: Animals; Cell Line; Cell Membrane; Cytoplasmic Vesicles; Green Fluorescent Proteins; Heterocyclic Compounds, 4 or More Rings; Kinesins; Lysosomes; Membrane Fusion; Membrane Proteins; Microtubules; Muscle Cells; Muscle Fibers, Skeletal; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies, Limb-Girdle; Myosin Type II; Nocodazole; Rats; Tubulin Modulators

Mesenchymal stromal cells (MSCs) have remarkable clinical potential for cell-based therapy. Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs) from umbilical cord share unique properties with both embryonic and adult stem cells. MSCs are found at low frequency in vivo, and their successful therapeutic application depends on rapid and efficient large-scale expansion in vitro. Non-muscle myosin II (NMII) has pivotal roles in different cellular activities, such as cell division, migration and differentiation. We performed this study to understand the role of NMII in proliferation and cell cycle progression in WJ-MSCs.. WJ-MSCs were cultured in the presence of blebbistatin, and cell cycle analysis was performed using flow cytometry, proliferation kinetics, senescence assay and gene expression profile using polymerase chain reaction array.. When cultured in the presence of blebbistatin, an inhibitor of NMII adenosine triphosphatase activity, WJ-MSCs exhibited dose-dependent reduction in proliferative potential along with increase in cell size and induction of early senescence. Inhibition of NMII activity also affected cell cycle progression in WJ-MSCs and led to an increase in the percentage of cells in G0/G1 phase with a corresponding reduction in the percentage of cells in G2/M phase. Blebbistatin-induced G0/G1 arrest of WJ-MSCs was further associated with up-regulation of cell cycle inhibitory genes CDKN1A, CDKN2A and CDKN2B and down-regulation of numerous genes related to progression through S and M phases of the cell cycle.. Our study demonstrates that inhibition of NMII activity in WJ-MSCs leads to G0/G1 arrest and alteration in the expression levels of certain key cell cycle-related genes.

Topics: Cell Cycle Checkpoints; Cell Differentiation; Cells, Cultured; G1 Phase; Heterocyclic Compounds, 4 or More Rings; Humans; Mesenchymal Stem Cells; Myosin Type II; Resting Phase, Cell Cycle; Umbilical Cord; Wharton Jelly

Regulation of striated muscle contraction is achieved by Ca2+ -dependent steric modulation of myosin cross-bridge cycling on actin by the thin filament troponin-tropomyosin complex. Alterations in the complex can induce contractile dysregulation and disease. For example, mutations between or near residues 112 to 136 of cardiac troponin-T, the crucial TnT1 (N-terminal domain of troponin-T)-tropomyosin-binding region, cause cardiomyopathy. The Drosophila upheld(101) Glu/Lys amino acid substitution lies C-terminally adjacent to this phylogenetically conserved sequence.. Using a highly integrative approach, we sought to determine the molecular trigger of upheld(101) myofibrillar degeneration, to evaluate contractile performance in the mutant cardiomyocytes, and to examine the effects of the mutation on the entire Drosophila heart to elucidate regulatory roles for conserved TnT1 regions and provide possible mechanistic insight into cardiac dysfunction.. Live video imaging of Drosophila cardiac tubes revealed that the troponin-T mutation prolongs systole and restricts diastolic dimensions of the heart, because of increased numbers of actively cycling myosin cross-bridges. Elevated resting myocardial stiffness, consistent with upheld(101) diastolic dysfunction, was confirmed by an atomic force microscopy-based nanoindentation approach. Direct visualization of mutant thin filaments via electron microscopy and 3-dimensional reconstruction resolved destabilized tropomyosin positioning and aberrantly exposed myosin-binding sites under low Ca2+ conditions.. As a result of troponin-tropomyosin dysinhibition, upheld(101) hearts exhibited cardiac dysfunction and remodeling comparable to that observed during human restrictive cardiomyopathy. Thus, reversal of charged residues about the conserved tropomyosin-binding region of TnT1 may perturb critical intermolecular associations required for proper steric regulation, which likely elicits myopathy in our Drosophila model.

Topics: Amino Acid Sequence; Animals; Calcium; Cardiomyopathies; Diastole; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Excitation Contraction Coupling; Female; Genotype; Heterocyclic Compounds, 4 or More Rings; Male; Microscopy, Electron; Microscopy, Video; Molecular Sequence Data; Mutation; Myofibrils; Phenotype; Systole; Tropomyosin; Troponin T; Ventricular Dysfunction; Ventricular Function; Ventricular Remodeling

Self-renewal and differentiation of stem cells depend on asymmetric division and polarized motility processes that in other cell types are modulated by nonmuscle myosin-II (MII) forces and matrix mechanics. Here, mass spectrometry-calibrated intracellular flow cytometry of human hematopoiesis reveals MIIB to be a major isoform that is strongly polarized in hematopoietic stem cells and progenitors (HSC/Ps) and thereby downregulated in differentiated cells via asymmetric division. MIIA is constitutive and activated by dephosphorylation during cytokine-triggered differentiation of cells grown on stiff, endosteum-like matrix, but not soft, marrow-like matrix. In vivo, MIIB is required for generation of blood, while MIIA is required for sustained HSC/P engraftment. Reversible inhibition of both isoforms in culture with blebbistatin enriches for long-term hematopoietic multilineage reconstituting cells by 5-fold or more as assessed in vivo. Megakaryocytes also become more polyploid, producing 4-fold more platelets. MII is thus a multifunctional node in polarized division and niche sensing.

Topics: Apoptosis; Blotting, Western; Cell Culture Techniques; Cell Differentiation; Cell Lineage; Cell Movement; Cell Proliferation; Flow Cytometry; Hematopoiesis; Hematopoietic Stem Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Muscle Contraction; Nonmuscle Myosin Type IIA; Nonmuscle Myosin Type IIB; Phosphorylation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stem Cell Niche

Contraction regulates heart development via a complex mechanotransduction process controlled by various mechanical forces. Here, we exploit zebrafish embryos as an in vivo animal model to discern the contribution from different mechanical forces and identify the underlying mechanotransductive signaling pathways of cardiogenesis. We treated 2 days postfertilization zebrafish embryos with Blebbistatin, a myosin II inhibitor, to stop cardiac contraction, which induces a response termed cessation of contraction-induced cardiomyocyte (CM) enlargement (CCE). Accompanying the CCE, lateral fusion of myofibrils was attenuated within CMs. The CCE can be blunted by loss of blood in tail-docked zebrafish but not in cloche mutant fish, suggesting that transmural pressure rather than shear stress is accountable for the chamber enlargement. By screening a panel of small molecule inhibitors, our data suggested essential functions of phosphoinositide 3-kinase signaling and protein synthesis in CCE, which are independent of the sarcomere integrity. In summary, we defined a unique CCE response in genetically tractable zebrafish embryos. A panel of assays was established to verify the contribution from extrinsic forces and interrogate underlying signaling pathways.

Topics: Animals; Cell Differentiation; Heart; Heterocyclic Compounds, 4 or More Rings; Mechanotransduction, Cellular; Muscle Development; Mutation; Myocardial Contraction; Myocardium; Myocytes, Cardiac; Zebrafish; Zebrafish Proteins

A substrate fabrication process is developed to pattern both the extracellular matrix (ECM) and rigidity at sub-cellular spatial resolution. When growing cells on these substrates, it is found that cells respond locally in their cytoskeleton assembly. The presented method allows unique insight into the biological interpretation of mechanical signals, whereas photolithography-based fabrication is amenable to integration with complex microfabricated substructures.

Topics: 3T3 Cells; Acrylamide; Actins; Animals; Cell Adhesion; Cell Culture Techniques; Cell Shape; Culture Media; Cytoskeleton; Dimethylpolysiloxanes; Elastic Modulus; Elasticity; Epoxy Resins; Extracellular Matrix; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Mechanotransduction, Cellular; Mice; Microtechnology; Myosin Type II

Physical cues exist across all biological scales, from the geometries of molecules to the shapes of complex organisms. While their roles have been identified across a range of scales, i.e. the arrangements of biomolecules and the form and function of tissues, less is known in some intermediate lengths. Particularly, at the cell scale, there is emerging evidence demonstrating the impact of mechanical signals, such as substrate stiffness and confinement, on many critical biological processes and malignancies, especially cancer dissemination. In the context of cell invasion, it is currently unclear how cells select from accessible mechanical paths that result in migratory patterns observed in physiological environments. Here, we devise microchannel decision trees to explore how fundamental and ubiquitous mechanical factors, specifically dimensionality and directionality, affect migratory cell decision making. We then implement strategies based purely on mechanical asymmetries to induce repetitive, non-disseminating motions, in a phenomenon we call iteratio ad nauseam.

Topics: Actomyosin; Cell Line, Tumor; Cell Movement; Cell Shape; Decision Trees; Dimethylpolysiloxanes; Heterocyclic Compounds, 4 or More Rings; Humans; Microfluidic Analytical Techniques; Single-Cell Analysis

Cardiac and skeletal muscle function depends on the proper formation of myofibrils, which are tandem arrays of highly organized actomyosin contractile units called sarcomeres. How the architecture of these colossal molecular assemblages is established during development and maintained over the lifetime of an animal is poorly understood. We investigate the potential roles in myofibril formation and repair of formin proteins, which are encoded by 15 different genes in mammals. Using quantitative real-time PCR analysis, we find that 13 formins are differentially expressed in mouse hearts during postnatal development. Seven formins immunolocalize to sarcomeres in diverse patterns, suggesting that they have a variety of functional roles. Using RNA interference silencing, we find that the formins mDia2, DAAM1, FMNL1, and FMNL2 are required nonredundantly for myofibrillogenesis. Knockdown phenotypes include global loss of myofibril organization and defective sarcomeric ultrastructure. Finally, our analysis reveals an unanticipated requirement specifically for FMNL1 and FMNL2 in the repair of damaged myofibrils. Together our data reveal an unexpectedly large number of formins, with diverse localization patterns and nonredundant roles, functioning in myofibril development and maintenance, and provide the first evidence of actin assembly factors being required to repair myofibrils.

Topics: Actins; Animals; Animals, Newborn; Bridged Bicyclo Compounds, Heterocyclic; Cell Differentiation; Formins; Gene Expression Regulation, Developmental; Heterocyclic Compounds, 4 or More Rings; Intracellular Signaling Peptides and Proteins; Mice; Mice, Inbred C57BL; Microfilament Proteins; Microtubule-Associated Proteins; Muscle Development; Myocardium; Myocytes, Cardiac; NADPH Dehydrogenase; Primary Cell Culture; Protein Isoforms; rho GTP-Binding Proteins; RNA, Small Interfering; Sarcomeres; Thiazolidines; Wound Healing

The cell nucleus functions amidst active cytoskeletal filaments, but its response to their contractile stresses is largely unexplored. We study the dynamics of the nuclei of single fibroblasts, with cell migration suppressed by plating onto micro-fabricated patterns. We find the nucleus undergoes noisy but coherent rotational motion. We account for this observation through a hydrodynamic approach, treating the nucleus as a highly viscous inclusion residing in a less viscous fluid of orientable filaments endowed with active stresses. Lowering actin contractility selectively by introducing blebbistatin at low concentrations drastically reduced the speed and coherence of the angular motion of the nucleus. Time-lapse imaging of actin revealed a correlated hydrodynamic flow around the nucleus, with profile and magnitude consistent with the results of our theoretical approach. Coherent intracellular flows and consequent nuclear rotation thus appear to be an intrinsic property of cells.

Topics: Actin Cytoskeleton; Actins; Actomyosin; Animals; Cell Movement; Cell Nucleus; Cytoskeleton; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Mice; Muscle Contraction; NIH 3T3 Cells; Time-Lapse Imaging

Tolerogenic dendritic cells (tDCs) play important roles in immune tolerance, autoimmune disease, tissue transplantation, and the tumor micro-environment. Factors that induce tDCs have been reported, however the intracellular mechanisms involved are rarely discussed.. Circulating CD14(+)CD16(+) of breast cancer patients and induced CD14(+)CD16(+) DCs were identified as tDCs by treating CD14(+) monocytes with galectin-1 and cancer cell-derived medium combined with IL-4 and GM-CSF. In addition, the 4T1 breast cancer syngeneic xenograft model was used to investigate the effect of galectin-1 in vivo.. The CD14(+)CD16(+) tDC population in the breast cancer patients was comparatively higher than that in the healthy donors, and both the MDA-MB-231 conditioned medium and galectin-1 could induce tDC differentiation. In a BALB/c animal model, the 4T1 breast cancer cell line enhanced IL-10 expression in CD11c(+) DCs which was down-regulated after knocking down the galectin-1 expression of 4T1 cells. Analysis of galectin-1 interacting proteins showed that myosin IIa was a major target of galectin-1 after internalization through a caveolin-dependent endocytosis. Myosin IIa specific inhibitor could diminish the effects of galectin-1 on monocyte-derived tDCs and also block the 4T1 cell induced CD11c(+)/Ly6G(+)/IL-10(+) in the BALB/c mice.. Galectin-1 can induce tDCs after internalizing into CD14(+) monocytes through the caveolae-dependent pathway and activating myosin IIa. For the breast cancer patients with a high galectin-1 expression, blebbistatin and genistein show potential in immune modulation and cancer immunotherapy.. Myosin IIa activation and galectin-1 endocytosis are important in tumor associated tDC development.

Topics: Animals; Breast Neoplasms; Cell Differentiation; Dendritic Cells; Endocytosis; Female; Galectin 1; Heterocyclic Compounds, 4 or More Rings; Humans; Immune Tolerance; Mice; Mice, Inbred BALB C; Nonmuscle Myosin Type IIA

Speckle microscopy directly visualizes the retrograde actin flow, which is believed to promote cell-edge protrusion when linked to focal adhesions (FAs). However, it has been argued that, due to rapid actin turnover, the use of green fluorescent protein-actin, the lack of appropriate analysis algorithms, and technical difficulties, speckle microscopy does not necessarily report the flow velocities of entire actin populations. In this study, we developed a new, user-friendly single-molecule speckle (SiMS) microscopy using DyLight dye-labeled actin. Our new SiMS method enables in vivo nanometer-scale displacement analysis with a low localization error of ±8-8.5 nm, allowing accurate flow-velocity measurement for actin speckles with lifetime <5 s. In lamellipodia, both short- and long-lived F-actin molecules flow with the same speed, indicating they are part of a single actin network. These results do not support coexistence of F-actin populations with different flow speeds, which is referred to as the lamella hypothesis. Mature FAs, but not nascent adhesions, locally obstruct the retrograde flow. Interestingly, the actin flow in front of mature FAs is fast and biased toward FAs, suggesting that mature FAs attract the flow in front and actively remodel the local actin network.

Topics: Actin Cytoskeleton; Actins; Animals; Cytoplasm; Electroporation; Focal Adhesions; Heterocyclic Compounds, 4 or More Rings; Humans; Intracellular Signaling Peptides and Proteins; Light; Lysine; Microscopy; Microtubule-Associated Proteins; Myosin Type II; Nanoparticles; Particle Size; Pseudopodia; Rabbits; Regression Analysis; Staining and Labeling; Stress Fibers; Time Factors

Biomaterial approaches for engineering orthopedic interfaces such as the tendon-bone junction (TBJ) are limited by a lack of understanding of how insoluble (microstructure, composition) and soluble regulators of stem cell fate work in concert to promote bioactivity and differentiation. One strategy for regenerating the interface is to design biomaterials containing spatially graded structural properties sufficient to induce divergent mesenchymal stem cell (MSC) differentiation into multiple interface-specific phenotypes. This work explores the hypothesis that selective structural modification to a 3D collagen-glycosaminoglycan (CG) scaffold combined with biochemical supplementation can drive human bone-marrow-derived MSC differentiation down tenogenic, osteogenic, and chondrogenic lineages. Tenogenic differentiation is enhanced in geometrically anisotropic scaffolds versus a standard isotropic control. Notably, blebbistatin treatment abrogates this microstructurally driven effect. Further, enhanced osteogenic differentiation and new mineral synthesis are achieved by incorporation of a calcium phosphate mineral phase within the CG scaffold along with the use of osteogenic induction media. Finally, chondrogenic differentiation is optimally driven by combining chondrogenic induction media with a reduced density scaffold that promotes increased cellular condensation, significantly higher expression of chondrogenic genes, and increased GAG deposition. Together these data provide critical insight regarding design rules for elements of an integrated biomaterial platform for orthopedic interface regeneration.

Topics: Cell Differentiation; Cell Shape; Cells, Cultured; Collagen; Extracellular Matrix Proteins; Gene Expression Profiling; Heterocyclic Compounds, 4 or More Rings; Histocytochemistry; Humans; Mesenchymal Stem Cells; Osteogenesis; Tissue Scaffolds

Microenvironment stiffening plays a crucial role in tumorigenesis. While filopodia are generally thought to be one of the cellular mechanosensors for probing environmental stiffness, the effects of environmental stiffness on filopodial activities of cancer cells remain unclear. In this work, we investigated the filopodial activities of human lung adenocarcinoma cells CL1-5 cultured on substrates of tunable stiffness using a novel platform. The platform consists of an optical system called structured illumination nano-profilometry, which allows time-lapsed visualization of filopodial activities without fluorescence labeling. The culturing substrates were composed of polyvinyl chloride mixed with an environmentally friendly plasticizer to yield Young's modulus ranging from 20 to 60 kPa. Cell viability studies showed that the viability of cells cultured on the substrates was similar to those cultured on commonly used elastomers such as polydimethylsiloxane. Time-lapsed live cell images were acquired and the filopodial activities in response to substrates with varying degrees of stiffness were analyzed. Statistical analyses revealed that lung cancer cells cultured on softer substrates appeared to have longer filopodia, higher filopodial densities with respect to the cellular perimeter, and slower filopodial retraction rates. Nonetheless, the temporal analysis of filopodial activities revealed that whether a filopodium decides to extend or retract is purely a stochastic process without dependency on substrate stiffness. The discrepancy of the filopodial activities between lung cancer cells cultured on substrates with different degrees of stiffness vanished when the myosin II activities were inhibited by treating the cells with blebbistatin, which suggests that the filopodial activities are closely modulated by the adhesion strength of the cells. Our data quantitatively relate filopodial activities of lung cancer cells with environmental stiffness and should shed light on the understanding and treatment of cancer progression and metastasis.

Topics: Cell Adhesion; Cell Line, Tumor; Cell Survival; Heterocyclic Compounds, 4 or More Rings; Humans; Lung Neoplasms; Pseudopodia; Tumor Cells, Cultured

Haemolytic uraemic syndrome caused by Shiga toxin-producing E. coli (STEC) is dependent on release of Shiga toxins (Stxs) during intestinal infection and subsequent absorption into the bloodstream. An understanding of Stx-related events in the human gut is limited due to lack of suitable experimental models. In this study, we have used a vertical diffusion chamber system with polarized human colon carcinoma cells to simulate the microaerobic (MA) environment in the human intestine and investigate its influence on Stx release and translocation during STEC O157:H7 and O104:H4 infection. Stx2 was the major toxin type released during infection. Whereas microaerobiosis significantly reduced bacterial growth as well as Stx production and release into the medium, Stx translocation across the epithelial monolayer was enhanced under MA versus aerobic conditions. Increased Stx transport was dependent on STEC infection and occurred via a transcellular pathway other than macropinocytosis. While MA conditions had a similar general effect on Stx release and absorption during infection with STEC O157:H7 and O104:H4, both serotypes showed considerable differences in colonization, Stx production, and Stx translocation which suggest alternative virulence strategies. Taken together, our study suggests that the MA environment in the human colon may modulate Stx-related events and enhance Stx absorption during STEC infection.

Topics: Anaerobiosis; Animals; Cell Line, Tumor; Chlorocebus aethiops; Colonic Diseases; Cytochalasin D; Escherichia coli Infections; Gastrointestinal Tract; Hemolytic-Uremic Syndrome; Heterocyclic Compounds, 4 or More Rings; Humans; Oxygen; Pinocytosis; Shiga Toxin; Shiga-Toxigenic Escherichia coli; Vero Cells

Non-muscle myosin II (NM II) regulates a wide range of cellular functions, including neuronal differentiation, which requires precise spatio-temporal activation of Rho GTPases. The molecular mechanism underlying the NM II-mediated activation of Rho GTPases is poorly understood. The present study explored the possibility that NM II regulates neuronal differentiation, particularly morphological changes in growth cones and the distal axon, through guanine nucleotide exchange factors (GEFs) of the Dbl family.. NM II colocalized with GEFs, such as βPIX, kalirin and intersectin, in growth cones. Inactivation of NM II by blebbistatin (BBS) led to the increased formation of short and thick filopodial actin structures at the periphery of growth cones. In line with these observations, FRET analysis revealed enhanced Cdc42 activity in BBS-treated growth cones. BBS treatment also induced aberrant targeting of various GEFs to the distal axon where GEFs were seldom observed under physiological conditions. As a result, numerous protrusions and branches were generated on the shaft of the distal axon. The disruption of the NM II-GEF interactions by overexpression of the DH domains of βPIX or Tiam1, or by βPIX depletion with specific siRNAs inhibited growth cone formation and induced slender axons concomitant with multiple branches in cultured hippocampal neurons. Finally, stimulation with nerve growth factor induced transient dissociation of the NM II-GEF complex, which was closely correlated with the kinetics of Cdc42 and Rac1 activation.. Our results suggest that NM II maintains proper morphology of neuronal growth cones and the distal axon by regulating actin dynamics through the GEF-Rho GTPase signaling pathway.

Topics: Actins; Animals; Axons; cdc42 GTP-Binding Protein; Cells, Cultured; Female; Growth Cones; Heterocyclic Compounds, 4 or More Rings; Hippocampus; Models, Biological; Nerve Growth Factors; Neurites; Neurons; Nonmuscle Myosin Type IIB; p21-Activated Kinases; Protein Binding; Protein Structure, Tertiary; Pseudopodia; rac1 GTP-Binding Protein; Rats, Sprague-Dawley; Rho Guanine Nucleotide Exchange Factors; Signal Transduction

Contraction of 3D collagen matrices by fibroblasts frequently is used as an in vitro model of wound closure. Different iterations of the model - all conventionally referred to as "contraction" - involve different morphological patterns. During floating matrix contraction, cells initially are round without stress fibers and subsequently undergo spreading. During stressed matrix contraction, cells initially are spread with stress fibers and subsequently undergo shortening. In the current studies, we used siRNA silencing of myosin IIA (MyoIIA) and myosin IIB (MyoIIB) to test the roles of myosin II isoforms in fibroblast interactions with 3D collagen matrices and collagen matrix contraction. We found that MyoIIA but not MyoIIB was required for cellular global inward contractile force, formation of actin stress fibers, and morphogenic cell clustering. Stressed matrix contraction required MyoIIA but not MyoIIB. Either MyoIIA or MyoIIB was sufficient for floating matrix contraction (FMC) stimulated by platelet-derived growth factor. Neither MyoIIA or MyoIIB was necessary for FMC stimulated by serum. Our findings suggest that myosin II-dependent motor mechanisms for collagen translocation during extracellular matrix remodeling differ depending on cell tension and growth factor stimulation.

Topics: Biomechanical Phenomena; Cell Adhesion; Cell Aggregation; Cell Line; Cell Movement; Collagen; Extracellular Matrix; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Humans; Nonmuscle Myosin Type IIA; Nonmuscle Myosin Type IIB; RNA, Small Interfering; Wound Healing

Epithelial scattering occurs when cells disassemble cell-cell junctions, allowing individual epithelial cells to act in a solitary manner. Epithelial scattering occurs frequently in development, where it accompanies epithelial-mesenchymal transitions and is required for individual cells to migrate and invade. While migration and invasion have received extensive research focus, how cell-cell junctions are detached remains poorly understood. An open debate has been whether disruption of cell-cell interactions occurs by remodeling of cell-cell adhesions, increased traction forces through cell substrate adhesions, or some combination of both processes. Here we seek to examine how changes in adhesion and contractility are coupled to drive detachment of individual epithelial cells during hepatocyte growth factor (HGF)/scatter factor-induced EMT. We find that HGF signaling does not alter the strength of cell-cell adhesion between cells in suspension, suggesting that changes in cell-cell adhesion strength might not accompany epithelial scattering. Instead, cell-substrate adhesion seems to play a bigger role, as cell-substrate adhesions are stronger in cells treated with HGF and since rapid scattering in cells treated with HGF and TGFβ is associated with a dramatic increase in focal adhesions. Increases in the pliability of the substratum, reducing cells ability to generate traction on the substrate, alter cells׳ ability to scatter. Further consistent with changes in substrate adhesion being required for cell-cell detachment during EMT, scattering is impaired in cells expressing both active and inactive RhoA mutants, though in different ways. In addition to its roles in driving assembly of both stress fibers and focal adhesions, RhoA also generates myosin-based contractility in cells. We therefore sought to examine how RhoA-dependent contractility contributes to cell-cell detachment. Inhibition of Rho kinase or myosin II induces the same effect on cells, namely an inhibition of cell scattering following HGF treatment. Interestingly, restoration of myosin-based contractility in blebbistatin-treated cells results in cell scattering, including global actin rearrangements. Scattering is reminiscent of HGF-induced epithelial scattering without a concomitant increase in cell migration or decrease in adhesion strength. This scattering is dependent on RhoA, as blebbistatin-induced scattering is reduced in cells expressing dominant-negative RhoA mutants. This suggests that

Topics: Actins; Animals; Cell Adhesion; Cell Line; Cell Movement; Dogs; Epithelial Cells; Epithelial-Mesenchymal Transition; Focal Adhesions; Hepatocyte Growth Factor; Heterocyclic Compounds, 4 or More Rings; Intercellular Junctions; Mutation; Myosin Type II; rhoA GTP-Binding Protein; Signal Transduction; Stress Fibers

It has been unknown whether cells retain their mechanical properties after fixation. Therefore, this study was designed to compare the stiffness properties of the cell cortex (the 50-100 nm thick zone below the plasma membrane) before and after fixation. Atomic force microscopy was used to acquire force indentation curves from which the nanomechanical cell properties were derived. Cells were pretreated with different concentrations of actin destabilizing agent cytochalasin D, which results in a gradual softening of the cell cortex. Then cells were studied 'alive' or 'fixed'. We show that the cortical stiffness of fixed endothelial cells still reports functional properties of the actin web qualitatively comparable to those of living cells. Myosin motor protein activity, tested by blebbistatin inhibition, can only be detected, in terms of cortical mechanics, in living but not in fixed cells. We conclude that fixation interferes with motor proteins while maintaining a functional cortical actin web. Thus, fixation of cells opens up the prospect of differentially studying the actions of cellular myosin and actin.

Topics: Actin Cytoskeleton; Animals; Biomechanical Phenomena; Cattle; Cell Line; Cytochalasin D; Endothelial Cells; Heterocyclic Compounds, 4 or More Rings; Microscopy, Atomic Force; Myosins; Tissue Fixation

Precise control of the formation and development of dendritic spines is critical for synaptic plasticity. Consequently, abnormal spine development is linked to various neurological disorders. The actin cytoskeleton is a structural element generating specific changes in dendritic spine morphology. Although mechanisms underlying dendritic filopodia elongation and spine head growth are relatively well understood, it is still not known how spine heads are enlarged and stabilized during dendritic spine maturation. By using rat hippocampal neurons, we demonstrate that the size of the stable actin pool increases during the neuronal maturation process. Simultaneously, the treadmilling rate of the dynamic actin pool increases. We further show that myosin IIb controls dendritic spine actin cytoskeleton by regulating these two different pools of F-actin via distinct mechanisms. The findings indicate that myosin IIb stabilizes the stable F-actin pool through actin cross-linking. Simultaneously, activation of myosin IIb contractility increases the treadmilling rate of the dynamic pool of actin. Collectively, these data show that myosin IIb has a major role in the regulation of actin filament stability in dendritic spines, and elucidate the complex mechanism through which myosin IIb functions in this process. These new insights into the mechanisms underlying dendritic spine maturation further the model of dendritic spine morphogenesis.

Topics: Actin Cytoskeleton; Actins; Animals; Cells, Cultured; Dendritic Spines; Embryo, Mammalian; Enzyme Inhibitors; Green Fluorescent Proteins; Heterocyclic Compounds, 4 or More Rings; Hippocampus; Luminescent Proteins; Marine Toxins; Microtubule-Associated Proteins; Models, Biological; Neurons; Nonmuscle Myosin Type IIB; Oxazoles; Rats; Rats, Wistar; Red Fluorescent Protein; RNA, Small Interfering; Time Factors

Blebbistatin, the best characterized myosin II-inhibitor, is commonly used to study the biological roles of various myosin II isoforms. Despite its popularity, the use of blebbistatin is greatly hindered by its blue-light sensitivity, resulting in phototoxicity and photoconversion of the molecule. Additionally, blebbistatin has serious cytotoxic side effects even in the absence of irradiation, which may easily lead to the misinterpretation of experimental results since the cytotoxicity-derived phenotype could be attributed to the inhibition of the myosin II function. Here we report the synthesis as well as the in vitro and in vivo characterization of a photostable, C15 nitro derivative of blebbistatin with unaffected myosin II inhibitory properties. Importantly, para-nitroblebbistatin is neither phototoxic nor cytotoxic, as shown by cellular and animal tests; therefore it can serve as an unrestricted and complete replacement of blebbistatin both in vitro and in vivo.

Topics: Heterocyclic Compounds, 4 or More Rings; Myosin Type II; Photochemical Processes

An endocytic vesicle is formed from a flat plasma membrane patch by a sequential process of invagination, bud formation and fission. The scission step requires the formation of a tubular membrane neck (the fission pore) that connects the endocytic vesicle with the plasma membrane. Progress in vesicle fission can be measured by the formation and closure of the fission pore. Live-cell imaging and sensitive biophysical measurements have provided various glimpses into the structure and behaviour of the fission pore. In the present study, the role of non-muscle myosin II (NM-2) in vesicle fission was tested by analyzing the kinetics of the fission pore with perforated-patch clamp capacitance measurements to detect single vesicle endocytosis with millisecond time resolution in peritoneal mast cells. Blebbistatin, a specific inhibitor of NM-2, dramatically increased the duration of the fission pore and also prevented closure during large endocytic events. Using the fluorescent markers FM1-43 and pHrodo Green dextran, we found that NM-2 inhibition greatly arrested vesicle fission in a late phase of the scission event when the pore reached a final diameter of ∼ 5 nm. Our results indicate that loss of the ATPase activity of myosin II drastically reduces the efficiency of membrane scission by making vesicle closure incomplete and suggest that NM-2 might be especially relevant in vesicle fission during compound endocytosis.

Topics: Animals; Cell Membrane; Endocytosis; Exocytosis; Heterocyclic Compounds, 4 or More Rings; Mast Cells; Mice; Myosin Type II; Protein Binding; Pyridinium Compounds; Quaternary Ammonium Compounds; Transport Vesicles

A key feature of Shigella pathogenesis is the ability to spread from cell-to-cell post-invasion. This is dependent on the bacteria's ability to initiate de novo F-actin tail polymerisation, followed by protrusion formation, uptake of bacteria-containing protrusion and finally, lysis of the double membrane vacuole in the adjacent cell. In epithelial cells, cytoskeletal tension is maintained by the actin-myosin II networks. In this study, the role of myosin II and its specific kinase, myosin light chain kinase (MLCK), during Shigella intercellular spreading was investigated in HeLa cells. Inhibition of MLCK and myosin II, as well as myosin IIA knockdown, significantly reduced Shigella plaque and infectious focus formation. Protrusion formation and intracellular bacterial growth was not affected. Low levels of myosin II were localised to the Shigella F-actin tail. HeLa cells were also infected with Shigella strains defective in cell-to-cell spreading. Unexpectedly loss of myosin IIA labelling was observed in HeLa cells infected with these mutant strains. This phenomenon was not observed with WT Shigella or with the less abundant myosin IIB isoform, suggesting a critical role for myosin IIA.

Topics: Actins; Bacterial Proteins; DNA-Binding Proteins; Gene Knockdown Techniques; HeLa Cells; Heterocyclic Compounds, 4 or More Rings; Host-Pathogen Interactions; Humans; Lipopolysaccharides; Mutation; Myosin-Light-Chain Kinase; Nonmuscle Myosin Type IIA; RNA, Small Interfering; Shigella flexneri; Transcription Factors; Virulence

Microglia are resident macrophages in the central nervous system (CNS) and the primary cells that contribute to CNS inflammation in many pathological conditions. Upon any signs of brain damage, microglia become activated and undergo tremendous cellular reorganization to adopt appropriate phenotypes. They migrate to lesion areas, accumulate, phagocytose cells or cellular debris, and produce a large array of inflammatory mediators like cytokines, chemokines, reactive oxygen species, and other mediators. To cope with the extreme cellular rearrangements during activation, microglia have to be highly dynamic. One major component of the cytoskeleton in nonmuscle cells is nonmuscle myosin II (NM II). This study was aimed to examine the functional role of NM II in resting and activated microglia. Using immunohistochemistry, we demonstrate strong expression of NM II isoform B (NM IIB) in microglia during cuprizone-induced demyelination as well as in cultured microglia. Treatment with the NM II inhibitor blebbistatin prevented the morphological shaping of microglial cells, led to functional deficits during chemokine-directed migration and phagocytosis, induced NM IIB redistribution, and affected actin microfilament patterning. In addition, inhibition of NM II led to an attenuated release of nitric oxide (NO), while TNFα secretion was not altered. In conclusion, we propose a pivotal role of NM II in cytoskeleton organization during microglial activation. This is of great importance to understand the mechanisms of microglial action in inflammatory CNS diseases.

Topics: Animals; Animals, Newborn; Brain; Cell Movement; Cells, Cultured; Chemokine CCL2; Cuprizone; Demyelinating Diseases; Disease Models, Animal; Dose-Response Relationship, Drug; Heterocyclic Compounds, 4 or More Rings; Male; Mice, Inbred C57BL; Microglia; Monoamine Oxidase Inhibitors; Myosin Heavy Chains; Nonmuscle Myosin Type IIB; Phagocytosis; Rats; Rats, Sprague-Dawley

After induction, the inner ear is transformed from a superficially located otic placode into an epithelial vesicle embedded in the mesenchyme of the head. Invagination of this epithelium is biphasic: phase 1 involves the expansion of the basal aspect of the otic cells, and phase 2, the constriction of their apices. Apical constriction is important not only for otic invagination, but also the invagination of many other epithelia; however, its molecular basis is still poorly understood. Here we show that phase 2 otic morphogenesis, like phase 1 morphogenesis, results from the activation of myosin-II. However unlike the actin depolymerising activity observed basally, active myosin-II results in actomyosin contractility. Myosin-II activation is triggered by the accumulation of the planar cell polarity (PCP) core protein, Celsr1 in apical junctions (AJ). Apically polarized Celsr1 orients and recruits the Rho Guanine exchange factor (GEF) ArhGEF11 to apical junctions, thus restricting RhoA activity to the junctional membrane where it activates the Rho kinase ROCK. We suggest that myosin-II and RhoA activation results in actomyosin dependent constriction in an apically polarised manner driving otic epithelium invagination.

Topics: Animals; Azepines; Blotting, Western; Cadherins; Chick Embryo; Ear, Inner; Electroporation; Gene Expression Regulation, Developmental; Heterocyclic Compounds, 4 or More Rings; Image Processing, Computer-Assisted; Immunohistochemistry; Morphogenesis; Naphthalenes; Peptides; rho-Associated Kinases; rhoA GTP-Binding Protein; RNA Interference

The molecular and cellular mechanisms governing cell motility and directed migration in response to the neuropeptide bradykinin are largely unknown. Here, we demonstrate that human glioma cells whose migration is guided by bradykinin generate bleb-like protrusions. We found that activation of the B2 receptor leads to a rise in free Ca(2+) from internal stores that activates actomyosin contraction and subsequent cytoplasmic flow into protrusions forming membrane blebs. Furthermore Ca(2+) activates Ca(2+)-dependent K(+) and Cl(-) channels, which participate in bleb regulation. Treatment of gliomas with bradykinin in situ increased glioma growth by increasing the speed of cell migration at the periphery of the tumour mass. To test if bleb formation is related to bradykinin-promoted glioma invasion we blocked glioma migration with blebbistatin, a blocker of myosin kinase II, which is necessary for proper bleb retraction. Our findings suggest a pivotal role of bradykinin during glioma invasion by stimulating amoeboid migration of glioma cells.

Topics: Animals; Bradykinin; Brain; Calcium; Cell Line, Tumor; Cell Movement; Cell Surface Extensions; Cytoskeleton; Glioma; Heterocyclic Compounds, 4 or More Rings; Humans; Mice; Mice, Inbred BALB C; Mice, SCID; Neoplasm Invasiveness; Neurons; Potassium Channels; Sodium Channels

The super-relaxed state of myosin (SRX), in which the myosin ATPase activity is strongly inhibited, has been observed in a variety of muscle types. It has been proposed that myosin heads in this state are inhibited by binding to the core of the thick filament in a structure known as the interacting-heads motif. The myosin inhibitor blebbistatin has been shown in structural studies to stabilize the binding of myosin heads to the thick filament, and here we have utilized measurements of single ATP turnovers to show that blebbistatin also stabilizes the SRX in both fast and slow skeletal muscle, providing further support for the proposal that myosin heads in the SRX are also in the interacting-heads motif. We find that the SRX is stabilized using blebbistatin even in conditions that normally destabilize it, e.g., rigor ADP. Using blebbistatin we show that spin-labeled nucleotides bound to myosin have an oriented spectrum in the SRX in both slow and fast skeletal muscle. This is to our knowledge the first observation of oriented spin probes on the myosin motor domain in relaxed skeletal muscle fibers. The spectra for skeletal muscle with blebbistatin are similar to those observed in relaxed tarantula fibers in the absence of blebbistatin, demonstrating that the structure of the SRX is similar in different muscle types and in the presence and absence of blebbistatin. The mobility of spin probes attached to nucleotides bound to myosin shows that the conformation of the nucleotide site is closed in the SRX.

Topics: Adenosine Triphosphate; Animals; Binding Sites; Heterocyclic Compounds, 4 or More Rings; Kinetics; Muscle Relaxation; Muscle, Skeletal; Myosins; Protein Conformation; Protein Stability; Rabbits; Spin Labels

Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin-Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization.

Topics: Actomyosin; Animals; Cell Line, Tumor; Cell Membrane; Cell Polarity; Cell Proliferation; Dogs; Epithelial Cells; Epithelium; HeLa Cells; Hepatocyte Growth Factor; Heterocyclic Compounds, 4 or More Rings; Humans; Intercellular Junctions; Madin Darby Canine Kidney Cells; MCF-7 Cells; Microvilli; Myosin Type II

Rigidity sensing and durotaxis are thought to be important elements in wound healing, tissue formation, and cancer treatment. It has been challenging, however, to study the underlying mechanism due to difficulties in capturing cells during the transient response to a rigidity interface. We have addressed this problem by developing a model experimental system that confines cells to a micropatterned area with a rigidity border. The system consists of a rigid domain of one large adhesive island, adjacent to a soft domain of small adhesive islands grafted on a nonadhesive soft gel. This configuration allowed us to test rigidity sensing away from the cell body during probing and spreading. NIH 3T3 cells responded to the micropatterned rigidity border similarly to cells at a conventional rigidity border, by showing a strong preference for staying on the rigid side. Furthermore, cells used filopodia extensions to probe substrate rigidity at a distance in front of the leading edge and regulated their responses based on the strain of the intervening substrate. Soft substrates inhibited focal adhesion maturation and promoted cell retraction, whereas rigid substrates allowed stable adhesions and cell spreading. Myosin II was required for not only the generation of probing forces but also the retraction in response to soft substrates. We suggest that a myosin II-driven, filopodia-based probing mechanism ahead of the leading edge allows cells to migrate efficiently, by sensing physical characteristics before moving over a substrate to avoid backtracking.

Topics: Animals; Cell Adhesion; Cell Movement; Cell Physiological Phenomena; Cellular Microenvironment; Fibroblasts; Focal Adhesions; Heterocyclic Compounds, 4 or More Rings; Hydrogels; Mice; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Models, Biological; Myosin Type II; NIH 3T3 Cells; Pseudopodia; Surface Properties; Time-Lapse Imaging

Cell migration is fundamental to a variety of physiological processes, including tissue development, homeostasis, and regeneration. Migration has been extensively studied with cells on 2-dimensional (2D) substrates, but much less is known about cell migration in 3D environments. Tissues and organs are 3D, which is the native environment of cells in vivo, pointing to a need to understand migration and the mechanisms that regulate it in 3D environments. To investigate cell migration in 3D environments, we developed microfluidic devices that afford a controlled, reproducible platform for generating 3D matrices. Using these devices, we show that the Rho family guanine nucleotide exchange factor (GEF) Asef2 inhibits cell migration in 3D type I collagen (collagen I) matrices. Treatment of cells with the myosin II (MyoII) inhibitor blebbistatin abolished the decrease in migration by Asef2. Moreover, Asef2 enhanced MyoII activity as shown by increased phosphorylation of serine 19 (S19). Furthermore, Asef2 increased activation of Rac, which is a Rho family small GTPase, in 3D collagen I matrices. Inhibition of Rac activity by treatment with the Rac-specific inhibitor NSC23766 abrogated the Asef2-promoted increase in S19 MyoII phosphorylation. Thus, our results indicate that Asef2 regulates cell migration in 3D collagen I matrices through a Rac-MyoII-dependent mechanism.

Topics: Cell Line; Cell Movement; Collagen; Guanine Nucleotide Exchange Factors; Heterocyclic Compounds, 4 or More Rings; Humans; Immunohistochemistry; Myosin Type II

Actin cytoskeletons, aggregated with myosin II, generate intracellular cytoskeletal tension, which is induced to cell attaching substrate as cell traction forces. It is thought that cytoskeletal tension links closely to cell functions. The present study examined quantitative relationships between cytoskeleton tension and the balance of cell metabolism of tenocytes. Using micromachining techniques, micropillar substrates were prepared with polydimethylsiloxane, having three different values of substrate elasticity (6, 18 and 33 kPa) by changing the micropillar height. After 24h incubation of bovine tenocytes on these micropillar substrates, cell traction forces were determined. Gene expressions for type I collagen (anabolic marker) and MMP-1 (catabolic marker) from tenocytes on micropillars were also analyzed with qPCR. In addition, effects of an inhibition of myosin II activity on tenocyte cytoskeletal tension and metabolism were examined using the inhibitor, blebbistatin. It was exhibited that cell traction forces were significantly larger in tenocytes on 33 kPa substrates compared to those on 6 kPa substrates. This was associated with significant lower expression of MMP-1 mRNA on 33 kPa substrates. Cell traction forces were decreased significantly by the supplementation of blebbistatin in a dose-dependent manner. Indeed, there were significant smaller traction forces and higher expression for MMP-1 mRNA from tenocytes treated with 10 μM blebbistatin compared to their corresponding controls. Accordingly, tenocyte responses to substrate stiffness are associated with alterations in intracellular tension and catabolic gene expression. On the other hand, tenocyte anabolism, as measured by type I collagen mRNA expression, was not altered with substrate stiffness.

Topics: Actin Cytoskeleton; Animals; Biomarkers; Cattle; Cells, Cultured; Collagen Type I; Dimethylpolysiloxanes; Elasticity; Gene Expression Regulation, Enzymologic; Heterocyclic Compounds, 4 or More Rings; Matrix Metalloproteinase 1; Matrix Metalloproteinase Inhibitors; Myosin Type II; RNA, Messenger; Stress, Physiological; Tendons

There is increasing evidence to suggest that physical parameters, including substrate rigidity, topography, and cell geometry, play an important role in cell migration. As there are significant differences in cell behavior when cultured in 1D, 2D, or 3D environments, we hypothesize that migrating cells are also able to sense the dimension of the environment as a guidance cue. NIH 3T3 fibroblasts were cultured on micropatterned substrates where the path of migration alternates between 1D lines and 2D rectangles. We found that 3T3 cells had a clear preference to stay on 2D rather than 1D substrates. Cells on 2D surfaces generated stronger traction stress than did those on 1D surfaces, but inhibition of myosin II caused cells to lose their sensitivity to substrate dimension, suggesting that myosin-II-dependent traction forces are the determining factor for dimension sensing. Furthermore, oncogene-transformed fibroblasts are defective in mechanosensing while generating similar traction forces on 1D and 2D surfaces. Dimension sensing may be involved in guiding cell migration for both physiological functions and tissue engineering, and for maintaining normal cells in their home tissue.

Topics: Acrylic Resins; Animals; Cell Movement; Cell Transformation, Neoplastic; Fibroblasts; Focal Adhesions; Genes, ras; Heterocyclic Compounds, 4 or More Rings; Hydrogel, Polyethylene Glycol Dimethacrylate; Mice; Myosin Type II; NIH 3T3 Cells; Stress Fibers; Stress, Physiological

The Ca(2+) sensitivity of the myofilaments is increased in hypertrophic cardiomyopathy and other heart diseases and may contribute to a higher risk for sudden cardiac death. Ca(2+) sensitization increases susceptibility to reentrant ventricular tachycardia in animal models, but the underlying mechanism is unknown.. To investigate how myofilament Ca(2+) sensitization creates reentrant arrhythmia susceptibility.. Using hypertrophic cardiomyopathy mouse models (troponinT-I79N) and a Ca(2+) sensitizing drug (EMD57033), here we identify focal energy deprivation as a direct consequence of myofilament Ca(2+) sensitization. To detect ATP depletion and thus energy deprivation, we measured accumulation of dephosphorylated Connexin 43 (Cx43) isoform P0 and AMP kinase activation by Western blotting and immunostaining. No differences were detected between groups at baseline, but regional accumulation of Connexin 43 isoform P0 occurred within minutes in all Ca(2+)-sensitized hearts, in vivo after isoproterenol challenge and in isolated hearts after rapid pacing. Lucifer yellow dye spread demonstrated reduced gap junctional coupling in areas with Connexin 43 isoform P0 accumulation. Optical mapping revealed that selectively the transverse conduction velocity was slowed and anisotropy increased. Myofilament Ca(2+) desensitization with blebbistatin prevented focal energy deprivation, transverse conduction velocity slowing, and the reentrant ventricular arrhythmias.. Myofilament Ca(2+) sensitization rapidly leads to focal energy deprivation and reduced intercellular coupling during conditions that raise arrhythmia susceptibility. This is a novel proarrhythmic mechanism that can increase arrhythmia susceptibility in structurally normal hearts within minutes and may, therefore, contribute to sudden cardiac death in diseases with increased myofilament Ca(2+) sensitivity.

Topics: Adenosine Triphosphate; Adenylate Kinase; Animals; Arrhythmias, Cardiac; Calcium; Cardiomyopathy, Hypertrophic; Cardiotonic Agents; Connexin 43; Disease Models, Animal; Disease Susceptibility; Electrocardiography; Energy Metabolism; Female; Gap Junctions; Heterocyclic Compounds, 4 or More Rings; Male; Mice; Mice, Inbred Strains; Mice, Transgenic; Myofibrils; Quinolines; Thiadiazines

Sensing of an electric field (EF) by cells-galvanotaxis-is important in wound healing [1], development [2], cell division, nerve growth, and angiogenesis [3]. Different cell types migrate in opposite directions in EFs [4], and the same cell can switch the directionality depending on conditions [5]. A tug-of-war mechanism between multiple signaling pathways [6] can direct Dictyostelium cells to either cathode or anode. Mechanics of motility is simplest in fish keratocytes, so we turned to keratocytes to investigate their migration in EFs. Keratocytes sense electric fields and migrate to the cathode [7, 8]. Keratocyte fragments [9, 10] are the simplest motile units. Cell fragments from leukocytes are able to respond to chemotactic signals [11], but whether cell fragments are galvanotactic was unknown. We found that keratocyte fragments are the smallest motile electric field-sensing unit: they migrate to the anode, in the opposite direction of whole cells. Myosin II was essential for the direction sensing of fragments but not for parental cells, while PI3 kinase was essential for the direction sensing of whole cells but not for fragments. Thus, two signal transduction pathways, one depending on PI3K, another on myosin, compete to orient motile cells in the electric field. Galvanotaxis is not due to EF force and does not depend on cell or fragment size. We propose a "compass" model according to which protrusive and contractile actomyosin networks self-polarize to the front and rear of the motile cell, respectively, and the electric signal orients both networks toward cathode with different strengths.

Topics: Animals; Cell Movement; Chromones; Cichlids; Corneal Keratocytes; Dimethyl Sulfoxide; Electric Stimulation; Electricity; Electromagnetic Fields; Heterocyclic Compounds, 4 or More Rings; Hydrogen-Ion Concentration; Microtubules; Models, Biological; Morpholines; Myosins; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction

Skin wound healing is an intricate process involving various cell types and molecules. In granulation tissue, fibroblasts proliferate and differentiate into myofibroblasts and generate mechanical tension for wound closure and contraction. Actin stress fibers formed in these cells, especially those containing α-smooth muscle actin (α-SMA), are the central machinery for contractile force generation. In the present study, calponin 3 (CNN3), which has a phosphorylation-dependent actin-binding property, was identified in the molecular mechanism underlying stress fiber formation. CNN3 was expressed by fibroblasts/myofibroblasts in the proliferation phase of wound healing, and was associated with α-SMA in stress fibers formed by cultured dermal fibroblasts. CNN3 expression was post-transcriptionally regulated by tension, as demonstrated by disruption of actin filament organization under floating culture or blebbistatin treatment. CNN3 knockdown in primary fibroblasts impaired stress fiber formation, resulting in a phenotype of decreased cellular dynamics such as cell motility and contractile ability. These findings indicate that CNN3 participates in actin stress fiber remodeling, which is required for cell motility and contraction of dermal fibroblasts in the wound healing process.

Topics: Actin Cytoskeleton; Actins; Animals; Calcium-Binding Proteins; Calponins; Cell Movement; Cells, Cultured; Dermis; Fibroblasts; Granulation Tissue; Heterocyclic Compounds, 4 or More Rings; Mice; Microfilament Proteins; Muscle Contraction; Myofibroblasts; RNA Processing, Post-Transcriptional; RNA, Small Interfering; Stress Fibers; Wound Healing

Nonmuscle myosin II (NMII) is thought to be the master integrator of force within epithelial apical junctions, mediating epithelial tissue morphogenesis and tensional homeostasis. Mutations in NMII are associated with a number of diseases due to failures in cell-cell adhesion. However, the organization and the precise mechanism by which NMII generates and responds to tension along the intercellular junctional line are still not known. We discovered that periodic assemblies of bipolar NMII filaments interlace with perijunctional actin and α-actinin to form a continuous belt of muscle-like sarcomeric units (∼400-600 nm) around each epithelial cell. Remarkably, the sarcomeres of adjacent cells are precisely paired across the junctional line, forming an integrated, transcellular contractile network. The contraction/relaxation of paired sarcomeres concomitantly impacts changes in apical cell shape and tissue geometry. We show differential distribution of NMII isoforms across heterotypic junctions and evidence for compensation between isoforms. Our results provide a model for how NMII force generation is effected along the junctional perimeter of each cell and communicated across neighboring cells in the epithelial organization. The sarcomeric network also provides a well-defined target to investigate the multiple roles of NMII in junctional homeostasis as well as in development and disease.

Topics: Actin Cytoskeleton; Actinin; Actins; Animals; Heterocyclic Compounds, 4 or More Rings; Intercellular Junctions; Mice; Microscopy, Fluorescence; Myosin Heavy Chains; Myosin Type II; Organ of Corti; Rats

Myocardial infarction (MI) is a major risk for ventricular arrhythmia. Pause-triggered ventricular arrhythmia can be caused by increased myofilament Ca binding due to sarcomeric mutations or Ca-sensitizing compounds. Myofilament Ca sensitivity is also increased after MI. Here we hypothesize that MI increases risk for pause-triggered ventricular arrhythmias, which can be prevented by myofilament Ca-desensitization and contractile uncoupling. To test this hypothesis, we generated a murine chronic MI model using male B6SJLF1/J mice (n=40) that underwent permanent ligation of the left anterior descending coronary artery. 4 weeks post MI, cardiac structure, function and myofilament Ca sensitivity were evaluated. Pause-dependent arrhythmia susceptibility was quantified in isolated hearts with pacing trains of increasing frequency, followed by a pause and an extra stimulus. Coronary ligation resulted in a mean infarct size of 39.6±5.7% LV and fractional shortening on echocardiography was reduced by 40% compared to non-infarcted controls. Myofilament Ca sensitivity was significantly increased in post MI hearts (pCa50: Control=5.66±0.03; MI=5.84±0.05; P<0.01). Exposure to the Ca desensitizer/contractile uncoupler blebbistatin (BLEB, 3 μM) reduced myofilament Ca sensitivity of MI hearts to that of control hearts and selectively reduced the frequency of post-pause ectopic beats (MI 0.12±0.04 vs MI+BLEB 0.01±0.005 PVC/pause; P=0.02). BLEB also reduced the incidence of ventricular tachycardia in chronic MI hearts from 59% to 10% (P<0.05). We conclude that chronic MI hearts exhibit increased myofilament Ca sensitivity and pause-triggered ventricular arrhythmias, which can be prevented by blebbistatin. Decreasing myofilament Ca sensitivity may be a strategy to reduce arrhythmia burden after MI.

Topics: Animals; Calcium; Chronic Disease; Disease Models, Animal; Heterocyclic Compounds, 4 or More Rings; Male; Mice; Myocardial Contraction; Myocardial Infarction; Myocardium; Myofibrils; Tachycardia, Ventricular

Computational modeling of eukaryotic cells moving on substrates is an extraordinarily complex task: many physical processes, such as actin polymerization, action of motors, formation of adhesive contacts concomitant with both substrate deformation and recruitment of actin etc., as well as regulatory pathways are intertwined. Moreover, highly nontrivial cell responses emerge when the substrate becomes deformable and/or heterogeneous. Here we extended a computational model for motile cell fragments, based on an earlier developed phase field approach, to account for explicit dynamics of adhesion site formation, as well as for substrate compliance via an effective elastic spring. Our model displays steady motion vs. stick-slip transitions with concomitant shape oscillations as a function of the actin protrusion rate, the substrate stiffness, and the rates of adhesion. Implementing a step in the substrate's elastic modulus, as well as periodic patterned surfaces exemplified by alternating stripes of high and low adhesiveness, we were able to reproduce the correct motility modes and shape phenomenology found experimentally. We also predict the following nontrivial behavior: the direction of motion of cells can switch from parallel to perpendicular to the stripes as a function of both the adhesion strength and the width ratio of adhesive to non-adhesive stripes.

Topics: Actins; Cell Adhesion; Cell Movement; Cell Shape; Cells, Cultured; Elastic Modulus; Heterocyclic Compounds, 4 or More Rings; Humans; Keratinocytes; Marine Toxins; Models, Biological; Myosins; Oligopeptides; Oxazoles; Surface Properties

Epithelial cells of developing embryonic organs, such as salivary glands, can display substantial motility during branching morphogenesis. Their dynamic movements and molecules involved in their migration are not fully characterized.. We generated transgenic mice expressing photo-convertible KikGR and tracked the movements of individual cells highlighted by red fluorescence in different regions of developing salivary glands. Motility was highest for outer bud epithelial cells adjacent to the basement membrane, lower in inner bud cells, and lowest in duct cells. The highly motile outer cells contacting the basement membrane were pleomorphic, whereas inner cells were rounded. Peripheral cell motility was disrupted by antibodies inhibiting α6+β1 integrins and the nonmuscle myosin II inhibitor blebbistatin. Inner bud cell migration was unaffected by these inhibitors, but their rate of migration was stimulated by inhibiting E-cadherin.. Cell motility in developing salivary glands was highest in cells in contact with the basement membrane. The basement membrane-associated motility of these outer bud cells depended on integrins and myosin II, but not E-cadherin. In contrast, motility of inner bud cells was restrained by E-cadherin. These findings identify the importance of integrin-dependent basement membrane association for the morphology, tissue organization, and lateral motility of morphogenetic epithelial cells.

Topics: Animals; Antibodies; Cadherins; Cell Movement; Embryo, Mammalian; Epithelial Cells; Heterocyclic Compounds, 4 or More Rings; Integrin alpha6; Integrin beta1; Mice; Mice, Transgenic; Myosin Type II; Organogenesis; Salivary Glands

Astrocytes exhibit a complex, branched morphology, allowing them to functionally interact with numerous blood vessels, neighboring glial processes and neuronal elements, including synapses. They also respond to central nervous system (CNS) injury by a process known as astrogliosis, which involves morphological changes, including cell body hypertrophy and thickening of major processes. Following severe injury, astrocytes exhibit drastically reduced morphological complexity and collectively form a glial scar. The mechanistic details behind these morphological changes are unknown. Here, we investigate the regulation of the actin-nucleating Arp2/3 complex in controlling dynamic changes in astrocyte morphology. In contrast to other cell types, Arp2/3 inhibition drives the rapid expansion of astrocyte cell bodies and major processes. This intervention results in a reduced morphological complexity of astrocytes in both dissociated culture and in brain slices. We show that this expansion requires functional myosin II downstream of ROCK and RhoA. Knockdown of the Arp2/3 subunit Arp3 or the Arp2/3 activator N-WASP by siRNA also results in cell body expansion and reduced morphological complexity, whereas depleting WAVE2 specifically reduces the branching complexity of astrocyte processes. By contrast, knockdown of the Arp2/3 inhibitor PICK1 increases astrocyte branching complexity. Furthermore, astrocyte expansion induced by ischemic conditions is delayed by PICK1 knockdown or N-WASP overexpression. Our findings identify a new morphological outcome for Arp2/3 activation in restricting rather than promoting outwards movement of the plasma membrane in astrocytes. The Arp2/3 regulators PICK1, and N-WASP and WAVE2 function antagonistically to control the complexity of astrocyte branched morphology, and this mechanism underlies the morphological changes seen in astrocytes during their response to pathological insult.

Topics: Actin-Related Protein 2-3 Complex; Amides; Animals; Astrocytes; Carrier Proteins; Cells, Cultured; Central Nervous System; Colforsin; Enzyme Inhibitors; Fibroblasts; HEK293 Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Mice; Myosin Type II; Nuclear Proteins; Pyridines; Rats; rhoA GTP-Binding Protein; RNA Interference; RNA, Small Interfering; Thiazoles; Thiones; Uracil; Vasodilator Agents; Wiskott-Aldrich Syndrome Protein, Neuronal

Using a microchannel assay, we demonstrate that cells adopt distinct signaling strategies to modulate cell migration in different physical microenvironments. We studied α4β1 integrin-mediated signaling, which regulates cell migration pertinent to embryonic development, leukocyte trafficking, and melanoma invasion. We show that α4β1 integrin promotes cell migration through both unconfined and confined spaces. However, unlike unconfined (2D) migration, which depends on enhanced Rac1 activity achieved by preventing α4/paxillin binding, confined migration requires myosin II-driven contractility, which is increased when Rac1 is inhibited by α4/paxillin binding. This Rac1-myosin II cross talk mechanism also controls migration of fibroblast-like cells lacking α4β1 integrin, in which Rac1 and myosin II modulate unconfined and confined migration, respectively. We further demonstrate the distinct roles of myosin II isoforms, MIIA and MIIB, which are primarily required for confined and unconfined migration, respectively. This work provides a paradigm for the plasticity of cells migrating through different physical microenvironments.

Topics: 3T3 Cells; Animals; Cell Line, Tumor; Cell Movement; CHO Cells; Cricetinae; Cricetulus; Focal Adhesions; Heterocyclic Compounds, 4 or More Rings; Humans; Integrin alpha4; Integrin alpha4beta1; Jurkat Cells; Melanoma; Mice; Mutant Proteins; Mutation; Myosin Type II; Neoplasm Invasiveness; Paxillin; Phenotype; Protein Binding; rac1 GTP-Binding Protein; rhoA GTP-Binding Protein; Signal Transduction; Stress Fibers

As cardiomyocytes mature, their sarcomeres and Z-band widths increase in length in order for their myofibrils to produce stronger twitch forces during a contraction. In this study, we tested the hypothesis that tensional homeostasis is affected by altering myofibril forces. To assess this hypothesis, neonatal rat cardiomyocytes were cultured on arrays of microposts to measure cellular contractility. An optical line scanning technique was used to measure the deflections in the microposts with high temporal resolution, enabling the analysis of twitch force, twitch velocity, and twitch power. Myofibril force production was elevated by vector-mediated overexpression of ribonucleotide reductase (RR) to increase cellular dATP content or by adding the inotropic agent EMD 57033 (EMD). We found that RR and EMD treatment did not affect cardiomyocyte twitch force, but it did lead to reduced twitch velocity and twitch power. Immunofluorescent analysis of α-actinin revealed that RR-over-expressing cardiomyocytes and EMD-treated cardiomyocytes had lower spread area, sarcomere length, and Z-band width as compared to control cells. These results indicate a correlation between myofibril structure and cardiac power. This correlation was confirmed by exposing the cells to the myosin II inhibitor blebbistatin, and then subsequently washing it out. After wash-out, cardiomyocytes exhibited a reduction in twitch force, velocity, and power due to shorter sarcomere length and Z-band widths. Our results suggest that cardiac myofibril structure is regulated by tensional homeostasis. If myofibril-generated forces in cardiomyocytes are elevated, a state of tensional homeostasis is maintained by producing sufficient twitch forces with a lower degree myofibril structure.

Topics: Actinin; Animals; Animals, Newborn; Cardiotonic Agents; Cells, Cultured; Deoxyadenine Nucleotides; Green Fluorescent Proteins; Heterocyclic Compounds, 4 or More Rings; Homeostasis; Microscopy, Fluorescence; Muscle Contraction; Myocytes, Cardiac; Myofibrils; Optics and Photonics; Quinolines; Rats; Rats, Inbred F344; Sarcomeres; Signal Transduction; Stress, Mechanical; Thiadiazines

Cells migrate in vivo within three-dimensional (3D) extracellular matrices. Cells also migrate through 3D longitudinal channels formed between the connective tissue and the basement membrane of muscle, nerve, and epithelium. Although traction forces have been measured during 2D cell migration, no assay has been developed to probe forces during migration through confined microenvironments. We thus fabricated a novel microfluidic device consisting of deflectable PDMS microposts incorporated within microchannels of varying cross-sectional areas. Using NIH-3T3 fibroblasts and human osteosarcoma (HOS) cells as models, we found that the average traction forces per post decreased upon increasing confinement. Inhibition of myosin-II function by blebbistatin in HOS cells decreased traction forces in unconfined (wide) channels but failed to alter them in confined spaces. Myosin activation by calyculin A also failed to affect traction forces in confining channels but increased them in wide channels. These observations underlie the importance of the physical microenvironment in the regulation of cell migration and cellular traction forces.

Topics: Animals; Cell Culture Techniques; Cell Line, Tumor; Cell Movement; Dimethylpolysiloxanes; Heterocyclic Compounds, 4 or More Rings; Humans; Marine Toxins; Mice; Microfluidic Analytical Techniques; Myosins; NIH 3T3 Cells; Oxazoles

Mutations in the MYH9 gene cause autosomal dominant MYH9-related diseases (MYH9-RD) that associate macrothrombocytopenia with various other clinical conditions. The mechanisms giving rise to giant platelets remain poorly understood.. To study the proplatelet formation (PPF) derived from megakaryocytes (MKs) generated in vitro from 11 patients with MYH9-RD with different mutations, compared with controls.. Proplatelet formation from cultured patients' MKs was evaluated with or without blebbistatin or the ROCK inhibitor Y27632. Myosin IIA and actin distribution were studied in spreading MKs on different surfaces by immunoconfocal analysis. Kinetic studies of contractility were performed on spreading MKs and the impact of blebbistatin on the maturation of the patients' MKs was evaluated by electron microscopy.. We show that in vitro MKs of 11 patients formed significantly fewer proplatelets than controls. MKs from MYH9-RD displayed an abnormal spreading on polylysine, fibronectin and collagen, with a disorganized actin network and a marked increase in stress fiber formation. Traction force microscopy studies demonstrated an elevated level of contractile forces in adherent mutated MKs. The myosin II inhibitor blebbistatin and the ROCK inhibitor Y27632 both rescued the proplatelet formation defect and normalized the ultrastructural characteristics of MYH9-RD MKs. Altogether, our results show that in MYH9-RD, mutations modify the overall MYH9 function and provoke a proplatelet defect through an excess of actomyosin contractility in spreading MKs. These results may promote new therapeutic strategies aimed at reducing this actomyosin contractility.

Topics: Actomyosin; Blood Platelets; Cells, Cultured; Heterocyclic Compounds, 4 or More Rings; Humans; Molecular Motor Proteins; Mutation; Myosin Heavy Chains; Nonmuscle Myosin Type IIA; Thrombocytopenia

Human placental stem villi (PSV) present contractile properties. We studied the role of actin-myosin cross bridges (CBs) and the effects of NO-cGMP pathway modulators in the PSV contraction and relaxation.. In vitro contractile properties were investigated in 71 PSV from term human placentas studied according to their long axis. Contraction was induced by both KCl and electrical tetanic stimulation. Relaxation was induced by inhibiting the CB cycle with either 2,3-butanedione monoxime (BDM) or blebbistatin (BLE) and by activating the NO-cGMP pathway with isosorbide dinitrate (ISDN), sildenafil (SIL) or ISDN + SIL.. PSV tension slowly increased by 140% of the basal tone after KCl exposure and by 85% after tetanus. The addition of BDM, BLE, ISDN, SIL and ISDN + SIL induced a relaxation of PSV, the overall time course of relaxation (in s) was respectively (means ± SD) 3412 ± 1904, 14,250 ± 3095*, 3813 ± 1383, 2883 ± 1188 and 2440 ± 477; significantly longer in BLE compared with BDM, ISDN, SIL and ISDN + SIL:*p < 0.001). the overall time course of relaxation (in s) was respectively (means ± SD) 3412 ± 1904, 14,250 ± 3095*, 3813 ± 1383, 2883 ± 1188 and 2440 ± 477; significantly longer in BLE compared with BDM, ISDN, SIL and ISDN + SIL:*p < 0.001). These relaxation kinetics were particularly slow. Other relaxation parametres, i.e., maximum lengthening, -peak dT/dt, and resting tension, did not differ between these 5 subgroups.. Isolated human PSV were able to contract after both KCl exposure and tetanus. This increase in contractility was reversed by inhibiting the CB cycle with BDM or BLE and by stimulating the NO-cGMP pathway with ISDN or SIL. The association ISDN + SIL did not potentiate the relaxing processes.

Topics: Actins; Chorionic Villi; Cyclic GMP; Diacetyl; Electric Stimulation; Enzyme Inhibitors; Female; Heterocyclic Compounds, 4 or More Rings; Humans; In Vitro Techniques; Isosorbide Dinitrate; Kinetics; Myosins; Nitric Oxide; Nitric Oxide Donors; Piperazines; Pliability; Potassium Chloride; Pregnancy; Protein Structure, Quaternary; Purines; Second Messenger Systems; Sildenafil Citrate; Sulfones; Term Birth

In development and differentiation, morphological changes often accompany mechanical changes [1], but it is unclear whether or when cells in embryos sense tissue elasticity. The earliest embryo is uniformly pliable, while adult tissues vary widely in mechanics from soft brain and stiff heart to rigid bone [2]. However, cell sensitivity to microenvironment elasticity is debated based in part on results from complex three-dimensional culture models [3]. Regenerative cardiology provides strong motivation to clarify any cell-level sensitivities to tissue elasticity because rigid postinfarct regions limit pumping by the adult heart [4]. Here, we focus on the spontaneously beating embryonic heart and sparsely cultured cardiomyocytes, including cells derived from pluripotent stem cells. Tissue elasticity, Et, increases daily for heart to 1-2 kPa by embryonic day 4 (E4), and although this is ~10-fold softer than adult heart, the beating contractions of E4 cardiomyocytes prove optimal at ~Et,E4 both in vivo and in vitro. Proteomics reveals daily increases in a small subset of proteins, namely collagen plus cardiac-specific excitation-contraction proteins. Rapid softening of the heart's matrix with collagenase or stiffening it with enzymatic crosslinking suppresses beating. Sparsely cultured E4 cardiomyocytes on collagen-coated gels likewise show maximal contraction on matrices with native E4 stiffness, highlighting cell-intrinsic mechanosensitivity. While an optimal elasticity for striation proves consistent with the mathematics of force-driven sarcomere registration, contraction wave speed is linear in Et as theorized for excitation-contraction coupled to matrix elasticity. Pluripotent stem cell-derived cardiomyocytes also prove to be mechanosensitive to matrix and thus generalize the main observation that myosin II organization and contractile function are optimally matched to the load contributed by matrix elasticity.

Topics: Cardiac Myosins; Cell Differentiation; Cells, Cultured; Collagen; Collagenases; Elasticity; Embryonic Stem Cells; Extracellular Matrix Proteins; Heart; Heart Rate; Heterocyclic Compounds, 4 or More Rings; Humans; Induced Pluripotent Stem Cells; Myocardial Contraction; Myocytes, Cardiac; Myofibrils; Myosins; Sarcomeres

Blebbistatin, a potent inhibitor of myosin II, has inhibiting effects on Ca(2+)-induced contraction and contractile filament organization without affecting the Ca(2+)-sensitivity to the force and phosphorylation level of myosin regulatory light chain (MLC20) in skinned (cell membrane permeabilized) taenia cecum from the guinea pig (Watanabe et al., Am J Physiol Cell Physiol. 2010; 298: C1118-26). In the present study, we investigated blebbistatin effects on the contractile force of skinned tracheal muscle, in which myosin filaments organization is more labile than that in the taenia cecum. Blebbistatin at 10 μM or higher suppressed Ca(2+)-induced tension development at any given Ca(2+) concentration, but had little effects on the Ca(2+)- induced myosin light chain phosphorylation. Also blebbistatin at 10 μM and higher significantly suppressed GTP-γS-induced "sensitized" force development. Since the force inhibiting effects of blebbistatin on the skinned trachea were much stronger than those in skinned taenia cecum, blebbistatin might directly affect myosin filaments organization.

Topics: Animals; Calcium; Calcium Channel Blockers; Dose-Response Relationship, Drug; Guanosine 5'-O-(3-Thiotriphosphate); Guinea Pigs; Heterocyclic Compounds, 4 or More Rings; In Vitro Techniques; Muscle Contraction; Muscle, Smooth; Myosin Type II; Trachea

In response to a wound, astrocytes in culture extend microtubule-rich processes and polarize, orienting their centrosomes and Golgi apparatus woundside. β1 Integrin null astrocytes fail to extend processes toward the wound, and are disoriented, and often migrate away orthogonal, to the wound. The centrosome is unusually fragmented in β1 integrin null astrocytes. Expression of a β1 integrin cDNA in the null background yields cells with intact centrosomes that polarize and extend processes normally. Fragmented centrosomes rapidly assemble following integrin ligation and cell attachment. However, several experiments indicated that cell adhesion is not necessary. For example, astrocytes in suspension expressing a chimeric β1 subunit that can be activated by an antibody assemble centrosomes suggesting that β1 activation is sufficient to cause centrosome assembly in the absence of cell adhesion. siRNA knockdown of PCM1, a major centrosomal protein, inhibits cell polarization, consistent with the notion that centrosomes are necessary for polarity and that integrins regulate polarity via centrosome integrity. Screening inhibitors of molecules downstream of integrins indicate that neither FAK nor ILK is involved in regulation of centrosome integrity. In contrast, blebbistatin, a specific inhibitor of non-muscle myosin II (NMII), mimics the response of β1 integrin null astrocytes by disrupting centrosome integrity and cell polarization. Blebbistatin also inhibits integrin-mediated centrosome assembly in astrocytes attaching to fibronectin, consistent with the hypothesis that NMII functions downstream of integrins in regulating centrosome integrity.

Topics: Animals; Astrocytes; Cell Adhesion; Cell Cycle Proteins; Cell Polarity; Cells, Cultured; Centrosome; Chick Embryo; DNA, Complementary; Extracellular Matrix; Heterocyclic Compounds, 4 or More Rings; Humans; Integrin beta1; Mice; Nonmuscle Myosin Type IIB; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; Retina; RNA, Small Interfering; Suspensions; Wound Healing

Neutrophils are the primary effector cells in the pathogenesis of transfusion-related acute lung injury or multiple organ failure after blood transfusion.. We aimed to investigate the effect of fresh (1 day preparation) and aged (42 day preparation) PRBC-derived plasma on neutrophil morphology, migration and phagocytosis.. We evaluated the production of reactive oxygen species (ROS) and the expression of non-muscle myosin heavy chain IIA (MYH9) in neutrophils treated with PRBC-derived plasma. We used western blots and antibody arrays to evaluate changes in signal transduction pathways in plasma-treated neutrophils.. Aged PRBC-derived plasma elicited a stronger oxidative burst in neutrophils when compared with fresh PRBC-derived plasma (p < 0.05). Antibody arrays showed increased phosphorylation of NF-ĸB proteins (p105, p50 and Ikk) in aged PRBC-derived plasma-treated neutrophils. The expression of non-muscle myosin IIA (MYH9), a cytoskeleton protein involved in immune cell migration and morphological change, was also significantly upregulated in neutrophils treated with aged PRBC-derived plasma compared to fresh plasma (p < 0.05). Pretreatment of neutrophils with blebbistatin (a specific type II myosin inhibitor), ascorbic acid (an antioxidant), or staurosporine (a protein tyrosine kinase inhibitor), effectively abrogated the morphological changes, neutrophil migration, and phagocytosis induced by aged PRBC-derived plasma.. Upregulation of MYH9 in neutrophils treated with aged PRBC-derived plasma and abrogation of neutrophil migration in blebbistatin-treated neutrophils suggested a functional role of MYH9 in the directional migration of immune cells. Our data help elucidate the cellular and molecular mechanisms of transfusion-related injury.

Topics: Antioxidants; Ascorbic Acid; Cell Movement; Cytoskeletal Proteins; Erythrocytes; Heterocyclic Compounds, 4 or More Rings; Humans; Molecular Motor Proteins; Myosin Heavy Chains; Neutrophils; NF-kappa B; Nonmuscle Myosin Type IIA; Phagocytosis; Phosphorylation; Protein Kinase Inhibitors; Reactive Oxygen Species; Respiratory Burst; Signal Transduction; Staurosporine

Stem cells reside in a multi-factorial environment containing biochemical and mechanical signals. Changing biochemical signals in most scaffolds often leads to simultaneous changes in mechanical properties, which makes it difficult to elucidate the complex interplay between niche cues. Combinatorial studies on cell-material interactions have emerged as a tool to facilitate analyses of stem cell responses to various niche cues, but most studies to date have been performed on two-dimensional environments. Here we developed three-dimensional combinatorial hydrogels with independent control of biochemical and mechanical properties to facilitate analysis of interactive biochemical and mechanical signaling on adipose-derived stem cell osteogenesis in three dimensions. Our results suggest that scaffold biochemical and mechanical signals synergize only at specific combinations to promote bone differentiation. Leading compositions were identified to have intermediate stiffness (∼55kPa) and low concentration of fibronectin (10μg ml(-1)), which led to an increase in osteocalcin gene expression of over 130-fold. Our results suggest that scaffolds with independently tunable niche cues could provide a powerful tool for conducting mechanistic studies to decipher how complex niche cues regulate stem cell fate in three dimensions, and facilitate rapid identification of optimal niche cues that promote desirable cellular processes or tissue regeneration.

Topics: Adipose Tissue; Anthraquinones; Biomarkers; Calcium; Cell Differentiation; Cell Survival; Collagen Type I; Collagen Type II; Compressive Strength; Extracellular Matrix; Gene Expression Regulation; Heterocyclic Compounds, 4 or More Rings; Humans; Hydrogels; Mechanical Phenomena; Nonmuscle Myosin Type IIB; Osteocalcin; Osteogenesis; Signal Transduction; Staining and Labeling; Stem Cells

Left ventricular (LV) hypertrophy is often present in patients with diastolic heart failure. However, stiffness of hypertrophied cardiomyocytes in the transverse direction has not been fully elucidated. The aim of this study was to assess passive cardiomyocyte stiffness of hypertrophied hearts in the transverse direction and the influence of actin-myosin cross-bridge formation on the stiffness.. Wistar rats received a vehicle (control) or isoproterenol (ISO) subcutaneously. After 7 days, compared with the controls, ISO administration had significantly increased heart weight and LV wall thickness and had decreased peak early annular relaxation velocity (e') assessed by echocardiography. Elastic modulus of living cardiomyocytes in the transverse direction assessed by an atomic force microscope was significantly higher in the ISO group than in controls. We added butanedione monoxime (BDM), an inhibitor of actin-myosin interaction, and blebbistatin, a specific myosin II inhibitor, to the medium. BDM and blebbistatin significantly reduced the elastic modulus of cardiomyocytes in the ISO group. X-ray diffraction analysis showed that the reflection intensity ratio (I((1,0))/I((1,1))) at diastole was not different before and after treatment with BDM, which induces complete relaxation, in control hearts, but that I((1,0))/I((1,1)) was significantly increased after BDM treatment in the ISO group, indicating residual cross-bridge formation in hypertrophied hearts.. Passive cardiomyocyte stiffness in the transverse direction is increased in hearts with ISO-induced hypertrophy and this is caused by residual actin-myosin cross-bridge formation. 

Topics: Actins; Adrenergic beta-Agonists; Animals; Cardiomegaly; Cells, Cultured; Diacetyl; Disease Models, Animal; Elasticity; Enzyme Inhibitors; Heterocyclic Compounds, 4 or More Rings; Hypertrophy, Left Ventricular; Isoproterenol; Male; Microscopy, Atomic Force; Myocytes, Cardiac; Myosins; Organ Size; Papillary Muscles; Radiography; Rats; Rats, Wistar; Ultrasonography

Ca(2+) waves are initiated not only by Ca(2+) leak from the sarcoplasmic reticulum (SR), but also by Ca(2+) dissociation from the myofilaments in the myocardium with nonuniform contraction. We investigated whether contractile properties and the production of reactive oxygen species (ROS) affect Ca(2+) wave propagation. Trabeculae were obtained from 76 rat hearts. Force was measured with a strain gauge, sarcomere length with a laser diffraction technique, and [Ca(2+)](i) with fura-2 and a CCD camera (24°C, 2.0mmol/L [Ca(2+)](o)). ROS production was estimated from 2',7'-dichlorofluorescein (DCF) fluorescence. Trabeculae were regionally exposed to a jet of solution containing 1) 10mmol/L Ca(2+) to initiate Ca(2+) waves by SR Ca(2+) leak due to Ca(2+) overload within the jet-exposed region, and 2) 0.2mmol/L Ca(2+) or 5mmol/L caffeine to initiate such waves by Ca(2+) dissociation from the myofilaments due to nonuniform contraction. Ca(2+) waves were induced by stimulus trains for 7.5s. Ten-percent muscle stretch increased DCF fluorescence and accelerated Ca(2+) waves initiated due to both Ca(2+) overload and nonuniform contraction. Preincubation with 3μmol/L diphenyleneiodonium or 10μmol/L colchicine suppressed the increase in DCF fluorescence but suppressed acceleration of Ca(2+) waves initiated only due to Ca(2+) overload. Irrespective of preincubation with colchicine, reduction of force after the addition of 10μmol/L blebbistatin did not decelerate Ca(2+) waves initiated due to Ca(2+) overload, while it did decelerate waves initiated due to nonuniform contraction. These results suggest that Ca(2+) wave propagation is modulated by ROS production through an intact microtubule network only during stretch and may be additionally modulated by Ca(2+) dissociated from the myofilaments in the case of nonuniform contraction.

Topics: Animals; Caffeine; Calcium Signaling; Cells, Cultured; Colchicine; Heterocyclic Compounds, 4 or More Rings; Myocardial Contraction; Myocardium; Myofibrils; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Tubulin Modulators

Blebbistatin (BS) is a recently discovered inhibitor of the myosin II isoform and has been adopted as the mechanical uncoupler of choice for optical mapping, because previous studies suggest that BS has no significant cardiac electrophysiological effects in a number of species. The aim of this study was to determine whether BS affects cardiac electrophysiology in isolated New Zealand White rabbit hearts. Langendorff-perfused hearts (n=39) in constant-flow mode had left ventricular monophasic action potential duration (MAPD) measured at apical and basal regions during constant pacing (300 ms cycle length). Standard action potential duration restitution was obtained using the single extrastimulus method with measurement of the maximal restitution slope. Ventricular fibrillation threshold was measured as the minimal current inducing sustained ventricular fibrillation with burst pacing (30 stimuli, at 30 ms intervals). Optical action potentials were recorded using the voltage-sensitive dye di-4-ANEPPS. Measurements were taken at baseline and after 60 min perfusion with BS (5 μm). Blebbistatin significantly prolonged left ventricular apical (mean±SEM; from 129.9±2.9 to 170.7±4.1 ms, P<0.001, n=8) and basal MAPD (from 135.0±2.3 to 163.3±5.6 ms, P<0.001) and effective refractory period (from 141.3±4.8 to 175.6±3.7 ms, P<0.001) whilst increasing the maximal slope of restitution (apex, from 0.79±0.09 to 1.57±0.16, P<0.001; and base, from 0.71±0.06 to 1.44±0.24, P<0.001) and ventricular fibrillation threshold (from 5.3±1.1 to 17.0±2.9 mA, P<0.001). In other hearts, blebbistatin significantly prolonged optically recorded action potentials (from 136.5±6.3 to 173.0±7.9 ms, P<0.05, n=4). In control experiments, the increase of MAPD with blebbistatin was present whether the hearts were perfused in constant-pressure mode (n=5) or in unloaded conditions (n=5). These data show that blebbistatin significantly affects cardiac electrophysiology. Its use in optical mapping studies should be treated with caution.

Topics: Action Potentials; Animals; Electrophysiological Phenomena; Heart; Heart Ventricles; Heterocyclic Compounds, 4 or More Rings; In Vitro Techniques; Male; Myocardial Contraction; Perfusion; Rabbits; Ventricular Fibrillation

Nonmuscle myosin heavy chain IIA (NMMHC IIA, gene code: MYH9) plays a critical role in physiological and pathological functions. A homology model of NMMHC IIA was constructed based on the crystal structure of smooth muscle myosin II. Blebbistatin, a myosin II ATPase inhibitor, had been found to bind to NMMHC IIA with Leu228 as the important amino acid residue and van der Waals contacts as the main force of the interaction. The final complex demonstrated that the destruction of the salt bridge occurred between the Arg204 and Glu427 residues when blebbistatin was present. Molecular dynamic simulation of the complex showed that the binding affinity of blebbistatin to NMMHC IIA was strongly sensitive to the nucleotide binding region and actin binding region. The disturbance of the two regions increased the enhancement of the binding cavity with blebbistatin and resulted in a slightly more expanded conformation in the nucleotide binding region and actin binding region. A combined pharmacophore- and docking-based virtual screening was performed to identify several saponins as potential inhibitors for NMMHC IIA. These findings introduce new insights on the binding mode of blebbistatin and NMMHC IIA and novel leading compounds from natural products for NMMHC IIA-related diseases.

Topics: Amino Acid Sequence; Binding Sites; Crystallography, X-Ray; Heterocyclic Compounds, 4 or More Rings; Humans; Ligands; Molecular Docking Simulation; Molecular Dynamics Simulation; Molecular Sequence Data; Nonmuscle Myosin Type IIA; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Sequence Alignment; Smooth Muscle Myosins; Static Electricity; Structural Homology, Protein

What governs tissue organization and movement? If molecular and genetic approaches are able to give some answers on these issues, more and more works are now giving a real importance to mechanics as a key component eventually triggering further signaling events. We chose embryonic cell aggregates as model systems for tissue organization and movement in order to investigate the origin of some mechanical constraints arising from cells organization. Steinberg et al. proposed a long time ago an analogy between liquids and tissues and showed that indeed tissues possess a measurable tissue surface tension and viscosity. We question here the molecular origin of these parameters and give a quantitative measurement of adhesion versus contractility in the framework of the differential interfacial tension hypothesis. Accompanying surface tension measurements by angle measurements (at vertexes of cell-cell contacts) at the cell/medium interface, we are able to extract the full parameters of this model: cortical tensions and adhesion energy. We show that a tunable surface tension and viscosity can be achieved easily through the control of cell-cell contractility compared to cell-medium one. Moreover we show that α-catenin is crucial for this regulation to occur: these molecules appear as a catalyser for the remodeling of the actin cytoskeleton underneath cell-cell contact, enabling a differential contractility between the cell-medium and cell-cell interface to take place.

Topics: Actin Cytoskeleton; alpha Catenin; Amides; Animals; Biomechanical Phenomena; Cell Adhesion; Cell Communication; Cell Line, Tumor; Cell Movement; Computer Simulation; Embryo, Mammalian; Gene Knockout Techniques; Heterocyclic Compounds, 4 or More Rings; Mechanotransduction, Cellular; Mice; Nocodazole; Pyridines; Surface Tension; Viscosity

Diabetes mellitus (DM) is a quite common chronic disease, and the prevalence of erectile dysfunction (ED) is three times higher in this large population. Although diabetes-related ED has been studied extensively, the actin-myosin contractile apparatus was not examined. The mRNAs encoding smooth muscle myosin (SMM) heavy chains (MHC) and essential light chains (LC(17)) exist as several different alternatively spliced isoforms with distinct contractile properties. Recently, we provided novel data that blebbistatin (BLEB), a specific myosin II inhibitor, potently relaxed corpus cavernosum smooth muscle (CCSM). In this study, we examine whether diabetes alters SMM expression, alternative splicing, and/or functional activities, including sensitivity to BLEB. By using streptozotocin (STZ)-induced 2-mo diabetic rats, functional activities were tested in vivo by intracavernous pressure (ICP) recording during cavernous nerve stimulation and in vitro via organ bath contractility studies. SMM isoform composition was analyzed by competitive RT-PCR and total SMM, myocardin, and embryonic SMM (SMemb) expression by real-time RT-PCR. Results revealed that the blood glucose level of STZ rats was 407.0 vs. 129.5 mg/dl (control). STZ rats exhibited ED confirmed by significantly increased CCSM contractile response to phenylephrine and decreased ICP response. For STZ rats, SM-B, LC(17a) and SM2 isoforms, total SMM, and myocardin expression increased, whereas SM-A, LC(17b), and SM1 isoforms were decreased, with SMemb unchanged. BLEB was significantly more effective in relaxing STZ CCSM both in vitro and in vivo. Thus we demonstrated a novel diabetes-specific effect on alternative splicing of the SMM heavy chain and essential light chain genes to a SMM isoform composition favoring a heightened contractility and ED. A switch to a more contractile phenotype was supported further by total SMM expression increase. Moreover, the change in CCSM phenotype was associated with an increased sensitivity to BLEB, which may serve as a novel pharmacotherapy for ED.

Topics: Alternative Splicing; Animals; Diabetes Mellitus, Type 1; Drug Resistance; Enzyme Inhibitors; Erectile Dysfunction; Gene Expression Regulation; Heterocyclic Compounds, 4 or More Rings; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; Myosin Heavy Chains; Myosin Light Chains; Myosin Type II; Nuclear Proteins; Penis; Protein Isoforms; Rats; Rats, Inbred F344; RNA, Messenger; Smooth Muscle Myosins; Streptozocin; Trans-Activators

Unlike other excitation-contraction uncouplers, blebbistatin has few electrophysiological side effects and has gained increasing acceptance as an excitation-contraction uncoupler in optical mapping experiments. However, the possible role of blebbistatin in ventricular arrhythmia has hitherto been unknown. Furthermore, experiments with blebbistatin and 2,3-butanedione monoxime (BDM) offer an opportunity to assess the contribution of dynamic instability and wavelength of impulse propagation to the induction and maintenance of ventricular arrhythmia. Recordings of monophasic action potentials were used to assess effects of blebbistatin in Langendorff-perfused rabbit hearts (n = 5). Additionally, panoramic optical mapping experiments were conducted in rabbit hearts (n = 7) that were sequentially perfused with BDM, then washed out, and subsequently perfused with blebbistatin. The susceptibility to arrhythmia was investigated using a shock-on-T protocol. We found that 1) application of blebbistatin did not change action potential duration (APD) restitution; 2) in contrast to blebbistatin, BDM flattened APD restitution curve and reduced the wavelength; and 3) incidence of sustained arrhythmia was much lower under blebbistatin than under BDM (2/123 vs. 23/99). While arrhythmias under BDM were able to stabilize, the arrhythmias under blebbistatin were unstable and terminated spontaneously. In conclusion, the lower susceptibility to arrhythmia under blebbistatin than under BDM indicates that blebbistatin has less effects on arrhythmia dynamics. A steep restitution slope under blebbistatin is associated with higher dynamic instability, manifested by the higher incidence of not only wave breaks but also wave extinctions. This relatively high dynamic instability leads to the self-termination of arrhythmia because of the sufficiently long wavelength under blebbistatin.

Topics: Action Potentials; Animals; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Cardiac Pacing, Artificial; Diacetyl; Disease Models, Animal; Excitation Contraction Coupling; Heart Conduction System; Heterocyclic Compounds, 4 or More Rings; Perfusion; Rabbits; Time Factors; Voltage-Sensitive Dye Imaging

Mammalian erythroblasts undergo enucleation, a process thought to be similar to cytokinesis. Although an assemblage of actin, non-muscle myosin II, and several other proteins is crucial for proper cytokinesis, the role of non-muscle myosin II in enucleation remains unclear. In this study, we investigated the effect of various cell-division inhibitors on cytokinesis and enucleation. For this purpose, we used human colony-forming unit-erythroid (CFU-E) and mature erythroblasts generated from purified CD34(+) cells as target cells for cytokinesis and enucleation assay, respectively. Here we show that the inhibition of myosin by blebbistatin, an inhibitor of non-muscle myosin II ATPase, blocks both cell division and enucleation, which suggests that non-muscle myosin II plays an essential role not only in cytokinesis but also in enucleation. When the function of non-muscle myosin heavy chain (NMHC) IIA or IIB was inhibited by an exogenous expression of myosin rod fragment, myosin IIA or IIB, each rod fragment blocked the proliferation of CFU-E but only the rod fragment for IIB inhibited the enucleation of mature erythroblasts. These data indicate that NMHC IIB among the isoforms is involved in the enucleation of human erythroblasts.

Topics: Amides; Aminoquinolines; Cells, Cultured; Cytokinesis; Enzyme Inhibitors; Erythroblasts; Erythroid Precursor Cells; Erythropoiesis; Green Fluorescent Proteins; Heterocyclic Compounds, 4 or More Rings; Humans; Microfilament Proteins; Myosins; Nonmuscle Myosin Type IIA; Nonmuscle Myosin Type IIB; Peptide Fragments; Protein Isoforms; Pyridines; Pyrimidines; rac1 GTP-Binding Protein; Recombinant Fusion Proteins; rho-Associated Kinases

When activated muscle fibers are stretched, there is a long-lasting increase in the force. This phenomenon, referred to as "residual force enhancement," has characteristics similar to those of the "static tension," a long-lasting increase in force observed when muscles are stretched in the presence of Ca(2+) but in the absence of myosin-actin interaction. Independent studies have suggested that these two phenomena have a common mechanism and are caused either by 1) a Ca(2+)-induced stiffening of titin or by 2) promoting titin binding to actin. In this study, we performed two sets of experiments in which activated fibers (pCa(2+) 4.5) treated with the myosin inhibitor blebbistatin were stretched from 2.7 to 2.8 μm at a speed of 40 L(o)/s, first, after partial extraction of TnC, which inhibits myosin-actin interactions, or, second, after treatment with gelsolin, which leads to the depletion of thin (actin) filaments. We observed that the static tension, directly related with the residual force enhancement, was not changed after treatments that inhibit myosin-actin interactions or that deplete fibers from troponin C and actin filaments. The results suggest that the residual force enhancement is caused by a stiffening of titin upon muscle activation but not with titin binding to actin. This finding indicates the existence of a Ca(2+)-regulated, titin-based stiffness in skeletal muscles.

Topics: Actins; Animals; Calcium; Cells, Cultured; Connectin; Gelsolin; Heterocyclic Compounds, 4 or More Rings; In Vitro Techniques; Muscle Contraction; Muscle Fibers, Skeletal; Muscle Proteins; Myosins; Protein Kinases; Rabbits; Sarcomeres; Stress, Physiological; Troponin C

Learning induces dynamic changes to the actin cytoskeleton that are required to support memory formation. However, the molecular mechanisms that mediate filamentous actin (F-actin) dynamics during learning and memory are poorly understood. Myosin II motors are highly expressed in actin-rich growth structures including dendritic spines, and we have recently shown that these molecular machines mobilize F-actin in response to synaptic stimulation and learning in the hippocampus. In this study, we report that Myosin II motors in the rat lateral amygdala (LA) are essential for fear memory formation. Pretraining infusions of the Myosin II inhibitor, blebbistatin (blebb), disrupted long term memory, while short term memory was unaffected. Interestingly, both post-training and pretesting infusions had no effect on memory formation, indicating that Myosin II motors operate during or shortly after learning to promote memory consolidation. These data support the idea that Myosin II motor-force generation is a general mechanism that supports memory consolidation in the mammalian CNS.

Topics: Amygdala; Animals; Association Learning; Conditioning, Classical; Dendritic Spines; Fear; Heterocyclic Compounds, 4 or More Rings; Hippocampus; Male; Memory; Nonmuscle Myosin Type IIB; Rats; Rats, Sprague-Dawley; Synapses

Chondroitin sulfate proteoglycans (CSPGs) are major components of the extracellular matrix in the CNS that inhibit axonal regeneration after CNS injury. Signaling pathways in neurons triggered by CSPGs are still largely unknown. In this study, using well-characterized in vitro assays for neurite outgrowth and neurite guidance, we demonstrate a major role for myosin II in the response of neurons to CSPGs. We found that the phosphorylation of myosin II regulatory light chains is increased by CSPGs. Specific inhibition of myosin II activity with blebbistatin allows growing neurites to cross onto CSPG-rich areas and increases the length of neurites of neurons growing on CSPGs. Using specific gene knockdown, we demonstrate selective roles for myosin IIA and IIB in these processes. Time lapse microscopy and immunocytochemistry demonstrated that CSPGs also inhibit cell adhesion and cell spreading. Inhibition of myosin II selectively accelerated neurite initiation without altering cell adhesion and spreading on CSPGs.

Topics: Actins; Analysis of Variance; Animals; Animals, Newborn; Cell Adhesion; Cell Movement; Cells, Cultured; Cerebellum; Chondroitin Sulfate Proteoglycans; Gene Expression Regulation; Heterocyclic Compounds, 4 or More Rings; Lysine; Mice; Mice, Inbred C57BL; Myosin Light Chains; Myosin Type II; Neurites; Neurons; Phosphorylation; RNA, Messenger; RNA, Small Interfering; Transfection

Actin retrograde flow and actomyosin II contraction have both been implicated in the inward movement of T cell receptor (TCR) microclusters and immunological synapse formation, but no study has integrated and quantified their relative contributions. Using Jurkat T cells expressing fluorescent myosin IIA heavy chain and F-tractin-a novel reporter for F-actin-we now provide direct evidence that the distal supramolecular activation cluster (dSMAC) and peripheral supramolecular activation cluster (pSMAC) correspond to lamellipodial (LP) and lamellar (LM) actin networks, respectively, as hypothesized previously. Our images reveal concentric and contracting actomyosin II arcs/rings at the LM/pSMAC. Moreover, the speeds of centripetally moving TCR microclusters correspond very closely to the rates of actin retrograde flow in the LP/dSMAC and actomyosin II arc contraction in the LM/pSMAC. Using cytochalasin D and jasplakinolide to selectively inhibit actin retrograde flow in the LP/dSMAC and blebbistatin to selectively inhibit actomyosin II arc contraction in the LM/pSMAC, we demonstrate that both forces are required for centripetal TCR microcluster transport. Finally, we show that leukocyte function-associated antigen 1 clusters accumulate over time at the inner aspect of the LM/pSMAC and that this accumulation depends on actomyosin II contraction. Thus actin retrograde flow and actomyosin II arc contraction coordinately drive receptor cluster dynamics at the immunological synapse.

Topics: Actins; Actomyosin; Cytochalasin D; Depsipeptides; Green Fluorescent Proteins; Heterocyclic Compounds, 4 or More Rings; Humans; Immunological Synapses; Jurkat Cells; Lymphocyte Function-Associated Antigen-1; Nonmuscle Myosin Type IIA; Receptors, Antigen, T-Cell; T-Lymphocytes

When skeletal muscles are activated and mechanically shortened, the force that is produced by the muscle fibers decreases in two phases, marked by two changes in slope (P₁ and P₂) that happen at specific lengths (L₁ and L₂). We tested the hypothesis that these force transients are determined by the amount of myosin cross-bridges attached to actin and by changes in cross-bridge strain due to a changing fraction of cross-bridges in the pre-power-stroke state. Three separate experiments were performed, using skinned muscle fibers that were isolated and subsequently (i) activated at different Ca²⁺ concentrations (pCa²⁺ 4.5, 5.0, 5.5, 6.0) (n = 13), (ii) activated in the presence of blebbistatin (n = 16), and (iii) activated in the presence of blebbistatin at varying velocities (n = 5). In all experiments, a ramp shortening was imposed (amplitude 10%L₀, velocity 1 L₀•sarcomere length (SL)•s⁻¹), from an initial SL of 2.5 µm (except by the third group, in which velocities ranged from 0.125 to 2.0 L₀•s⁻¹). The values of P₁, P₂, L₁, and L₂ did not change with Ca²⁺ concentrations. Blebbistatin decreased P₁, and it did not alter P₂, L₁, and L₂. We developed a mathematical cross-bridge model comprising a load-dependent power-stroke transition and a pre-power-stroke cross-bridge state. The P₁ and P₂ critical points as well as the critical lengths L₁ and L₂ were explained qualitatively by the model, and the effects of blebbistatin inhibition on P₁ were also predicted. Furthermore, the results of the model suggest that the mechanism by which blebbistatin inhibits force is by interfering with the closing of the myosin upper binding cleft, biasing cross-bridges into a pre-power-stroke state.

Topics: Animals; Biomechanical Phenomena; Calcium; Computer Simulation; Heterocyclic Compounds, 4 or More Rings; Hydrogen-Ion Concentration; In Vitro Techniques; Models, Biological; Muscle Contraction; Muscle Fibers, Skeletal; Rabbits; Sarcomeres

Alveolar epithelial cells (AECs) maintain integrity of the blood-gas barrier with actin-anchored intercellular tight junctions. Stretched type I-like AECs undergo magnitude- and frequency-dependent actin cytoskeletal remodeling into perijunctional actin rings. On the basis of published studies in human pulmonary artery endothelial cells (HPAECs), we hypothesize that RhoA activity, Rho kinase (ROCK) activity, and phosphorylation of myosin light chain II (MLC2) increase in stretched type I-like AECs in a manner that is dependent on stretch magnitude, and that RhoA, ROCK, or MLC2 activity inhibition will attenuate stretch-induced actin remodeling and preserve barrier properties. Primary type I-like AEC monolayers were stretched biaxially to create a change in surface area (ΔSA) of 12%, 25%, or 37% in a cyclic manner at 0.25 Hz for up to 60 min or left unstretched. Type I-like AECs were also treated with Rho pathway inhibitors (ML-7, Y-27632, or blebbistatin) and stained for F-actin or treated with the myosin phosphatase inhibitor calyculin-A and quantified for monolayer permeability. Counter to our hypothesis, ROCK activity and MLC2 phosphorylation decreased in type I-like AECs stretched to 25% and 37% ΔSA and did not change in monolayers stretched to 12% ΔSA. Furthermore, RhoA activity decreased in type I-like AECs stretched to 37% ΔSA. In contrast, MLC2 phosphorylation in HPAECs increased when HPAECs were stretched to 12% ΔSA but then decreased when they were stretched to 37% ΔSA, similar to type I-like AECs. Perijunctional actin rings were observed in unstretched type I-like AECs treated with the Rho pathway inhibitor blebbistatin. Myosin phosphatase inhibition increased MLC2 phosphorylation in stretched type I-like AECs but had no effect on monolayer permeability. In summary, stretch alters RhoA activity, ROCK activity, and MLC2 phosphorylation in a manner dependent on stretch magnitude and cell type.

Topics: Actins; Amides; Animals; Azepines; Cardiac Myosins; Cells, Cultured; Endothelial Cells; Enzyme Inhibitors; Epithelial Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Male; Mechanical Phenomena; Mechanotransduction, Cellular; Models, Biological; Myosin Light Chains; Naphthalenes; Phosphorylation; Pulmonary Alveoli; Pyridines; Rats, Sprague-Dawley; Respiration, Artificial; Respiratory Mucosa; rho-Associated Kinases; rhoA GTP-Binding Protein

Physical features of microenvironments such as matrix elasticity E can clearly influence cell morphology and cell phenotype, but many differences between model matrices raise questions as to whether a standard biological scale for E exists, especially in 3D as well as in 2D. An E-series of two distinct types of hydrogels are ligand-functionalized here with non-fibrous collagen and used to elucidate wide-ranging cell and cytoskeletal responses to E in both 2D and 3D matrix geometries. Cross-linked hyaluronic acid (HA) based matrices as well as standard polyacrylamide (PA) hydrogels show that, within hours of initial plating, the adhesion, asymmetric shape, and cytoskeletal order within mesenchymal stem cells generally depend on E nonmonotonically over a broad range of physiologically relevant E. In particular, with overlays of a second matrix the stiffer of the upper or lower matrix dominates key cell responses to 3D: the cell invariably takes an elongated shape that couples to E in driving cytoplasmic stress fiber assembly. In contrast, embedding cells in homogeneous HA matrices constrains cells to spherically symmetric shapes in which E drives the assembly of a predominantly cortical cytoskeleton. Non-muscle myosin II generates the forces required for key cell responses and is a target of a phospho-Tyrosine signaling pathway that likely regulates contractile assemblies and also depends nonmonotonically on E. The results can be understood in part from a theory for stress fiber polarization that couples to matrix elasticity as well as cell shape and accurately predicts cytoskeletal order in 2D and 3D, regardless of polymer system.

Topics: Acrylic Resins; Actins; Cell Adhesion; Cell Proliferation; Cell Shape; Cell Survival; Collagen Type I; Elastic Modulus; Elasticity; Extracellular Matrix; Gelatin; Heterocyclic Compounds, 4 or More Rings; Humans; Hyaluronic Acid; Hydrogels; Mesenchymal Stem Cells; Microscopy, Atomic Force; Microscopy, Fluorescence; Myosin Heavy Chains; Nonmuscle Myosin Type IIA; Phosphorylation; Phosphotyrosine; Stress Fibers; Vinculin

Transforming growth factor β1 is one of the main inducers of epithelial-to-mesenchymal transition (EMT). During EMT cells from an ordered epithelial state adopt a fibroblast-like shape combined with a reorganization of the cytoskeleton and altered cell-cell and cell-substrate interactions. Interestingly, an increased cellular motion lasting up to 9h after cytokine stimulation takes place. These changes in cellular shape and dynamics can be monitored by impedance spectroscopy. Analyzing impedance noise by means of variance and detrended fluctuation analysis provides information about the magnitude of vertical cellular micromotility and the long-term correlation of the impedance signal. Via preincubation with Rho kinase inhibitor Y-27632, blebbistatin, and the protein inhibitors rapamycin and cycloheximide before cytokine addition, we were able to assign the origin of the dynamic changes. Fluctuations upon TGF-β1 administration were diminished using cycloheximide, blebbistatin and rapamycin. Consequently, we conclude that mainly actin contractility and de novo protein synthesis leading to changes in actin polymerization/depolymerization processes are responsible for the detected alterations, whereas activation of Rho kinases (ROCK) is not involved. Importantly, none of the used agents affected the EMT phenotype, reflected in unchanged static impedance parameters, optical micrographs and unmodified correlations displayed in the impedance noise.

Topics: Actins; Amides; Animals; Cell Line; Cell Shape; Dielectric Spectroscopy; Epithelial-Mesenchymal Transition; Heterocyclic Compounds, 4 or More Rings; Mice; Microscopy, Fluorescence; Protein Kinase Inhibitors; Pyridines; rho-Associated Kinases; Transforming Growth Factor beta

Mitosis is a continuous process to separate replicated chromosomes into two daughter cells through prophase, metaphase, anaphase, and telophase. Although a number of methods have been established to synchronize cells at different phases of the cell cycle, it is difficult to synchronize cells at the specific phases, anaphase and telophase, during mitosis because of the short duration of anaphase. Here, we show that HeLa S3 cells in anaphase and in telophase are successfully enriched by treatment with a combination of low concentrations of the microtubule-depolymerizing agent nocodazole and the myosin II inhibitor blebbistatin. After 9-h release from thymidine block at G1/S phase, addition of nocodazole at 20 ng/ml but not 40 ng/ml ensures rapid release from the nocodazole arrest. Subsequently, the cells are cultured in the presence of 50 μM blebbistatin for 20 and 50 min to enrich cells in anaphase and telophase, respectively. Western blot analysis verifies down-regulation of phospho-histone H3-Ser10, phospho-Aurora A/B/C, and cyclin B1 during M-phase progression. Furthermore, we show how the electrophoretic mobility shifts of the Src-family kinases c-Yes and c-Src can change in each phase of mitosis. These results provide a useful synchronization method for biochemically examining protein dynamics during M-phase progression.

Topics: Anaphase; Cell Culture Techniques; Cell Cycle Checkpoints; HeLa Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Metaphase; Myosin Type II; Nocodazole; Telophase; Tubulin Modulators

An acquired mutation (T790M) in the epidermal growth factor receptor (EGFR) accounts for half of all relapses in non-small cell lung cancer (NSCLC) patients who initially respond to EGFR kinase inhibitors. In this study, we demonstrated for the first time that EGFR-T790M interacts with the cytoskeletal components, myosin heavy chain 9 (MYH9) and β-actin, in the nucleus of H1975 cells carrying the T790M-mutant EGFR. The interactions of EGFR with MYH9 and β-actin were reduced in the presence of blebbistatin, a specific inhibitor for the MYH9-β-actin interaction, suggesting that the EGFR interaction with MYH9 and β-actin is affected by the integrity of the cytoskeleton. These physical interactions among MYH9, β-actin, and EGFR were also impaired by CL-387,785, a kinase inhibitor for EGFR-T790M. Furthermore, CL-387,785 and blebbistatin interacted in a synergistic fashion to suppress cell proliferation and induce apoptosis in H1975 cells. The combination of CL-387,785 and blebbistatin enhanced the down-regulation of cyclooxygenase-2 (COX-2), a transcriptional target of nuclear EGFR. Overall, our findings demonstrate that disrupting EGFR interactions with the cytoskeletal components enhanced the anti-cancer effects of CL-387,785 against H1975 cells, suggesting a novel therapeutic approach for NSCLC cells that express the drug-resistant EGFR-T790M.

Topics: Actins; Apoptosis; Blotting, Western; Cell Line; Cell Nucleus; Cell Proliferation; Cyclooxygenase 2; ErbB Receptors; Flow Cytometry; Heterocyclic Compounds, 4 or More Rings; Humans; Immunoprecipitation; Molecular Motor Proteins; Myosin Heavy Chains; Myosin Type II; Protein Binding; Quinazolines; Real-Time Polymerase Chain Reaction; RNA Interference

During cytokinesis in eukaryotic cells, an actomyosin-based contractile ring (CR) is assembled along the equator of the cell. Myosin II ATPase activity is stimulated by the phosphorylation of the myosin II regulatory light chain (MRLC) in vitro, and phosphorylated MRLC localizes at the CR in various types of cells. Previous studies have determined that phosphorylated MRLC plays an important role in CR furrowing. However, the role of phosphorylated MRLC in CR assembly remains unknown. Here, we have used confocal microscopy to observe dividing HeLa cells expressing fluorescent protein-tagged MRLC mutants and actin during CR assembly near the cortex. Di-phosphomimic MRLC accumulated at the cell equator earlier than non-phosphorylatable MRLC and actin. Interestingly, perturbation of myosin II activity by non-phosphorylatable MRLC expression or treatment with blebbistatin, a myosin II inhibitor, did not alter the time of actin accumulation at the cell equator. Furthermore, inhibition of actin polymerization by treatment with latrunculin A had no effect on MRLC accumulation at the cell equator. Taken together, these data suggest that phosphorylated MRLC temporally controls its own accumulation, but not that of actin, in cultured mammalian cells.

Topics: Actin Cytoskeleton; Actins; Bridged Bicyclo Compounds, Heterocyclic; Cytokinesis; HeLa Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Myosin Light Chains; Myosin Type II; Phosphorylation; Thiazolidines

Megakaryocytes are unique mammalian cells that undergo polyploidization (endomitosis) during differentiation, leading to an increase in cell size and protein production that precedes platelet production. Recent evidence demonstrates that endomitosis is a consequence of a late failure in cytokinesis associated with a contractile ring defect. Here we show that the non-muscle myosin IIB heavy chain (MYH10) is expressed in immature megakaryocytes and specifically localizes in the contractile ring. MYH10 downmodulation by short hairpin RNA increases polyploidization by inhibiting the return of 4N cells to 2N, but other regulators, such as of the G1/S transition, might regulate further polyploidization of the 4N cells. Conversely, re-expression of MYH10 in the megakaryocytes prevents polyploidization and the transition of 2N to 4N cells. During polyploidization, MYH10 expression is repressed by the major megakaryocyte transcription factor RUNX1. Thus, RUNX1-mediated silencing of MYH10 is required for the switch from mitosis to endomitosis, linking polyploidization with megakaryocyte differentiation.

Topics: Animals; Antigens, CD34; Cell Line; Core Binding Factor Alpha 2 Subunit; Cytokinesis; Heterocyclic Compounds, 4 or More Rings; Humans; Megakaryocytes; Mice; Mice, Knockout; Mitosis; Myosin Heavy Chains; Nonmuscle Myosin Type IIB; Polyploidy; RNA Interference; RNA, Small Interfering

Synaptic vesicle exo- and endocytosis are usually driven by neuronal activity but can also occur spontaneously. The identity and differences between vesicles supporting evoked and spontaneous neurotransmission remain highly debated. Here we combined nanometer-resolution imaging with a transient motion analysis approach to examine the dynamics of individual synaptic vesicles in hippocampal terminals under physiological conditions. We found that vesicles undergoing spontaneous and stimulated endocytosis differ in their dynamic behavior, particularly in the ability to engage in directed motion. Our data indicate that such motional differences depend on the myosin family of motor proteins, particularly myosin II. Analysis of synaptic transmission in the presence of myosin II inhibitor confirmed a specific role for myosin II in evoked, but not spontaneous, neurotransmission and also suggested a functional role of myosin II-mediated vesicle motion in supporting vesicle mobilization during neural activity.

Topics: Analysis of Variance; Animals; Animals, Newborn; Azepines; Biophysics; Cells, Cultured; Electric Stimulation; Endocytosis; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Fluorescent Dyes; Heterocyclic Compounds, 4 or More Rings; Hippocampus; Microscopy, Fluorescence; Neurons; Nonlinear Dynamics; Organ Culture Techniques; Patch-Clamp Techniques; Rats; Sodium Channel Blockers; Statistics, Nonparametric; Synapses; Synaptic Vesicles; Tetrodotoxin; Time Factors

The cochlear spiral ligament is a connective tissue that plays diverse roles in normal hearing. Spiral ligament fibrocytes are classified into functional sub-types that are proposed to carry out specialized roles in fluid homeostasis, the mediation of inflammatory responses to trauma, and the fine tuning of cochlear mechanics. We derived a secondary sub-culture from guinea pig spiral ligament, in which the cells expressed protein markers of type III or "tension" fibrocytes, including non-muscle myosin II (nmII), α-smooth muscle actin (αsma), vimentin, connexin43 (cx43), and aquaporin-1. The cells formed extensive stress fibers containing αsma, which were also associated intimately with nmII expression, and the cells displayed the mechanically contractile phenotype predicted by earlier modeling studies. cx43 immunofluorescence was evident within intercellular plaques, and the cells were coupled via dye-permeable gap junctions. Coupling was blocked by meclofenamic acid (MFA), an inhibitor of cx43-containing channels. The contraction of collagen lattice gels mediated by the cells could be prevented reversibly by blebbistatin, an inhibitor of nmII function. MFA also reduced the gel contraction, suggesting that intercellular coupling modulates contractility. The results demonstrate that these cells can impart nmII-dependent contractile force on a collagenous substrate, and support the hypothesis that type III fibrocytes regulate tension in the spiral ligament-basilar membrane complex, thereby determining auditory sensitivity.

Topics: Actins; Animals; Aquaporin 1; Biomechanical Phenomena; Cell Communication; Cell Size; Cells, Cultured; Cochlea; Connexin 43; Gap Junctions; Guinea Pigs; Heterocyclic Compounds, 4 or More Rings; In Vitro Techniques; Meclofenamic Acid; Models, Animal; Myosin Type II; Spiral Ligament of Cochlea; Vimentin

The heart is the first functioning organ to form during development. During gastrulation, the cardiac progenitors reside in the lateral plate mesoderm but maintain close contact with the underlying endoderm. In amniotes, these bilateral heart fields are initially organized as a pair of flat epithelia that move towards the embryonic midline and fuse above the anterior intestinal portal (AIP) to form the heart tube. This medial motion is typically attributed to active mesodermal migration over the underlying endoderm. In this model, the role of the endoderm is twofold: to serve as a mechanically passive substrate for the crawling mesoderm and to secrete various growth factors necessary for cardiac specification and differentiation. Here, using computational modeling and experiments on chick embryos, we present evidence supporting an active mechanical role for the endoderm during heart tube assembly. Label-tracking experiments suggest that active endodermal shortening around the AIP accounts for most of the heart field motion towards the midline. Results indicate that this shortening is driven by cytoskeletal contraction, as exposure to the myosin-II inhibitor blebbistatin arrested any shortening and also decreased both tissue stiffness (measured by microindentation) and mechanical tension (measured by cutting experiments). In addition, blebbistatin treatment often resulted in cardia bifida and abnormal foregut morphogenesis. Moreover, finite element simulations of our cutting experiments suggest that the endoderm (not the mesoderm) is the primary contractile tissue layer during this process. Taken together, these results indicate that contraction of the endoderm actively pulls the heart fields towards the embryonic midline, where they fuse to form the heart tube.

Topics: Animals; Biomechanical Phenomena; Cell Movement; Chick Embryo; Endoderm; Heart; Heterocyclic Compounds, 4 or More Rings; Models, Biological; Morphogenesis; Tomography, Optical Coherence

Myocardial stretching is an arrhythmogenic factor. Optical techniques and mechanical uncouplers are used to study the mechanoelectric feedback. The aim of this study is to determine whether the mechanical uncouplers 2,3-butanedione monoxime and Blebbistatin hinder or modify the electrophysiological effects of acute mechanical stretch.. The ventricular fibrillation (VF) modifications induced by acute mechanical stretch were studied in 27 Langendorff-perfused rabbit hearts using epicardial multiple electrodes and mapping techniques under control conditions (n = 9) and during the perfusion of 2,3-butanedione monoxime (15 mM) (n = 9) or Blebbistatin (10 μm) (n = 9).. In the control series, myocardial stretch increased the complexity of the activation maps and the dominant frequency (DF) of VF from 13.1 ± 2.0 Hz to 19.1 ± 3.1 Hz (P < 0.001, 46% increment). At baseline, the activation maps showed less complexity in both the 2,3-butanedione monoxime and Blebbistatin series, and the DF was lower in the 2,3-butanedione monoxime series (11.4 ± 1.2 Hz; P < 0.05). The accelerating effect of mechanical stretch was abolished under 2,3-butanedione monoxime (maximum DF = 11.7 ± 2.4 Hz, 5% increment, ns vs baseline, P < 0.0001 vs. control series) and reduced under Blebbistatin (maximum DF = 12.9 ± 0.7 Hz, 8% increment, P < 0.01 vs. baseline, P < 0.0001 vs. control series). The variations in complexity of the activation maps under stretch were not significant in the 2,3-butanedione monoxime series and were significantly attenuated under Blebbistatin.. The accelerating effect and increased complexity of myocardial activation during VF induced by acute mechanical stretch are abolished under the action of 2,3-butanedione monoxime and reduced under the action of Blebbistatin.

Topics: Animals; Diacetyl; Enzyme Inhibitors; Feedback, Physiological; Heart; Heterocyclic Compounds, 4 or More Rings; Organ Culture Techniques; Rabbits

Blebbistatin is a new inhibitor of cell motility. It is used to study dynamics of cytokinesis machinery in cells. However, the potential of this inhibitor as an anticancer agent has not been studied so far.. Cytotoxicity of blebbistatin was evaluated in five human cell lines, FEMX-I melanoma, U87 glioma, androgen independent Du145 and androgen sensitive LNCaP prostate adenocarcinoma, and F11-hTERT immortalized fibroblasts. Phototoxicity of blebbistatin was assessed in these cell lines after their exposure to a blue light (390-470 nm). Photostability of blebbistatin and its reactive oxygen species (ROS) generating properties were measured during irradiation with the blue light.. Blebbistatin at a concentration range of 10-200 μmol/L was toxic to all studied cells. Toxic concentrations (TC) were about 10-25 μmol/L corresponding to TC10, 50-100 μmol/L to TC50 and 140-190 μmol/L to TC90. Only for the U87 glioma cells TC90 could not be measured as the highest studied concentration of 200 μmol/L gave around 70% toxicity. However, after exposure to the blue light blebbistatin exhibited phototoxicity on the cells, with a cytotoxicity enhancement ratio that was greatest for the FEMX-I cells (about 9) followed by LNCaP (5), Du145 (3), U87 (2) and F11-hTERT (1.7) cells.. Blebbistatin inhibits cell motility and viability. Under exposure to the blue light blebbistatin exhibits photodynamic action on human cancer cells. During the irradiation blebbistatin oxidizes dihydrorhodamine 123 but not Singlet Oxygen Sensor Green.. Our findings offer new possibilities for blebbistatin as a potential anticancer and photodynamic agent.

Topics: Apoptosis; Cell Movement; Cell Survival; Heterocyclic Compounds, 4 or More Rings; Humans; Light; Male; Myosins; Oxidation-Reduction; Photosensitizing Agents; Prostatic Neoplasms; Reactive Oxygen Species; Tumor Cells, Cultured

The actin cytoskeleton promotes clustering of proteins associated with cholesterol-dependent rafts, but its effect on lipid interactions that form and maintain rafts is not understood. We addressed this question by determining the effect of disrupting the cytoskeleton on co-clustering of dihexadecyl-(C(16))-anchored DiO and DiI, which co-enrich in ordered lipid environments such as rafts. Co-clustering was assayed by fluorescence resonance energy transfer (FRET) in labeled T cells, where rafts function in the phosphoregulation of the Src family kinase Lck. Our results show that probe co-clustering was sensitive to depolymerization of actin filaments with latrunculin B (Lat B), inhibition of myosin II with blebbistatin, and treatment with neomycin to sequester phosphatidylinositol 4,5-bisphosphate. Cytoskeletal effects on lipid interactions were not restricted to order-preferring label because co-clustering of C(16)-anchored DiO with didodecyl (C(12))-anchored DiI, which favors disordered lipids, was also reduced by Lat B and blebbistatin. Furthermore, conditions that disrupted probe co-clustering resulted in activation of Lck. These data show that the cytoskeleton globally modulates lipid interactions in the plasma membrane, and this property maintains rafts that function in Lck regulation.

Topics: Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cytoskeleton; Flow Cytometry; Fluorescence Resonance Energy Transfer; Heterocyclic Compounds, 4 or More Rings; Humans; Jurkat Cells; Lipids; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Neomycin; Phosphatidylinositol 4,5-Diphosphate; Thiazolidines

Research on human embryonic stem cells (hESCs) has attracted much attention given their great potential for tissue regenerative therapy and fundamental developmental biology studies. Yet, there is still limited understanding of how mechanical signals in the local cellular microenvironment of hESCs regulate their fate decisions. Here, we applied a microfabricated micromechanical platform to investigate the mechanoresponsive behaviors of hESCs. We demonstrated that hESCs are mechanosensitive, and they could increase their cytoskeleton contractility with matrix rigidity. Furthermore, rigid substrates supported maintenance of pluripotency of hESCs. Matrix mechanics-mediated cytoskeleton contractility might be functionally correlated with E-cadherin expressions in cell-cell contacts and thus involved in fate decisions of hESCs. Our results highlighted the important functional link between matrix rigidity, cellular mechanics, and pluripotency of hESCs and provided a novel approach to characterize and understand mechanotransduction and its involvement in hESC function.

Topics: Cadherins; Cell Communication; Cell Differentiation; Cytoskeleton; Dimethylpolysiloxanes; Embryonic Stem Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Mechanotransduction, Cellular; Nylons; Octamer Transcription Factor-3; Pluripotent Stem Cells

Stabilization of long-term potentiation (LTP) depends on reorganization of the dendritic spine actin cytoskeleton. The present study tested whether this involves activity-driven effects on the actin-regulatory protein cortactin, and whether such effects are disturbed in the Fmr1 knock-out (KO) model of fragile X syndrome, in which stabilization of both actin filaments and LTP is impaired. LTP induced by theta burst stimulation (TBS) in hippocampal slices from wild-type mice was associated with rapid, broadly distributed, and NMDA receptor-dependent decreases in synapse-associated cortactin. The reduction in cortactin content was blocked by blebbistatin, while basal levels were reduced by nocodazole, indicating that cortactin's movements into and away from synapses are regulated by microtubule and actomyosin motors, respectively. These results further suggest that synapse-specific LTP influences cytoskeletal elements at distant connections. The rapid effects of TBS on synaptic cortactin content were absent in Fmr1 KOs as was evidence for activity-driven phosphorylation of the protein or its upstream kinase, ERK1/2. Phosphorylation regulates cortactin's interactions with actin, and coprecipitation of the two proteins was reduced in the KOs. We propose that, in the KOs, excessive basal phosphorylation of ERK1/2 disrupts its interactions with cortactin, thereby blocking the latter protein's use of actomyosin transport systems. These impairments are predicted to compromise the response of the subsynaptic cytoskeleton to learning-related afferent activity, both locally and at distant sites.

Topics: Actins; Animals; Cortactin; Electric Stimulation; Enzyme Inhibitors; Fragile X Mental Retardation Protein; Heterocyclic Compounds, 4 or More Rings; Hippocampus; In Vitro Techniques; Long-Term Potentiation; MAP Kinase Signaling System; Mice; Mice, Knockout; Mice, Mutant Strains; Nocodazole; Phosphorylation; Protein Transport; Signal Transduction; Synapses; Tubulin Modulators

Myocardial ischemia results in metabolic changes, which collapse the mitochondrial network, that increase the vulnerability of the heart to ventricular fibrillation (VF). It has been demonstrated at the single cell level that uncoupling the mitochondria using carbonyl cyanide p-(tri-fluoromethoxy)phenyl-hydrazone (FCCP) at low concentrations can be cardioprotective. The aim of our study was to investigate the effect of FCCP on arrhythmogenesis during ischemia in the whole rabbit heart. We performed optical mapping of isolated rabbit hearts (n = 33) during control and 20 min of global ischemia and reperfusion, both with and without pretreatment with the mitochondrial uncoupler FCCP at 100, 50, or 30 nM. No hearts went into VF during ischemia under the control condition, with or without the electromechanical uncoupler blebbistatin. We found that pretreatment with 100 (n = 4) and 50 (n = 6) nM FCCP, with or without blebbistatin, leads to VF during ischemia in all hearts, whereas pretreatment with 30 nM of FCCP led to three out of eight hearts going into VF during ischemia. We demonstrated that 30 nM of FCCP significantly increased interventricular (but not intraventricular) action potential duration and conduction velocity heterogeneity in the heart during ischemia, thus providing the substrate for VF. We showed that wavebreaks during VF occurred between the right and left ventricular junction. We also demonstrated that no VF occurred in the heart pretreated with 10 μM glibenclamide, which is known to abolish interventricular heterogeneity. Our results indicate that low concentrations of FCCP, although cardioprotective at the single cell level, are arrhythmogenic at the whole heart level. This is due to the fact that FCCP induces different electrophysiological changes to the right and left ventricle, thus increasing interventricular heterogeneity and providing the substrate for VF.

Topics: Action Potentials; Animals; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Glyburide; Heterocyclic Compounds, 4 or More Rings; Mitochondria, Heart; Myocardial Ischemia; Proton Ionophores; Rabbits; Ventricular Fibrillation

A spectral analysis approach was developed for detailed study of time-resolved, dynamic changes in vascular smooth muscle cell (VSMC) elasticity and adhesion to identify differences in VSMC from young and aged monkeys. Atomic force microscopy (AFM) was used to measure Young's modulus of elasticity and adhesion as assessed by fibronectin (FN) or anti-beta 1 integrin interaction with the VSMC surface. Measurements demonstrated that VSMC cells from old vs. young monkeys had increased elasticity (21.6 kPa vs. 3.5 kPa or a 612% increase in elastic modulus) and adhesion (86 pN vs. 43 pN or a 200% increase in unbinding force). Spectral analysis identified three major frequency components in the temporal oscillation patterns for elasticity (ranging from 1.7 × 10(-3) to 1.9 × 10(-2) Hz in old and 8.4 × 10(-4) to 1.5 × 10(-2) Hz in young) and showed that the amplitude of oscillation was larger (P < 0.05) in old than in young at all frequencies. It was also observed that patterns of oscillation in the adhesion data were similar to the elasticity waveforms. Cell stiffness was reduced and the oscillations were inhibited by treatment with cytochalasin D, ML7 or blebbistatin indicating the involvement of actin-myosin-driven processes. In conclusion, these data demonstrate the efficacy of time-resolved analysis of AFM cell elasticity and adhesion measurements and that it provides a uniquely sensitive method to detect real-time functional differences in biomechanical and adhesive properties of cells. The oscillatory behavior suggests that mechanisms governing elasticity and adhesion are coupled and affected differentially during aging, which may link these events to changes in vascular stiffness.

Topics: Aging; Animals; Azepines; Cell Adhesion; Cells, Cultured; Cellular Senescence; Cytochalasin D; Elasticity; Extracellular Matrix; Fibronectins; Haplorhini; Heterocyclic Compounds, 4 or More Rings; Integrins; Male; Microscopy, Atomic Force; Muscle, Smooth, Vascular; Naphthalenes

The contractile system of nonmuscle cells consists of interconnected actomyosin networks and bundles anchored to focal adhesions. The initiation of the contractile system assembly is poorly understood structurally and mechanistically, whereas system's maturation heavily depends on nonmuscle myosin II (NMII). Using platinum replica electron microscopy in combination with fluorescence microscopy, we characterized the structural mechanisms of the contractile system assembly and roles of NMII at early stages of this process. We show that inhibition of NMII by a specific inhibitor, blebbistatin, in addition to known effects, such as disassembly of stress fibers and mature focal adhesions, also causes transformation of lamellipodia into unattached ruffles, loss of immature focal complexes, loss of cytoskeleton-associated NMII filaments and peripheral accumulation of activated, but unpolymerized NMII. After blebbistatin washout, assembly of the contractile system begins with quick and coordinated recovery of lamellipodia and focal complexes that occurs before reappearance of NMII bipolar filaments. The initial formation of focal complexes and subsequent assembly of NMII filaments preferentially occurred in association with filopodial bundles and concave actin bundles formed by filopodial roots at the lamellipodial base. Over time, accumulating NMII filaments help to transform the precursor structures, focal complexes and associated thin bundles, into stress fibers and mature focal adhesions. However, semi-sarcomeric organization of stress fibers develops at much slower rate. Together, our data suggest that activation of NMII motor activity by light chain phosphorylation occurs at the cell edge and is uncoupled from NMII assembly into bipolar filaments. We propose that activated, but unpolymerized NMII initiates focal complexes, thus providing traction for lamellipodial protrusion. Subsequently, the mechanical resistance of focal complexes activates a load-dependent mechanism of NMII polymerization in association with attached bundles, leading to assembly of stress fibers and maturation of focal adhesions.

Topics: Actin Cytoskeleton; Actins; Animals; Antibodies; Cattle; Fibroblasts; Focal Adhesions; Heterocyclic Compounds, 4 or More Rings; Models, Biological; Myosin Type II; Polymerization; Pseudopodia; Rats; Stress Fibers

Cilia-generated fluid flow in an 'organ of asymmetry' is critical for establishing the left-right body axis in several vertebrate embryos. However, the cell biology underlying how motile cilia produce coordinated flow and asymmetric signals is not well defined. In the zebrafish organ of asymmetry-called Kupffer's vesicle (KV)-ciliated cells are asymmetrically positioned along the anterior-posterior axis such that more cilia are placed in the anterior region. We previously demonstrated that Rho kinase 2b (Rock2b) is required for anteroposterior asymmetry and fluid flow in KV, but it remained unclear how the distribution of ciliated cells becomes asymmetric during KV development. Here, we identify a morphogenetic process we refer to as 'KV remodeling' that transforms initial symmetry in KV architecture into anteroposterior asymmetry. Live imaging of KV cells revealed region-specific cell shape changes that mediate tight packing of ciliated cells into the anterior pole. Mathematical modeling indicated that different interfacial tensions in anterior and posterior KV cells are involved in KV remodeling. Interfering with non-muscle myosin II (referred to as Myosin II) activity, which modulates cellular interfacial tensions and is regulated by Rock proteins, disrupted KV cell shape changes and the anteroposterior distribution of KV cilia. Similar defects were observed in Rock2b depleted embryos. Furthermore, inhibiting Myosin II at specific stages of KV development perturbed asymmetric flow and left-right asymmetry. These results indicate that regional cell shape changes control the development of anteroposterior asymmetry in KV, which is necessary to generate coordinated asymmetric fluid flow and left-right patterning of the embryo.

Topics: Animals; Body Patterning; Cell Shape; Cilia; Fluorescence; Heterocyclic Compounds, 4 or More Rings; Immunohistochemistry; In Situ Hybridization; Models, Biological; Morphogenesis; Morpholinos; Myosin Type II; rho-Associated Kinases; Zebrafish

FPD/AML is a familial platelet disorder characterized by platelet defects, predisposition to acute myelogenous leukemia (AML) and germ-line heterozygous RUNX1 alterations. Here we studied the in vitro megakaryopoiesis of 3 FPD/AML pedigrees. A 60% to 80% decrease in the output of megakaryocytes (MKs) from CD34(+) was observed. MK ploidy level was low and mature MKs displayed a major defect in proplatelet formation. To explain these defects, we focused on myosin II expression as RUNX1 has been shown to regulate MYL9 and MYH10 in an inverse way. In FPD/AML MKs, expression of MYL9 and MYH9 was decreased, whereas MYH10 expression was increased and the MYH10 protein was still present in the cytoplasm of mature MKs. Myosin II activity inhibition by blebbistatin rescued the ploidy defect of FPD/AML MKs. Finally, we demonstrate that MYH9 is a direct target of RUNX1 by chromatin immunoprecipitation and luciferase assays and we identified new RUNX1 binding sites in the MYL9 promoter region. Together, these results demonstrate that the defects in megakaryopoiesis observed in FPD/AML are, in part, related to a deregulation of myosin IIA and IIB expression leading to both a defect in ploidization and proplatelet formation.

Topics: Blood Platelet Disorders; Blotting, Western; Chromatin Immunoprecipitation; Core Binding Factor Alpha 2 Subunit; Female; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Heterocyclic Compounds, 4 or More Rings; Humans; Leukemia, Myeloid, Acute; Luciferases; Male; Megakaryocytes; Mutation; Nonmuscle Myosin Type IIA; Nonmuscle Myosin Type IIB; Pedigree; Ploidies; Prognosis; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Previous studies demonstrated the importance of substrate stiffness and topography on the phenotype of many different cell types including fibroblasts. Yet the interaction of these two physical parameters remains insufficiently characterized, in particular for cardiac fibroblasts. Most studies focusing on contact guidance use rigid patterned substrates. It is not known how the ability of cardiac fibroblasts to follow grooves and ridges changes as the substrate stiffness is decreased to match the range of stiffness found in native heart tissues. This report demonstrates a significant interactive effect of substrate stiffness and topography on cardiac fibroblast elongation and orientation using polyacrylamide substrates of different stiffness and topography.

Topics: Acrylic Resins; Animals; Animals, Newborn; Biocompatible Materials; Cell Survival; Cells, Cultured; Collagen; Elasticity; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Hydrogels; Microscopy, Electron, Scanning; Myocardium; Rats; Surface Properties; Tissue Engineering; Tissue Scaffolds

Blebbistatin is a recently discovered myosin II inhibitor. It is rapidly becoming a compound of choice to reduce motion artifacts during cardiac optical mapping, as well as to study cell motility and cell invasion. Although blebbistatin has a number of advantages over other electromechanical uncouplers, many of its properties have yet to be addressed. Here we describe several methodological issues associated with the use of blebbistatin, including its spectral properties, reversibility, and its effect on tissue metabolic state. We show that if precautions are not taken, perfusion with blebbistatin may result in blebbistatin precipitate that accumulates in the vasculature. Although such precipitate is fluorescent, it is not detectable within wavelength bands that are typically used for transmembrane voltage fluorescence imaging (i.e., emission wavelengths >600 nm). Therefore, blockage of the microcirculation by blebbistatin may cause data misinterpretation in studies that use voltage-sensitive dyes. Blebbistatin may also impact imaging of green fluorophores due to the spectral shift it causes in endogenous tissue fluorescence. 3D excitation-emission matrices of blebbistatin in precipitate form and in various solutions (DMSO, water, and 1 % aqueous albumin) revealed significant changes in the fluorescence of this molecule in different environments. Finally, we examined the reversibility of blebbistatin's uncoupling effect on cardiac contraction. Our findings provide important new information about the properties of this myosin II inhibitor, which will aid in the proper design and interpretation of studies that use this compound.

Topics: Animals; Epicardial Mapping; Heterocyclic Compounds, 4 or More Rings; In Vitro Techniques; Myocardial Perfusion Imaging; Rats; Rats, Sprague-Dawley; Voltage-Sensitive Dye Imaging

The difficulty in expanding cancer-initiating cells in vitro is one of major obstacles for their biochemical characterization. We found that Rho kinase (ROCK) inhibitors as well as blebbistatin, a myosin II inhibitor, greatly facilitated the establishment of spheroids from primary colon cancer. The spheroid cells expressed cancer stem cell markers, showed the ability to differentiate, and induced tumors in mice. The spheroids were composed of cells that express various levels of CD44, whereas CD44(high) cells were associated with increased sphere-forming ability, expression of the activating form of β-catenin, and elevated levels of glycolytic genes, CD44(-/low) cells showed increased levels of differentiation markers and apoptotic cells. The spheroid cells expressed variant forms of CD44 including v6, and the induction of the variants was associated with the activating phosphorylation of c-Met. As expected from the predicted hierarchy, CD44(high) cells differentiated into CD44(-/low) cells. Unexpectedly, a fraction of CD44(-/low) cells generated CD44(high) cells, and the ROCK inhibitor or blebbistatin primed the transition by inducing CD44 expression. We propose that the transition from CD44(-/low) to CD44(high) state helps to maintain a CD44(high) fraction and the tumorigenic diversity in colon cancer.

Topics: Amides; Animals; Apoptosis; Blotting, Western; Cell Proliferation; Colonic Neoplasms; Enzyme Inhibitors; Gene Expression Profiling; Hep G2 Cells; Heterocyclic Compounds, 4 or More Rings; HT29 Cells; Humans; Hyaluronan Receptors; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasms, Experimental; Neoplastic Stem Cells; Oligonucleotide Array Sequence Analysis; Pyridines; Reverse Transcriptase Polymerase Chain Reaction; rho-Associated Kinases; RNA Interference; Spheroids, Cellular; Transplantation, Heterologous; Tumor Cells, Cultured

Cellular self-organization is essential to physiological tissue and organ development. We previously observed that vascular mesenchymal cells, a multipotent subpopulation of aortic smooth muscle cells, self-organize into macroscopic, periodic patterns in culture. The patterns are produced by cells gathering into raised aggregates in the shape of nodules or ridges. To determine whether these patterns are accounted for by an oriented pattern of cell divisions or postmitotic relocation of cells, we acquired time-lapse, videomicrographic phase-contrast, and fluorescence images during self-organization. Cell division events were analyzed for orientation of daughter cells in mitoses during separation and their angle relative to local cell alignment, and frequency distribution of the mitotic angles was analyzed by both histographic and bin-free statistical methods. Results showed a statistically significant preferential orientation of daughter cells along the axis of local cell alignment as early as day 8, just before aggregate formation. This alignment of mitotic axes was also statistically significant at the time of aggregate development (day 11) and after aggregate formation was complete (day 15). Treatment with the nonmuscle myosin II inhibitor, blebbistatin, attenuated alignment of mitotic orientation, whereas Rho kinase inhibition eliminated local cell alignment, suggesting a role for stress fiber orientation in this self-organization. Inhibition of cell division using mitomycin C reduced the macroscopic pattern formation. Time-lapse monitoring of individual cells expressing green fluorescent protein showed postmitotic movement of cells into neighboring aggregates. These findings suggest that polarization of mitoses and postmitotic migration of cells both contribute to self-organization into periodic, macroscopic patterns in vascular stem cells.

Topics: Animals; Aorta; Cattle; Cell Division; Cell Polarity; Cells, Cultured; Enzyme Inhibitors; Heterocyclic Compounds, 4 or More Rings; Mesenchymal Stem Cells; Microscopy, Video; Mitomycin; Mitosis; Models, Animal; Muscle, Smooth, Vascular; rho-Associated Kinases; Time-Lapse Imaging

Blood clots form under flow during intravascular thrombosis or vessel leakage. Prevailing hemodynamics influence thrombus structure and may regulate contraction processes. A microfluidic device capable of flowing human blood over a side channel plugged with collagen (± tissue factor) was used to measure thrombus permeability (κ) and contraction at controlled transthrombus pressure drops.. The collagen (κ(collagen)=1.98 × 10(-11) cm(2)) supported formation of a 20-µm thick platelet layer, which unexpectedly underwent massive platelet retraction on flow arrest. This contraction resulted in a 5.34-fold increase in permeability because of collagen restructuring. Without stopping flow, platelet deposits (no fibrin) had a permeability of κ(platelet)=5.45 × 10(-14) cm(2) and platelet-fibrin thrombi had κ(thrombus)=2.71 × 10(-14) cm(2) for ΔP=20.7 to 23.4 mm Hg, the first ever measurements for clots formed under arterial flow (1130 s(-1) wall shear rate). Platelet sensing of flow cessation triggered a 4.6- to 6.5-fold (n=3, P<0.05) increase in contraction rate, which was also observed in a rigid, impermeable parallel-plate microfluidic device. This triggered contraction was blocked by the myosin IIA inhibitor blebbistatin and by inhibitors of thromboxane A2 (TXA(2)) and ADP signaling. In addition, flow arrest triggered platelet intracellular calcium mobilization, which was blocked by TXA(2)/ADP inhibitors. As clots become occlusive or blood pools following vessel leakage, the flow diminishes, consequently allowing full platelet retraction.. Flow dilution of ADP and thromboxane regulates platelet contractility with prevailing hemodynamics, a newly defined flow-sensing mechanism to regulate clot function.

Topics: Adenosine Diphosphate; Blood Coagulation; Blood Platelets; Calcium; Cardiovascular Physiological Phenomena; Collagen; Equipment Design; Hemodynamics; Heterocyclic Compounds, 4 or More Rings; Humans; In Vitro Techniques; Microfluidic Analytical Techniques; Nonmuscle Myosin Type IIA; Regional Blood Flow; Signal Transduction; Thrombosis; Thromboxane A2

The mechanical microenvironment is known to influence single-cell migration; however, the extent to which mechanical cues affect collective migration of adherent cells is not well understood. We measured the effects of varying substrate compliance on individual cell migratory properties in an epithelial wound-healing assay. Increasing substrate stiffness increased collective cell migration speed, persistence, and directionality as well as the coordination of cell movements. Dynamic analysis revealed that wounding initiated a wave of motion coordination from the wound edge into the sheet. This was accompanied by a front-to-back gradient of myosin-II activation and establishment of cell polarity. The propagation was faster and farther reaching on stiff substrates, indicating that substrate stiffness affects the transmission of directional cues. Manipulation of myosin-II activity and cadherin-catenin complexes revealed that this transmission is mediated by coupling of contractile forces between neighboring cells. Thus, our findings suggest that the mechanical environment integrates in a feedback with cell contractility and cell-cell adhesion to regulate collective migration.

Topics: Acrylic Resins; Blotting, Western; Breast; Cadherins; Catenins; Cell Adhesion; Cell Movement; Cell Polarity; Cell Proliferation; Cells, Cultured; Epithelial Cells; Female; Heterocyclic Compounds, 4 or More Rings; Humans; Muscle Contraction; Myosin Type II; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time-Lapse Imaging; Wound Healing

The stiffness of the extracellular matrix exerts powerful effects on cell proliferation and differentiation, but the mechanisms transducing matrix stiffness into cellular fate decisions remain poorly understood. Two widely reported responses to matrix stiffening are increases in actomyosin contractility and cell proliferation. To delineate their relationship, we modulated cytoskeletal tension in cells grown across a physiological range of matrix stiffnesses. On both synthetic and naturally derived soft matrices, and across a panel of cell types, we observed a striking reversal of the effect of inhibiting actomyosin contractility, switching from the attenuation of proliferation on rigid substrates to the robust promotion of proliferation on soft matrices. Inhibiting contractility on soft matrices decoupled proliferation from cytoskeletal tension and focal adhesion organization, but not from cell spread area. Our results demonstrate that matrix stiffness and actomyosin contractility converge on cell spreading in an unexpected fashion to control a key aspect of cell fate.

Topics: Actin Cytoskeleton; Actomyosin; Amides; Animals; Cantharidin; Cell Adhesion; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Dogs; Extracellular Matrix; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Humans; Madin Darby Canine Kidney Cells; Mesenchymal Stem Cells; Mice; NIH 3T3 Cells; Pyridines; Rats

OBJECTIVE To investigate the in vitro and in vivo effects of blebbistatin (a small cell-permeable molecule with high affinity and selectivity toward the myosin II contractile molecule) on bladder smooth muscle (SM) contractility, as antimuscarinic therapy is only 65-75% effective in treating overactive bladder (OAB) and is associated with considerable side-effects, with a < 25% continuation rate at 1 year. MATERIALS AND METHODS Bladder and aortic strips from adult male rats, and human bladder strips obtained from open prostatectomy, were used for organ-bath studies of blebbistatin. Awake cystometry was also used in rats in both the presence and absence of intravesically delivered blebbistatin. RESULTS Blebbistatin dose-dependently and completely relaxed both KCl- and carbachol-induced rat detrusor and endothelin-1-induced human bladder contraction. Pre-incubation with 10 µm blebbistatin attenuated carbachol responsiveness by ≈ 65% while blocking electrical field stimulation-induced bladder contraction reaching 50% inhibition at 32 Hz. The basal tone and amplitude of spontaneous contraction were also significantly diminished. Urodynamic variables were obviously altered by intravesical infusion with blebbistatin. CONCLUSION Our novel data show that blebbistatin strongly relaxes both rat and human bladder contraction induced by various physiological stimuli. Coupled with our in vivo data showing that nanomole doses of blebbistatin significantly alter urodynamic variables to produce a less active bladder, our results suggest the possibility of intravesically administered blebbistatin binding at myosin II being developed as a therapeutic treatment for OAB via a novel targeting of the SM contractile apparatus.

Topics: Animals; Endothelin-1; Heterocyclic Compounds, 4 or More Rings; Humans; Male; Muscle Contraction; Muscle, Smooth; Myosin Type II; Parasympatholytics; Rats; Rats, Sprague-Dawley; Urinary Bladder; Urinary Bladder, Overactive

Chikungunya virus (CHIKV) surprised medical workers by a massive outbreak in the Indian Ocean region, reaching Europe in 2007, with exceptional pathologies in infants and elderly patients. Although CHIKV was recently shown to persist in myoblasts, monocytes, and macrophages, we argued that robust antiviral mechanisms, including apoptosis, are essential to ward off the virus. Herein, we tested the capacity of CHIKV to mobilize the apoptotic machinery in HeLa cells as well as primary fibroblasts, making use of several inhibitors of caspases, cell blebbing, and engulfment of the apoptotic blebs by neighboring cells. CHIKV triggered apoptosis through intrinsic and extrinsic pathways. Bystander apoptosis was also evidenced in neighboring cells in a caspase-8-dependent manner. Remarkably, by hiding in apoptotic blebs, CHIKV was able to infect neighboring cells. In HeLa cells, these events were inhibited specifically by zVAD-fmk and DEVD-cho (caspase inhibitors), blebbistatin, Y-27632 (ROCK inhibitor), and genistein, annexin V, and cytochalasin B (inhibitors of blebbing and engulfment). These CHIKV-apoptotic blebs were also capable of infecting macrophages (primary cultures, MM6- and THP1-PMA differentiated cells) otherwise refractory to infection by CHIKV alone. Remarkably, viral replication in macrophages did not yield a proinflammatory response. We describe a novel infectious mechanism by which CHIKV invades host cells and escapes the host response.

Topics: Alphavirus Infections; Amides; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Bystander Effect; Caspase 8; Caspase Inhibitors; Cell Line; Cell Survival; Cells, Cultured; Chikungunya virus; Chlorocebus aethiops; Cytopathogenic Effect, Viral; Fibroblasts; Genistein; HeLa Cells; Heterocyclic Compounds, 4 or More Rings; Host-Pathogen Interactions; Humans; Macrophages; Oligopeptides; Pyridines; Vero Cells

Proliferation and migration of smooth muscle cells (SMC) require myosin II activity; thus, we examined whether blebbistatin, a cell-permeable selective inhibitor of myosin II ATP activity, would impair neointimal hyperplasia after vascular injury. Delivery of blebbistatin via a perivascular polymer cuff reduced neointimal formation by 73% and luminal obstruction by 75% after carotid denudation injury in C57BL/6 mice. Blebbistatin treatment was also associated with a reduction in cell density within the neointima; total number of cells (76 ± 7 to 27 ± 3 cells/high-powered field) and actin-positive cells (64 ± 4 to 24 ± 2 cells/high-powered field) in the neointima were reduced in blebbistatin-treated mice compared with vehicle-treated mice. In a model of vascular injury with an intact endothelium, implantation of a blebbistatin-secreting cuff after carotid ligation in FVB/N mice was associated with a 61% decrease in neointimal area and a significant decrease in luminal obstruction (88 ± 4% in vehicle-treated mice versus 36 ± 4% in blebbistatin-treated mice; p < 0.0001). In cultured rat aortic SMC, blebbistatin disrupted cellular morphology and actin cytoskeleton structure, and these effects were rapid and completely reversible. Blebbistatin had a dose-dependent inhibitory effect on DNA replication and cell proliferative responses to platelet-derived growth factor-BB, angiotensin II, and α-thrombin, migratory responses to serum, and migratory responses after blunt injury. In summary, perivascular delivery of blebbistatin reduced neointimal hyperplasia after carotid injury in the mouse.

Topics: Animals; Carotid Artery Injuries; Cell Movement; Cells, Cultured; Drug Delivery Systems; Heterocyclic Compounds, 4 or More Rings; Hyperplasia; Male; Mice; Mice, Inbred C57BL; Neointima; Random Allocation; Rats; Rats, Sprague-Dawley; Tunica Intima

Signaling through cell adhesion complexes plays a critical role in coordinating cytoskeletal remodeling necessary for efficient cell migration. During embryonic development, normal morphogenesis depends on a series of concerted cell movements; but the roles of cell adhesion signaling during these movements are poorly understood. The transparent zebrafish embryo provides an excellent system to study cell migration during development. Here, we have identified zebrafish git2a and git2b, two new members of the GIT family of genes that encode ArfGAP proteins associated with cell adhesions. Loss-of-function studies revealed an essential role for Git2a in zebrafish cell movements during gastrulation. Time-lapse microscopy analysis demonstrated that antisense depletion of Git2a greatly reduced or arrested cell migration towards the vegetal pole of the embryo. These defects were rescued by expression of chicken GIT2, indicating a specific and conserved role for Git2 in controlling embryonic cell movements. Git2a knockdown embryos showed defects in cell morphology that were associated with reduced cell contractility. We show that Git2a is required for phosphorylation of myosin light chain (MLC), which regulates myosin II-mediated cell contractility. Consistent with this, embryos treated with Blebbistatin-a small molecule inhibitor for myosin II activity-exhibited cell movement defects similar to git2a knockdown embryos. These observations provide in vivo evidence of a physiologic role for Git2a in regulating cell morphogenesis and directed cell migration via myosin II activation during zebrafish embryonic development.

Topics: Animals; Base Sequence; Cell Adhesion Molecules; Cell Movement; Embryonic Development; Gene Knockdown Techniques; GTPase-Activating Proteins; Heterocyclic Compounds, 4 or More Rings; Immunoblotting; Immunohistochemistry; In Situ Hybridization; Molecular Sequence Data; Morphogenesis; Myosin Light Chains; Phosphorylation; Phylogeny; Sequence Analysis, DNA; Signal Transduction; Time-Lapse Imaging; Zebrafish; Zebrafish Proteins

A robust protocol for the CuI-catalysed arylation of amidines is presented. Whilst the initially identified conditions were useful for benzamidine-derived substrates, difficulties were encountered with more complex substrates. This problem was overcome following a change in ligand type, enabling the synthesis of analogues of the chemical tool, blebbistatin.

Topics: Amidines; Catalysis; Copper; Heterocyclic Compounds, 4 or More Rings

Cell spreading is a critical component of numerous physiological phenomena including cancer metastasis, embryonic development, and mitosis. We have previously illustrated that cellular blebs appear after abrupt cell-substrate detachment and play a critical role in regulating membrane tension; however, the dynamics of bleb-substrate interactions during spreading remains unclear. Here we explore the role of blebs during endothelial cell spreading using chemical and osmotic modifications to either induce or inhibit bleb formation. We track cell-substrate dynamics as well as individual blebs using surface sensitive microscopic techniques. Blebbing cells (both control and chemically induced) exhibit increased lag times prior to fast growth. Interestingly, lamellae appear later for blebbing compared to non-blebbing cells, and in all cases, lamellae signal the start of fast spreading. Our results indicate that cellular blebs play a key role in the early stage of cell spreading, first by controling the initial cell adhesion and then by presenting a dynamic inhibition of cell spreading until a lamella appears and fast spreading ensues.

Topics: Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Cattle; Cell Adhesion; Cell Membrane; Cell Movement; Cell Shape; Cell Surface Extensions; Cells, Cultured; Cytoskeleton; Endothelial Cells; Heterocyclic Compounds, 4 or More Rings; Nocodazole; Osmotic Pressure; Thiazolidines

Myosin II is an actin-binding protein composed of MHC (myosin heavy chain) IIs, RLCs (regulatory light chains) and ELCs (essential light chains). Myosin II expressed in non-muscle tissues plays a central role in cell adhesion, migration and division. The regulation of myosin II activity is known to involve the phosphorylation of RLCs, which increases the Mg2+-ATPase activity of MHC IIs. However, less is known about the details of RLC-MHC II interaction or the loss-of-function phenotypes of non-muscle RLCs in mammalian cells. In the present paper, we investigate three highly conserved non-muscle RLCs of the mouse: MYL (myosin light chain) 12A (referred to as MYL12A), MYL12B and MYL9 (MYL12A/12B/9). Proteomic analysis showed that all three are associated with the MHCs MYH9 (NMHC IIA) and MYH10 (NMHC IIB), as well as the ELC MYL6, in NIH 3T3 fibroblasts. We found that knockdown of MYL12A/12B in NIH 3T3 cells results in striking changes in cell morphology and dynamics. Remarkably, the levels of MYH9, MYH10 and MYL6 were reduced significantly in knockdown fibroblasts. Comprehensive interaction analysis disclosed that MYL12A, MYL12B and MYL9 can all interact with a variety of MHC IIs in diverse cell and tissue types, but do so optimally with non-muscle types of MHC II. Taken together, our study provides direct evidence that normal levels of non-muscle RLCs are essential for maintaining the integrity of myosin II, and indicates that the RLCs are critical for cell structure and dynamics.

Topics: Amides; Amino Acid Sequence; Animals; Cell Movement; Enzyme Inhibitors; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Humans; Mice; Molecular Sequence Data; Myosin Light Chains; Myosin Type II; NIH 3T3 Cells; Pyridines; RNA Interference

Most tissue cells evolve in vivo in a three-dimensional (3D) microenvironment including complex topographical patterns. Cells exert contractile forces to adhere and migrate through the extracellular matrix (ECM). Although cell mechanics has been extensively studied on 2D surfaces, there are too few approaches that give access to the traction forces that are exerted in 3D environments. Here, we describe an approach to measure dynamically the contractile forces exerted by fibroblasts while they spread within arrays of large flexible micropillars coated with ECM proteins. Contrary to very dense arrays of microposts, the density of the micropillars has been chosen to promote cell adhesion in between the pillars. Cells progressively impale onto the micropatterned substrate. They first adhere on the top of the pillars without applying any detectable forces. Then, they spread along the pillar sides, spanning between the elastic micropillars and applying large forces on the substrate. Interestingly, the architecture of the actin cytoskeleton and the adhesion complexes vary over time as cells pull on the pillars. In particular, we observed less stress fibers than for cells spread on flat surfaces. However, prominent actin stress fibers are observed at cell edges surrounding the micropillars. They generate increasing contractile forces during cell spreading. Cells treated with blebbistatin, a myosin II inhibitor, relax their internal tension, as observed by the release of pillar deformations. Moreover, cell spreading on pillars coated with ECM proteins only on their tops are not able to generate significant traction forces. Taken together, these findings highlight the dynamic relationship between cellular forces and acto-myosin contractility in 3D environments, the influence of cytoskeletal network mechanics on cell shape, as well as the importance of cell-ECM contact area in the generation of traction forces.

Topics: Actins; Animals; Biomechanical Phenomena; Cell Adhesion; Cell Line; Cell Shape; Cytoskeleton; Elasticity; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Mechanical Phenomena; Microtechnology; Myosins; Rats; Single-Cell Analysis; Time Factors

A carbon fiber-based cell attachment and force measurement system was used to measure the diastolic stress-sarcomere length (SL) relation of mouse intact cardiomyocytes, before and after the addition of actomyosin inhibitors (2,3-butanedione monoxime [BDM] or blebbistatin). Stress was measured during the diastolic interval of twitching myocytes that were stretched at 100% base length/second. Diastolic stress increased close to linear from 0 at SL 1.85 µm to 4.2 mN/mm(2) at SL 2.1 µm. The actomyosin inhibitors BDM and blebbistatin significantly lowered diastolic stress by ∼1.5 mN/mm(2) (at SL 2.1 µm, ∼30% of total), suggesting that during diastole actomyosin interaction is not fully switched off. To test this further, calcium sensitivity of skinned myocytes was studied under conditions that simulate diastole: 37°C, presence of Dextran T500 to compress the myofilament lattice to the physiological level, and [Ca(2+)] from below to above 100 nM. Mean active stress was significantly increased at [Ca(2+)] > 55 nM (pCa 7.25) and was ∼0.7 mN/mm(2) at 100 nM [Ca(2+)] (pCa 7.0) and ∼1.3 mN/mm(2) at 175 nM Ca(2+) (pCa 6.75). Inhibiting active stress in intact cells attached to carbon fibers at their resting SL and stretching the cells while first measuring restoring stress (pushing outward) and then passive stress (pulling inward) made it possible to determine the passive cell's mechanical slack SL as ∼1.95 µm and the restoring stiffness and passive stiffness of the cells around the slack SL each as ∼17 mN/mm(2)/µm/SL. Comparison between the results of intact and skinned cells shows that titin is the main contributor to restoring stress and passive stress of intact cells, but that under physiological conditions, calcium sensitivity is sufficiently high for actomyosin interaction to contribute to diastolic stress. These findings are relevant for understanding diastolic function and for future studies of diastolic heart failure.

Topics: Actin Cytoskeleton; Actomyosin; Animals; Blood Pressure; Calcium; Carbon; Carbon Fiber; Connectin; Diacetyl; Heart; Heterocyclic Compounds, 4 or More Rings; Male; Mice; Mice, Inbred C57BL; Muscle Proteins; Myocardial Contraction; Myocytes, Cardiac; Protein Kinases; Sarcomeres; Stress, Mechanical

We show that in melanoma cells oncogenic BRAF, acting through MEK and the transcription factor BRN2, downregulates the cGMP-specific phosphodiesterase PDE5A. Although PDE5A downregulation causes a small decrease in proliferation, its major impact is to stimulate a dramatic increase in melanoma cell invasion. This is because PDE5A downregulation leads to an increase in cGMP, which induces an increase in cytosolic Ca(2+), stimulating increased contractility and inducing invasion. PDE5A downregulation also this leads to an increase in short-term and long-term colonization of the lungs by melanoma cells. We do not observe this pathway in NRAS mutant melanoma or BRAF mutant colorectal cells. Thus, we show that in melanoma cells oncogenic BRAF induces invasion through downregulation of PDE5A.

Topics: Animals; Calcimycin; Calcium; Cardiac Myosins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Down-Regulation; Gene Expression; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 4 or More Rings; Homeodomain Proteins; Humans; Lung Neoplasms; Melanoma; Mice; Mice, Nude; Myosin Light Chains; Neoplasm Invasiveness; Phosphodiesterase 5 Inhibitors; Phosphorylation; POU Domain Factors; Promoter Regions, Genetic; Protein Binding; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; RNA, Small Interfering; Transplantation, Heterologous

The aryl hydrocarbon receptor (AhR) is one of the best known ligand-activated transcription factors and it induces Cyp1a1 transcription by binding with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Recent focus has been on the relationship of AhR with signaling pathways that modulate cell shape and migration. In nonmuscle cells, nonmuscle myosin II is one of the key determinants of cell morphology, but it has not been investigated whether its function is related to Cyp1a1 induction. In this study, we observed that (-)-blebbistatin, which is a specific inhibitor of nonmuscle myosin II, increased the level of CYP1A1-mRNA in Hepa-1 cells. Comparison of (-)-blebbistatin with (+)-blebbistatin, which is an inactive enantiomer, indicated that the increase of CYP1A1-mRNA was due to nonmuscle myosin II inhibition. Subsequent knockdown experiments observed that reduction of nonmuscle myosin IIA, which is only an isoform of nonmuscle myosin II expressed in Hepa-1 cells, was related to the enhancement of TCDD-dependent Cyp1a1 induction. Moreover, chromatin immunoprecipitation assay indicated that the increase of Cyp1a1 induction was the result of transcriptional activation due to increased binding of AhR and RNA polymerase II to the enhancer and proximal promoter regions of Cyp1a1, respectively. These findings provide a new insight into the correlation between the function of nonmuscle myosin II and gene induction.

Topics: Animals; Cytochrome P-450 CYP1A1; Enzyme Induction; Gene Expression Regulation, Enzymologic; Gene Knockdown Techniques; Heterocyclic Compounds, 4 or More Rings; Mice; NIH 3T3 Cells; Nonmuscle Myosin Type IIA; Polychlorinated Dibenzodioxins; Promoter Regions, Genetic; Protein Binding; Protein Isoforms; Receptors, Aryl Hydrocarbon; RNA Polymerase II; RNA, Messenger; Transcription, Genetic

Myosin II ATPase activity is enhanced by the phosphorylation of MRLC (myosin II regulatory light chain) in non-muscle cells. It is well known that pMRLC (phosphorylated MRLC) co-localizes with F-actin (filamentous actin) in the CR (contractile ring) of dividing cells. Recently, we reported that HeLa cells expressing non-phosphorylatable MRLC show a delay in the speed of furrow ingression, suggesting that pMRLC plays an important role in the control of furrow ingression. However, it is still unclear how pMRLC regulates myosin II and F-actin at the CR to control furrow ingression during cytokinesis. In the present study, to clarify the roles of pMRLC, we measured the turnover of myosin II and actin at the CR in dividing HeLa cells expressing fluorescent-tagged MRLCs and actin by FRAP (fluorescence recovery after photobleaching). A myosin II inhibitor, blebbistatin, caused an enhancement of the turnover of MRLC and actin at the CR, which induced a delay in furrow ingression. Furthermore, only non-phosphorylatable MRLC and a Rho-kinase inhibitor, Y-27632, accelerated the turnover of MRLC and actin at the CR. Interestingly, the effect of Y-27632 was cancelled in the cell expressing phosphomimic MRLCs. Taken together, these results reveal that pMRLC reduces the turnover of myosin II and also actin at the CR. In conclusion, we show that the enhancement of myosin II and actin turnover at the CR induced slower furrowing in dividing HeLa cells.

Topics: Actins; Amides; Cell Membrane; Gene Expression Regulation; HeLa Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Mitosis; Myosin Light Chains; Myosin Type II; Protein Transport; Pyridines

Endocrine cells, such as H295R have been widely used to study secretion of steroid and other hormones. Exocytosis-dependent hormone release is accompanied by an increase in plasma membrane surface area and a decrease in vesicle content. Recovery of vesicles and decrease in plasma membrane area is achieved by endocytotic processes. These changes in the extent of the surface area lead to morphological changes which can be determined by label-free real-time impedance measurements. Exo- and endocytosis have been described to be triggered by activation of L-type Ca(2+) channels. The present study demonstrates that activation of L-type calcium channels induces prolonged oscillating changes in cellular impedance. The data support the hypothesis that a tight regulation of the intracellular Ca(2+) concentration is a prerequisite for the observed cellular impedance oscillations. Furthermore evidence is presented for a mechanism in which the oscillations depend on a Ca(2+)-triggered calmodulin-dependent cascade involving myosin light chain kinase, nonmuscle myosin II and ultimately actin polymerization, a known determinant for cell shape changes and exocytosis in secretory cells. The described assay provides a method to determine continuously prolonged changes in cellular morphology such as exo/endocytosis cycles. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adrenal Cortex; Angiotensin II; Calcium; Calcium Channels, L-Type; Calmodulin; Cell Line; Cell Shape; Electric Impedance; Heterocyclic Compounds, 4 or More Rings; Humans; Imaging, Three-Dimensional; Intracellular Space; Ouabain; RNA, Small Interfering; Thapsigargin; Time Factors

The cellular response to external mechanical forces has important effects on numerous biological phenomena. The sequences of molecular events that underlie the observed changes in cellular properties have yet to be elucidated in detail. Here we have detected the responses of a cultured cell against locally applied cyclic stretching and compressive forces, after creating an artificial focal adhesion under a glass bead attached to the cantilever of an atomic force microscope. The cell tension initially increased in response to the tensile stress and then decreased within ∼1 min as a result of viscoelastic properties of the cell. This relaxation was followed by a gradual increase in tension extending over several minutes. The slow recovery of tension ceased after several cycles of force application. This tension-recovering activity was inhibited when cells were treated with cytochalasin D, an inhibitor of actin polymerization, or with (-)-blebbistatin, an inhibitor of myosin II ATPase activity, suggesting that the activity was driven by actin-myosin interaction. To our knowledge, this is the first quantitative analysis of cellular mechanical properties during the process of adaptation to locally applied cyclic external force.

Topics: Animals; Cells, Cultured; Cytochalasin D; Elasticity; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Humans; Microscopy, Atomic Force; Rats; Stress, Mechanical; Viscosity

The enhanced migration found in tumor cells is often caused by external stimuli and the sequential participation of cytoskeleton-related signaling molecules. However, until now, the molecular connection between the lysophosphatidic acid (LPA) receptor and nonmuscle myosin II (NM II) has not been analyzed in detail for LPA-induced migration. Here, we demonstrate that LPA induces migration by activating the LPA(1) receptor which promotes phosphorylation of the 20 kDa NM II light chain through activation of Rho kinase (ROCK). We show that LPA-induced migration is insensitive to pertussis toxin (PTX) but does require the LPA(1) receptor as determined by siRNA and receptor antagonists. LPA activates ROCK and also increases GTP-bound RhoA activity, concomitant with the enhanced membrane recruitment of RhoA. LPA-induced migration and invasion are attenuated by specific inhibitors including C3 cell-permeable transferase and Y-27632. We demonstrate that NM II plays an important role in LPA-induced migration and invasion by inhibiting its cellular function with blebbistatin and shRNA lentivirus directed against NM II-A or II-B. Inhibition or loss of either NM II-A or NM II-B in 4T1 cells results in a decrease in migration and invasion. Restoration of the expression of NM II-A or NM II-B also rescued LPA-induced migration. Taken together, these results suggest defined pathways for signaling through the LPA(1) receptor to promote LPA-mediated NM II activation and subsequent cell migration in 4T1 breast cancer cells.

Topics: Amides; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Enzyme Inhibitors; Female; Heterocyclic Compounds, 4 or More Rings; Lysophospholipids; Mice; Myosin Light Chains; Pertussis Toxin; Phosphorylation; Pyridines; Receptors, Lysophosphatidic Acid; rho-Associated Kinases; rhoA GTP-Binding Protein; RNA, Small Interfering; Signal Transduction

Epithelial invagination in many model systems is driven by apical cell constriction, mediated by actin and myosin II contraction regulated by GTPase activity. Here we investigate apical constriction during chick lens placode invagination. Inhibition of actin polymerization and myosin II activity by cytochalasin D or blebbistatin prevents lens invagination. To further verify if lens placode invaginate through apical constriction, we analyzed the role of Rho-ROCK pathway. Rho GTPases expression at the apical portion of the lens placode occurs with the same dynamics as that of the cytoskeleton. Overexpression of the pan-Rho inhibitor C3 exotoxin abolished invagination and had a strong effect on apical myosin II enrichment and a mild effect on apical actin localization. In contrast, pharmacological inhibition of ROCK activity interfered significantly with apical enrichment of both actin and myosin. These results suggest that apical constriction in lens invagination involves ROCK but apical concentration of actin and myosin are regulated through different pathways upstream of ROCK. genesis 49:368-379, 2011.

Topics: Actins; Actomyosin; ADP Ribose Transferases; Amides; Animals; Botulinum Toxins; Chick Embryo; Chickens; Cytochalasin D; Cytoskeleton; Ectoderm; Enzyme Inhibitors; Female; Fluorescent Antibody Technique; Heterocyclic Compounds, 4 or More Rings; Immunohistochemistry; Lens, Crystalline; Myosin Type II; Nucleic Acid Synthesis Inhibitors; Pyridines; rho GTP-Binding Proteins; rho-Associated Kinases; Signal Transduction

A characteristic of integrins is their ability to transfer chemical and mechanical signals across the plasma membrane. Force generated by myosin II makes cells able to sense substrate stiffness and induce maturation of nascent adhesions into focal adhesions. In this paper, we present a comprehensive proteomic analysis of nascent and mature adhesions. The purification of integrin adhesion complexes combined with quantitative mass spectrometry enabled the identification and quantification of known and new adhesion-associated proteins. Furthermore, blocking adhesion maturation with the myosin II inhibitor blebbistatin markedly impaired the recruitment of LIM domain proteins to integrin adhesion sites. This suggests a common recruitment mechanism for a whole class of adhesion-associated proteins, involving myosin II and the zinc-finger-type LIM domain.

Topics: Animals; Cell Adhesion; Cells, Cultured; Extracellular Matrix Proteins; Focal Adhesions; Heterocyclic Compounds, 4 or More Rings; Integrins; Intracellular Signaling Peptides and Proteins; Mass Spectrometry; Mice; Myosin Type II; Proteomics; Signal Transduction; Zinc Fingers

Cell elongation is a fundamental process that allows cells and tissues to adopt new shapes and functions. During notochord tubulogenesis in the ascidian Ciona intestinalis, a dramatic elongation of individual cells takes place that lengthens the notochord and, consequently, the entire embryo. We find a novel dynamic actin- and non-muscle myosin II-containing constriction midway along the anteroposterior aspect of each notochord cell during this process. Both actin polymerization and myosin II activity are required for the constriction and cell elongation. Discontinuous localization of myosin II in the constriction indicates that the actomyosin network produces local contractions along the circumference. This reveals basal constriction by the actomyosin network as a novel mechanism for cell elongation. Following elongation, the notochord cells undergo a mesenchymal-epithelial transition and form two apical domains at opposite ends. Extracellular lumens then form at the apical surfaces. We show that cortical actin and Ciona ezrin/radixin/moesin (ERM) are essential for lumen formation and that a polarized network of microtubules, which contributes to lumen development, forms in an actin-dependent manner at the apical cortex. Later in notochord tubulogenesis, when notochord cells initiate a bi-directional crawling movement on the notochordal sheath, the microtubule network rotates 90° and becomes organized as parallel bundles extending towards the leading edges of tractive lamellipodia. This process is required for the correct organization of actin-based protrusions and subsequent lumen coalescence. In summary, we establish the contribution of the actomyosin and microtubule networks to notochord tubulogenesis and reveal extensive crosstalk and regulation between these two cytoskeleton components.

Topics: Animals; Bridged Bicyclo Compounds, Heterocyclic; Ciona intestinalis; Cytoskeleton; Heterocyclic Compounds, 4 or More Rings; Immunohistochemistry; Microtubules; Morphogenesis; Nocodazole; Notochord; Thiazolidines

Focal adhesions (FAs) have key roles in the interaction of cells with the extracellular matrix (ECM) and in adhesion-mediated signaling. These dynamic, multi-protein structures sense the ECM both chemically and physically, and respond to external and internal forces by changing their size and signaling activity. However, this mechanosensitivity is still poorly understood at the molecular level. Here, we present direct evidence that actomyosin contractility regulates the molecular kinetics of FAs. We show that the molecular turnover of proteins within FAs is primarily regulated by their dissociation rate constant (k(off)), which is sensitive to changes in forces applied to the FA. We measured the early changes in k(off) values for three FA proteins (vinculin, paxillin and zyxin) upon inhibition of actomyosin-generated forces using two methods - high temporal resolution FRAP and direct measurement of FA protein dissociation in permeabilized cells. When myosin II contractility was inhibited, the k(off) values for all three proteins changed rapidly, in a highly protein-specific manner: dissociation of vinculin from FAs was facilitated, whereas dissociation of paxillin and zyxin was attenuated. We hypothesize that these early kinetic changes initiate FA disassembly by affecting the molecular turnover of FAs and altering their composition.

Topics: Actomyosin; Cell Line, Tumor; Cytoskeletal Proteins; Fluorescence Recovery After Photobleaching; Focal Adhesions; Glycoproteins; Heterocyclic Compounds, 4 or More Rings; Humans; Marine Toxins; Oxazoles; Paxillin; Vinculin; Zyxin

After cardiac injury, activated cardiac myofibroblasts can influence tissue electrophysiology. Because mechanical coupling through adherens junctions provides a route for intercellular communication, we tested the hypothesis that myofibroblasts exert tonic contractile forces on the cardiomyocytes and affect electric propagation via a process of mechanoelectric feedback.. The role of mechanoelectric feedback was examined in transforming growth factor-β-treated monolayers of cocultured myofibroblasts and neonatal rat ventricular cells by inhibiting myofibroblast contraction and blocking mechanosensitive channels. Untreated (control) and transforming growth factor-β-treated (fibrotic) anisotropic monolayers were optically mapped for electrophysiological comparison. Longitudinal conduction velocity, transverse conduction velocity, and normalized action potential upstroke velocity (dV/dt(max)) significantly decreased in fibrotic monolayers (14.4 ± 0.7 cm/s [mean ± SEM], 4.1 ± 0.3 cm/s [n=53], and 3.1 ± 0.2% per ms [n=14], respectively) compared with control monolayers (27.2 ± 0.8 cm/s, 8.5 ± 0.4 cm/s [n=40], and 4.9 ± 0.1% per ms [n=12], respectively). Application of the excitation-contraction uncoupler blebbistatin or the mechanosensitive channel blocker gadolinium or streptomycin dramatically increased longitudinal conduction velocity, transverse conduction velocity, and dV/dt(max) in fibrotic monolayers (35.9 ± 1.5 cm/s, 10.3 ± 0.6 cm/s [n=17], and 4.5 ± 0.1% per ms [n=14], respectively). Similar results were observed with connexin43-silenced cardiac myofibroblasts. Spiral-wave induction in fibrotic monolayers also decreased after the aforementioned treatments. Finally, traction force measurements of individual myofibroblasts showed a significant increase with transforming growth factor-β, a decrease with blebbistatin, and no change with mechanosensitive channel blockers.. These observations suggest that myofibroblast-myocyte mechanical interactions develop during cardiac injury, and that cardiac conduction may be impaired as a result of increased mechanosensitive channel activation owing to tension applied to the myocyte by the myofibroblast.

Topics: Animals; Animals, Newborn; Biomechanical Phenomena; Cell Communication; Cells, Cultured; Coculture Techniques; Electric Conductivity; Feedback, Physiological; Fibrosis; Gadolinium; Heterocyclic Compounds, 4 or More Rings; Intercellular Junctions; Models, Animal; Myocardium; Myocytes, Cardiac; Myofibroblasts; Rats; Rats, Sprague-Dawley; Streptomycin; Transforming Growth Factor beta

Assessment of mitochondrial ADP-stimulated respiratory kinetics in PmFBs (permeabilized fibre bundles) is increasingly used in clinical diagnostic and basic research settings. However, estimates of the Km for ADP vary considerably (~20-300 μM) and tend to overestimate respiration at rest. Noting that PmFBs spontaneously contract during respiration experiments, we systematically determined the impact of contraction, temperature and oxygenation on ADP-stimulated respiratory kinetics. BLEB (blebbistatin), a myosin II ATPase inhibitor, blocked contraction under all conditions and yielded high Km values for ADP of >~250 and ~80 μM in red and white rat PmFBs respectively. In the absence of BLEB, PmFBs contracted and the Km for ADP decreased ~2-10-fold in a temperature-dependent manner. PmFBs were sensitive to hyperoxia (increased Km) in the absence of BLEB (contracted) at 30 °C but not 37 °C. In PmFBs from humans, contraction elicited high sensitivity to ADP (Km<100 μM), whereas blocking contraction (+BLEB) and including a phosphocreatine/creatine ratio of 2:1 to mimic the resting energetic state yielded a Km for ADP of ~1560 μM, consistent with estimates of in vivo resting respiratory rates of <1% maximum. These results demonstrate that the sensitivity of muscle to ADP varies over a wide range in relation to contractile state and cellular energy charge, providing evidence that enzymatic coupling of energy transfer within skeletal muscle becomes more efficient in the working state.

Topics: Adenosine Diphosphate; Adult; Animals; Creatine; Heterocyclic Compounds, 4 or More Rings; Humans; Male; Mitochondria, Muscle; Muscle Contraction; Muscle Fibers, Skeletal; Muscle, Skeletal; Myosins; Phosphocreatine; Rats; Respiration; Temperature

It is becoming increasingly evident that the micromechanics of cells and their environment determine cell fate and function as much as soluble molecular factors do. We hypothesized that extracellular matrix proteolysis by membrane type 1 matrix metalloproteinase (MT1-MMP) depends on adhesion, force generation and rigidity sensing of the cell. Melanoma cells (MV3 clone) stably transfected with MT1-MMP, or the empty vector as a control, served as the model system. α2β1 integrins (cell adhesion), actin and myosin II (force generation and rigidity sensing) were blocked by their corresponding inhibitors (α2β1 integrin antibodies, Cytochalasin D, blebbistatin). A novel, anisotropic matrix array of parallel, fluorescently labeled collagen-I fibrils was used. Cleavage and bundling of the collagen-I fibrils, and spreading and durotaxis of the cells on this matrix array could be readily discerned and quantified by a combined set-up for fluorescence and atomic force microscopy. In short, expression of the protease resulted in the generation of structural matrix defects, clearly indicated by gaps in the collagen lattice and loose fiber bundles. This key feature of matrix remodeling depended essentially on the functionality of α2β1 integrin, the actin filament network and myosin II motor activity. Interference with any of these negatively impacted matrix cleavage and three-dimensional matrix entanglement of cells.

Topics: Anisotropy; Cell Adhesion; Cell Line, Tumor; Collagen Type I; Cytochalasin D; Extracellular Matrix; Heterocyclic Compounds, 4 or More Rings; Humans; Integrin alpha2beta1; Matrix Metalloproteinase 14; Matrix Metalloproteinase Inhibitors; Microscopy, Atomic Force; Microscopy, Fluorescence; Myosin Type II; Stress, Mechanical

Blebbistatin, a myosin II inhibitor, interferes with myosin-actin interaction and microtubule assembly. By influencing cytoskeletal dynamics blebbistatin counteracts apoptosis of several types of nucleated cells. Even though lacking nuclei and mitochondria, erythrocytes may undergo suicidal cell death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include energy depletion and osmotic shock, which enhance cytosolic Ca(2+) activity with subsequent Ca(2+)-sensitive cell shrinkage and cell membrane scrambling. The present study explored the effect of blebbistatin on eryptosis. Cell membrane scrambling was estimated from binding of annexin V to phosphatidylserine at the erythrocyte surface, cell volume from forward scatter in fluorescence-activated cell sorting analysis and cytosolic Ca(2+) concentration from Fluo3 fluorescence. Exposure to blebbistatin on its own (1-50 μM) did not significantly modify cytosolic Ca(2+) concentration, forward scatter, or annexin V binding. Glucose depletion (48 h) was followed by a significant increase of Fluo3 fluorescence and annexin V binding, effects significantly blunted by blebbistatin (Fluo3 fluorescence ≥ 25 μM, annexin V binding ≥ 10 μM). Osmotic shock (addition of 550 mM sucrose) again significantly increased Fluo3 fluorescence and annexin binding, effects again significantly blunted by blebbistatin (Fluo3 fluorescence ≥ 25 μM, annexin V binding ≥ 25 μM). The present observations disclose a novel effect of blebbistatin, i.e., an influence on Ca(2+) entry and suicidal erythrocyte death following energy depletion and osmotic shock.

Topics: Analysis of Variance; Annexin A5; Blood Glucose; Calcium; Cell Death; Cell Separation; Dose-Response Relationship, Drug; Energy Metabolism; Erythrocyte Membrane; Erythrocytes; Flow Cytometry; Heterocyclic Compounds, 4 or More Rings; Humans; Microscopy, Confocal; Osmotic Pressure; Phosphatidylserines

We investigate the effect of myosin II inhibition on cell shape and nuclear motility in cultures of mouse radial glia-like neural progenitor and rat glioma C6 cells. Instead of reducing nucleokinesis, the myosin II inhibitor blebbistatin provokes an elongated bipolar morphology and increased nuclear motility in both cell types. When myosin II is active, time-resolved traction force measurements indicate a pulling force between the leading edge and the nucleus of C6 cells. In the absence of myosin II activity, traction forces during nucleokinesis are diminished below the sensitivity threshold of our assay. By visualizing the centrosome position in C6 cells with GFP-centrin, we show that in the presence or absence of myosin II activity, the nucleus tends to overtake or lag behind the centrosome, respectively. We interpret these findings with the help of a simple viscoelastic model of the cytoskeleton consisting active contractile and passive compressed elements.

Topics: Actins; Animals; Cell Nucleus; Cell Polarity; Cell Shape; Cells, Cultured; Centrosome; Cytoskeleton; Elasticity; Heterocyclic Compounds, 4 or More Rings; Mice; Microtubules; Myosin Type II; Neural Stem Cells; Neuroglia; Rats; Stress, Mechanical

Cancer cells are defined by their ability to invade through the basement membrane, a critical step during metastasis. While increased secretion of proteases, which facilitates degradation of the basement membrane, and alterations in the cytoskeletal architecture of cancer cells have been previously studied, the contribution of the mechanical properties of cells in invasion is unclear. Here, we applied a magnetic tweezer system to establish that stiffness of patient tumor cells and cancer cell lines inversely correlates with migration and invasion through three-dimensional basement membranes, a correlation known as a power law. We found that cancer cells with the highest migratory and invasive potential are five times less stiff than cells with the lowest migration and invasion potential. Moreover, decreasing cell stiffness by pharmacologic inhibition of myosin II increases invasiveness, whereas increasing cell stiffness by restoring expression of the metastasis suppressor TβRIII/betaglycan decreases invasiveness. These findings are the first demonstration of the power-law relation between the stiffness and the invasiveness of cancer cells and show that mechanical phenotypes can be used to grade the metastatic potential of cell populations with the potential for single cell grading. The measurement of a mechanical phenotype, taking minutes rather than hours needed for invasion assays, is promising as a quantitative diagnostic method and as a discovery tool for therapeutics. By showing that altering stiffness predictably alters invasiveness, our results indicate that pathways regulating these mechanical phenotypes are novel targets for molecular therapy of cancer.

Topics: Actomyosin; Ascites; Cell Line, Tumor; Cell Movement; Cell Shape; Collagen; Compliance; Drug Combinations; Drug Design; Female; Heterocyclic Compounds, 4 or More Rings; Humans; Laminin; Magnetics; Micromanipulation; Microscopy, Atomic Force; Microspheres; Molecular Targeted Therapy; Myosin Type II; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Ovarian Neoplasms; Proteoglycans; Receptors, Transforming Growth Factor beta; Tumor Cells, Cultured

Viscosity is proposed to modulate diastolic function, but only limited understanding of the source(s) of viscosity exists. In vitro experiments have shown that the proline-glutamic acid-valine-lysine (PEVK) rich element of titin interacts with actin, causing a viscous force in the sarcomere. It is unknown whether this mechanism contributes to viscosity in vivo. We tested the hypothesis that PEVK-actin interaction causes cardiac viscosity and is important in vivo via an integrative physiological study on a unique PEVK knockout (KO) model. Both skinned cardiomyocytes and papillary muscle fibers were isolated from wildtype (WT) and PEVK KO mice and passive viscosity was examined using stretch-hold-release and sinusoidal analysis. Viscosity was reduced by ~60% in KO myocytes and ~50% in muscle fibers at room temperature. The PEVK-actin interaction was not modulated by temperature or diastolic calcium, but was increased by lattice compression. Stretch-hold and sinusoidal frequency protocols on intact isolated mouse hearts showed a smaller, 30-40% reduction in viscosity, possibly due to actomyosin interactions, and showed that microtubules did not contribute to viscosity. Transmitral Doppler echocardiography similarly revealed a 40% decrease in LV chamber viscosity in the PEVK KO in vivo. This integrative study is the first to quantify the influence of a specific molecular (PEVK-actin) viscosity in vivo and shows that PEVK-actin interactions are an important physiological source of viscosity.

Topics: Actins; Actomyosin; Animals; Connectin; Heart Ventricles; Heterocyclic Compounds, 4 or More Rings; Male; Mice; Mice, Knockout; Muscle Proteins; Myocardium; Protein Binding; Protein Kinases; Sarcomeres; Viscosity

Cell division, membrane rigidity, and strong adhesion to a rigid matrix are all promoted by myosin-II, and so multinucleated cells with distended membranes--typical of megakaryocytes (MKs)--seem predictable for low myosin activity in cells on soft matrices. Paradoxically, myosin mutations lead to defects in MKs and platelets. Here, reversible inhibition of myosin-II is sustained over several cell cycles to produce 3- to 10-fold increases in polyploid MK and a number of other cell types. Even brief inhibition generates highly distensible, proplatelet-like projections that fragment readily under shear, as seen in platelet generation from MKs in vivo. The effects are maximized with collagenous matrices that are soft and 2D, like the perivascular niches in marrow rather than 3D or rigid, like bone. Although multinucleation of other primary hematopoietic lineages helps to generalize a failure-to-fission mechanism, lineage-specific signaling with increased polyploidy proves possible and novel with phospho-regulation of myosin-II heavy chain. Label-free mass spectrometry quantitation of the MK proteome uses a unique proportional peak fingerprint (ProPF) analysis to also show upregulation of the cytoskeletal and adhesion machinery critical to platelet function. Myosin-inhibited MKs generate more platelets in vitro and also in vivo from the marrows of xenografted mice, while agonist stimulation activates platelet spreading and integrin αIIbβ3. Myosin-II thus seems a central, matrix-regulated node for MK-poiesis and platelet generation.

Topics: Animals; Blood Platelets; Cell Culture Techniques; Collagen; Heterocyclic Compounds, 4 or More Rings; Humans; Megakaryocytes; Mice; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; Nonmuscle Myosin Type IIA; Phosphorylation; Polyploidy; Proteome; Thrombopoiesis

Cellular motility is the major driving force of numerous biological phenomena including wound healing, immune response, embryogenesis, cancer formation, and metastasis. We studied the response of epithelial FaDu monolayers cultured on gold electrodes of an acoustic resonator (quartz crystal microbalance, QCM) and impedance sensor (electric cell-substrate impedance sensing, ECIS) to externally applied chemical stimuli interfering with cytoskeleton organization. Epithelial cell motility of confluent monolayers is characterized by subtle cell shape changes and variations in the cell-substrate as well as cell-cell distance without net directionality of individual cells. The impact of small molecules such as cytochalasin D, phalloidin, and blebbistatin as well as paclitaxel, nocodazol, and colchicin on actin and microtubules organization was quantified by conventional sensors' readouts and by comparing the noise pattern of the signals which is attributed to cellular dynamics. The responsiveness of noninvasive and label-free techniques relying on cellular dynamics is compared to classical viability assays and changes of the overall impedance of ultrasmall electrodes or acoustic loads of a thickness shear mode resonator. Depending on the agent used, a distinct sensor response was found, which can be used as a fingerprint of the cellular response. Cytoskeletal rearrangements and nuclear integrity were corroborated by fluorescence microscopy and correlated to the readouts of QCM and ECIS.

Topics: Actins; Biosensing Techniques; Cell Line, Tumor; Cell Movement; Cytochalasin D; Cytoskeleton; Electric Impedance; Epithelial Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Paclitaxel; Phalloidine; Quartz Crystal Microbalance Techniques; Tubulin Modulators

Viral particle binding to plasma membrane receptors elicits virus motions, recruits signaling proteins, and triggers membrane bending and fission, finally resulting in endocytic virus uptake. Here we analyze how human adenovirus engages its receptor coxsackievirus adenovirus receptor (CAR) and coreceptor αv integrin to move on the plasma membrane. Virus binding to CAR through fiber knobs gave rise to diffusive motions and actomyosin-2-dependent drifts, while integrin-targeted viruses were spatially more confined. Diffusions, drifts, and confined motions were specifically observed with viral particles that were subsequently internalized. CAR-mediated drifts together with integrin binding supported fiber shedding from adenovirus particles, leading to exposure of the membrane-lytic internal virion protein VI and enhanced viral escape from endosomes. Our results show that adenovirus uncoating is initiated at the plasma membrane by CAR drifting motion and binding to immobile integrins.

Topics: Actins; Actomyosin; Adenoviruses, Human; Animals; Capsid Proteins; Cell Membrane; Coxsackie and Adenovirus Receptor-Like Membrane Protein; Cryoelectron Microscopy; Endocytosis; Fluorescent Antibody Technique; HeLa Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Integrin alphaV; Mice; Microscopy, Confocal; Pseudopodia; Receptors, Virus; Virus Attachment; Virus Internalization; Virus Release; Virus Uncoating

Re-epithelialization in skin wound healing is a process in which epidermal sheets grow and close the wound. Although the actin-myosin system is thought to have a pivotal role in re-epithelialization, its role is not clear. In fish skin, re-epithelialization occurs around 500 μm/h and is 50 times faster than in mammalian skin. We had previously reported that leading-edge cells of the epidermal outgrowth have both polarized large lamellipodia and "purse string"-like actin filament cables in the scale-skin culture system of medaka fish, Oryzias latipes (Cell Tissue Res, 2007). The actin purse-string (APS) is a supracellular contractile machinery in which adherens junctions (AJs) link intracellular myosin II-including actin cables between neighboring cells. In this study, we developed a modified "face-to-face" scale-skin culture system as an ex vivo model to study epidermal wound healing, and examined the role of the actin-myosin system in the rapid re-epithelialization using a myosin II ATPase inhibitor, blebbistatin. A low level of blebbistatin suppressed the formation of APS and induced the dissociation of keratocytes from the leading edge without attenuating the growth of the epidermal sheet or the migration rate of solitary keratocytes. AJs in the superficial layer showed no obvious changes elicited by blebbistatin. However, two epidermal sheets without APSs did not make a closure with each other, which was confirmed by inhibiting the connecting AJs between the superficial layers. These results suggest that myosin II activity is required for functional leading-edge cells and for epidermal closure.

Topics: Actins; Animals; Cell Movement; Epidermis; Heterocyclic Compounds, 4 or More Rings; Microscopy, Confocal; Microscopy, Fluorescence; Myosin Type II; Organ Culture Techniques; Oryzias; Phenotype; Pseudopodia; Time Factors; Vinculin; Wound Healing

Mutations in the MYH9 gene, coding for the non-muscle myosin heavy chain IIA (NMHC-IIA), are responsible for syndromes characterized by macrothrombocytopenia associated with deafness, cataracts, and severe glomerular disease. Electron microscopy of renal biopsies from these patients found glomerular abnormalities characterized by alterations in mesangial cells, podocytes, and thickening of the glomerular basement membrane. Knockout of NMHC-IIA in mice is lethal, and therefore little is known about the glomerular-related functions of Myh9. Here, we use zebrafish as a model to study the role and function of zNMHC-IIA in the glomerulus. Knockdown of zNMHC-IIA resulted in malformation of the glomerular capillary tuft characterized by few and dilated capillaries of the pronephros. In zNMHC-IIA morphants, endothelial cells failed to develop fenestrations, mesangial cells were absent or reduced, and the glomerular basement membrane appeared nonuniformly thickened. Knockdown of zNMHC-IIA did not impair the formation of podocyte foot processes or slit diaphragms; however, podocyte processes were less uniform in these morphants compared to controls. In vivo clearance of fluorescent dextran indicated that the glomerular barrier function was not compromised by zNMHC-IIA knockdown; however, glomerular filtration was significantly reduced. Thus, our results demonstrate an important role of zNMHC-IIA for the proper formation and function of the glomerulus in zebrafish.

Topics: Animals; Blood Platelets; Dextrans; Edema; Endothelial Cells; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Genotype; Glomerular Basement Membrane; Glomerular Filtration Rate; Heterocyclic Compounds, 4 or More Rings; Kidney Glomerulus; Larva; Nonmuscle Myosin Type IIA; Permeability; Phenotype; Podocytes; Recombinant Proteins; Time Factors; Zebrafish; Zebrafish Proteins

Mature mammalian oocytes are poised for completing meiosis II (MII) on fertilization by positioning the spindle close to an actomyosin-rich cortical cap. Here, we show that the Arp2/3 complex localizes to the cortical cap in a Ran-GTPase-dependent manner and nucleates actin filaments in the cortical cap and a cytoplasmic actin network. Inhibition of Arp2/3 activity leads to rapid dissociation of the spindle from the cortex. Live-cell imaging and spatiotemporal image correlation spectroscopy analysis reveal that actin filaments flow continuously away from the Arp2/3-rich cortex, driving a cytoplasmic streaming expected to exert a net pushing force on the spindle towards the cortex. Arp2/3 inhibition not only diminishes this actin flow and cytoplasmic streaming but also enables a reverse streaming driven by myosin-II-based cortical contraction, moving the spindle away from the cortex. Thus, the asymmetric MII spindle position is dynamically maintained as a result of balanced forces governed by the Arp2/3 complex.

Topics: Actin Cytoskeleton; Actin-Related Protein 2; Actin-Related Protein 2-3 Complex; Actin-Related Protein 3; Animals; Cells, Cultured; Cytoplasmic Streaming; Female; Fourier Analysis; Heterocyclic Compounds, 4 or More Rings; Indoles; Kymography; Meiosis; Mice; Microscopy, Confocal; Microscopy, Video; Morpholinos; Myosin Type II; Nocodazole; Oocytes; Protein Transport; ran GTP-Binding Protein; Recombinant Fusion Proteins; Spindle Apparatus; Time Factors

Partial bladder outlet obstruction (PBOO), a common urologic pathology mostly caused by benign prostatic hyperplasia, can coexist in 40-45% of patients with overactive bladder (OAB) and is associated with detrusor overactivity (DO). PBOO that induces DO results in alteration in bladder myosin II type and isoform composition. Blebbistatin (BLEB) is a myosin II inhibitor we recently demonstrated potently relaxed normal detrusor smooth muscle (SM) and reports suggest varied BLEB efficacy for different SM myosin (SMM) isoforms and/or SMM vs nonmuscle myosin (NMM). We hypothesize BLEB inhibition of myosin II as a novel contraction protein targeted strategy to regulate DO. Using a surgically-induced male rat PBOO model, organ bath contractility, competitive and Real-Time-RT-PCR were performed. It was found that obstructed-bladder weight significantly increased 2.74-fold while in vitro contractility of detrusor to various stimuli was impaired ∼50% along with decreased shortening velocity. Obstruction also altered detrusor spontaneous activities with significantly increased amplitude but depressed frequency. PBOO switched bladder from a phasic-type to a more tonic-type SM. Expression of 5' myosin heavy chain (MHC) alternatively spliced isoform SM-A (associated with tonic-type SM) increased 3-fold while 3' MHC SM1 and essential light chain isoform MLC(17b) also exhibited increased relative expression. Total SMMHC expression was decreased by 25% while the expression of NMM IIB (SMemb) was greatly increased by 4.5-fold. BLEB was found to completely relax detrusor strips from both sham-operated and PBOO rats pre-contracted with KCl, carbachol or electrical field stimulation although sensitivity was slightly decreased (20%) only at lower doses for PBOO. Thus we provide the first thorough characterization of the response of rat bladder myosin to PBOO and demonstrate complete BLEB-induced PBOO bladder SM relaxation. Furthermore, the present study provides valuable evidence that BLEB may be a novel type of potential therapeutic agent for regulation of myogenic and nerve-evoked DO in OAB.

Topics: Animals; Carbachol; Disease Models, Animal; Gene Expression Regulation; Heterocyclic Compounds, 4 or More Rings; In Vitro Techniques; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Myosin Type II; Protein Isoforms; Rats; Urinary Bladder; Urinary Bladder Neck Obstruction

Maintaining proper adhesion between neighboring cells depends on the ability of cells to mechanically respond to tension at cell-cell junctions through the actin cytoskeleton. Thus, identifying the molecules involved in responding to cell tension would provide insight into the maintenance, regulation, and breakdown of cell-cell junctions during various biological processes. Vinculin, an actin-binding protein that associates with the cadherin complex, is recruited to cell-cell contacts under increased tension in a myosin II-dependent manner. However, the precise role of vinculin at force-bearing cell-cell junctions and how myosin II activity alters the recruitment of vinculin at quiescent cell-cell contacts have not been demonstrated.. We generated vinculin knockdown cells using shRNA specific to vinculin and MDCK epithelial cells. These vinculin-deficient MDCK cells form smaller cell clusters in a suspension than wild-type cells. In wound healing assays, GFP-vinculin accumulated at cell-cell junctions along the wound edge while vinculin-deficient cells displayed a slower wound closure rate compared to vinculin-expressing cells. In the presence of blebbistatin (myosin II inhibitor), vinculin localization at quiescent cell-cell contacts was unaffected while in the presence of jasplakinolide (F-actin stabilizer), vinculin recruitment increased in mature MDCK cell monolayers.. These results demonstrate that vinculin plays an active role at adherens junctions under increased tension at cell-cell contacts where vinculin recruitment occurs in a myosin II activity-dependent manner, whereas vinculin recruitment to the quiescent cell-cell junctions depends on F-actin stabilization.

Topics: Actins; Animals; Antineoplastic Agents; Cadherins; Cell Adhesion; Cell Line; Cytoskeleton; Depsipeptides; Dogs; Heterocyclic Compounds, 4 or More Rings; Myosin Type II; Protein Transport; RNA Interference; RNA, Small Interfering; Vinculin

Human MCF-7/6 breast cancer cells differ from their MCF-7/AZ counterparts by their invasiveness in a number of assays in vitro, such as invasion of MCF-7 spheroids into embryonic chick heart fragments or type I collagen gels. Comparative proteomic analysis of these two variants revealed an identical pattern, except for a 230 kDa protein present in the invasive MCF-7/6 variant, but hardly detectable in the non-invasive MCF-7/AZ one. This protein appeared to be the non-muscle myosin IIA heavy chain (NMIIA), also coined MYH9. Experimental inhibition of NMIIA by reducing either its expression (via stable shRNA transduction) or its function (via the specific ATPase inhibitor blebbistatin) underpinned the decisive role of NMIIA in MCF-7 cell invasion. Inhibition of NMIIA indeed blocked the invasion of MCF-7/6 cells in three-dimensional invasion substrata such as embryonic chick heart fragments and type I collagen gels. Invasiveness of MCF-7/6 cells has been related to poor formation and compaction of aggregates, due to a functionally defective E-cadherin/catenin complex. Both genetic and pharmacological inhibition of NMIIA stimulated MCF-7/6 cell aggregation. Together, these data indicate that NMIIA is a decisive protein for MCF-7 cells to invade, indicating that this molecule is a candidate for targeted anti-invasive treatment.

Topics: Animals; Base Sequence; Breast Neoplasms; Cell Aggregation; Cell Line, Tumor; Chick Embryo; Female; Gene Knockdown Techniques; Heterocyclic Compounds, 4 or More Rings; Humans; Molecular Motor Proteins; Myosin Heavy Chains; Neoplasm Invasiveness; RNA, Small Interfering; Spheroids, Cellular; Tumor Stem Cell Assay

The membrane proximal α helix of integrin β subunit cytoplasmic tails plays an important functional role by interacting with various intracellular proteins, namely talin, α-actinin or skelemin. This study was designed to investigate the functional role of 5 highly conserved charged amino acids (R(724), K(725), E(726), E(731), E(733)) within this α helix by site-directed mutagenesis. The result showed that CHO cells expressing the αIIbβ3E726Q mutant had the most prominent phenotype and characterized by defective cell spreading on immobilized fibrinogen. In addition, this E726Q mutation induced membrane blebbing in cells adherent on fibrinogen, and this blebbing could be inhibited by the myosin light chain ATPase inhibitor blebbistatin. It is concluded that the membrane proximal α-helix of integrin β3 subunit is important in linking the phospholipid membrane to the submembraneous actin cortex.

Topics: Animals; Cell Surface Extensions; CHO Cells; Cricetinae; Cricetulus; Heterocyclic Compounds, 4 or More Rings; Integrin beta3; Mutagenesis, Site-Directed; Mutation; Protein Structure, Tertiary

We have used optical tweezers to identify the elementary events underlying force generation in neuronal lamellipodia. When an optically trapped bead seals on the lamellipodium membrane, Brownian fluctuations decrease revealing the underlying elementary events. The distribution of bead velocities has long tails with frequent large positive and negative values associated to forward and backward jumps occurring in 0.1-0.2 ms with varying amplitudes up to 20 nm. Jump frequency and amplitude are reduced when actin turnover is slowed down by the addition of 25 nM Jasplakinolide. When myosin II is inhibited by the addition of 20 μM Blebbistatin, jump frequency is reduced but to a lesser extent than by Jasplainolide. These jumps constitute the elementary events underlying force generation.

Topics: Actins; Animals; Biomechanical Phenomena; Biophysical Phenomena; Cell Movement; Depsipeptides; Ganglia, Spinal; Heterocyclic Compounds, 4 or More Rings; Myosin Type II; Neurons; Optical Tweezers; Pseudopodia; Rats; Rats, Wistar

The function and mechanism of macropinocytosis in cells outside of the immune system remain poorly understood. We used a neuroblastoma cell line, Neuro-2a, to study macropinocytosis in neuronal cells. We found that phorbol 12-myristate 13-acetate (PMA) and insulin-like growth factor 1 (IGF-1) induced two distinct types of macropinocytosis in the Neuro-2a cells. IGF-1-induced macropinocytosis occurs mostly around the cell bodies and requires phosphoinositide 3-kinase (PI3K), while PMA-induced macropinocytosis occurs predominantly in the neurites and is independent of PI3K activity. Both types of macropinocytosis were inhibited by a specific inhibitor of nonmuscle myosin II, blebbistatin. siRNA knockdown of nonmuscle myosin II isoforms, -IIA and -IIB, resulted in opposite effects on macropinocytosis induced by PMA or IGF. Myosin IIA knockdown significantly increased, whereas myosin IIB knockdown significantly decreased, macropinocytosis with correlating changes in membrane ruffle formation.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Fluorescent Antibody Technique; Heterocyclic Compounds, 4 or More Rings; Insulin-Like Growth Factor I; Mice; Nonmuscle Myosin Type IIB; Pinocytosis; Polymethacrylic Acids; Protein Isoforms

Three-dimensional (3D) cell-matrix cultures provide a useful model to analyze and dissect the structural, functional, and mechanical aspects of cell-matrix interactions and motile behavior important for cell and tissue morphogenesis. In the current studies we tested the effects of serum and physiological growth factors on the morphogenetic behavior of human fibroblasts cultured on the surfaces of 3D collagen matrices. Fibroblasts in medium containing serum contracted into clusters, whereas cells in medium containing platelet-derived growth factor (PDGF) were observed to migrate as individuals. The clustering activity of serum appeared to depend on lysophosphatidic acid, required cell contraction based on inhibition by blocking Rho kinase or myosin II, and was reversed upon switching to PDGF. Oncogenic Ras transformed human fibroblasts did not exhibit serum-stimulated cell clustering. Our findings emphasize the importance of cell-specific promigratory and procontractile growth factor environments in the differential regulation of cell motile function and cell morphogenesis.

Topics: Actins; Amides; Cell Aggregation; Cell Culture Techniques; Cell Line, Transformed; Cell Movement; Cell Shape; Collagen; Enzyme Inhibitors; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Humans; Intercellular Signaling Peptides and Proteins; Lysophospholipids; Male; Myosin Type II; Oncogene Protein p21(ras); Platelet-Derived Growth Factor; Pyridines; rho-Associated Kinases; Serum; Sphingosine; Vinculin

We have studied the spatiotemporal pattern of blebbistatin-induced anomalous electrical activities in isolated rat atrial preparations using the optical mapping of excitation spread. Atrial preparations including the right or left auricle were dissected from adult rat hearts. Each preparation was then stained with a fast merocyanine-rhodanine voltage-sensitive dye (NK2761). Using a multi-element (16 x 16) photodiode array, we assessed the spread of excitation optically by timing the initiation of the action potential-related extrinsic absorption changes. The contraction-related optical signals were suppressed by adding (S)-(-)-blebbistatin (10-100 miocroM) to the bathing solution. Blebbistatin had an effective delay time of about 1.5 h following its application, at which time anomalous electrical activities occurred. These took the form of triggered activities and rhythmical spontaneous excitations. We optically mapped the spatiotemporal patterns of the excitation spread during these anomalous electrical activities. When the triggered activities occurred, the site of ectopic focus, where the triggered action potential first appeared, and the area of excitation spread varied in every event. When the rhythmical spontaneous excitations occurred, the excitation spread from the anomalous pacemaker and, occasionally, their spatial shift was observed. In addition, the combination pattern of the spontaneous excitations and triggered activities was also observed. We suggest that these phenomena are due to the disturbed intracellular calcium dynamics induced by the application of blebbistatin.

Topics: Action Potentials; Animals; Arrhythmias, Cardiac; Body Surface Potential Mapping; Female; Heart Atria; Heart Conduction System; Heterocyclic Compounds, 4 or More Rings; Male; Microscopy, Fluorescence; Rats; Rats, Wistar

Purpose. To determine the role of actin cytoskeleton in the disassembly and reformation of adherens junctions (AJs) and tight junctions (TJs) in bovine corneal endothelial monolayers. Methods. Disassembly and reformation of AJs and TJs were induced by extracellular Ca(2+) depletion and subsequent add-back of Ca(2+), respectively. Resultant changes in the transendothelial electrical resistance (TER), an indicator of integrity of TJs, were measured based on electrical cell-substrate impedance. Phosphorylated myosin light chain (ppMLC), a biochemical measure of actomyosin contraction, and activation of its upstream regulatory molecule RhoA-GTP were assessed by Western blot analysis. Results. Extracellular Ca(2+) depletion led to activation of RhoA, increase in ppMLC, decrease in TER, contraction of the perijunctional actomyosin ring (PAMR), and redistribution of zonula occludens-1 (ZO-1) and cadherins. These effects were reversed on Ca(2+) add-back. Pretreatment with Y-27632 and blebbistatin (as inhibitors of actomyosin contraction) reduced the rate of decline in TER, opposed the contraction of the PAMR, and blocked the redistribution of ZO-1 and cadherins. Both drugs reduced the recovery in TER and opposed the normal redistribution of ZO-1 and cadherins on Ca(2+) add-back. Cytochalasin D, which led to dissolution of the PAMR, also reduced the recovery of TER on Ca(2+) add-back. Conclusions. The (Ca(2+) depletion)-induced disassembly of AJs accelerates the breakdown of TJs through a concomitant increase in the actomyosin contraction of the PAMR. However, these data on reassembly show that a contractile tone of the PAMR is essential for assembly of the apical junctional complex.

Topics: Actins; Actomyosin; Adherens Junctions; Amides; Animals; Blotting, Western; Cadherins; Calcium; Cattle; Cell Culture Techniques; Electric Impedance; Endothelium, Corneal; Fluorescent Antibody Technique, Indirect; Heterocyclic Compounds, 4 or More Rings; Membrane Proteins; Myosin Light Chains; Phosphoproteins; Phosphorylation; Pyridines; rhoA GTP-Binding Protein; Tight Junctions; Zonula Occludens-1 Protein

To study how the dynamic subcellular mechanical properties of the heart relate to the fundamental underlying process of actin-myosin cross-bridge cycling, we developed a novel atomic force microscope elastography technique for mapping spatiotemporal stiffness of isolated, spontaneously beating neonatal rat cardiomyocytes. Cells were indented repeatedly at a rate close but unequal to their contractile frequency. The resultant changes in pointwise apparent elastic modulus cycled at a predictable envelope frequency between a systolic value of 26.2 +/- 5.1 kPa and a diastolic value of 7.8 +/- 4.1 kPa at a representative depth of 400 nm. In cells probed along their major axis, spatiotemporal changes in systolic stiffness displayed a heterogeneous pattern, reflecting the banded sarcomeric structure of underlying myofibrils. Treatment with blebbistatin eliminated contractile activity and resulted in a uniform apparent modulus of 6.5 +/- 4.8 kPa. This study represents the first quantitative dynamic mechanical mapping of beating cardiomyocytes. The technique provides a means of probing the micromechanical effects of disease processes and pharmacological treatments on beating cardiomyocytes, providing new insights and relating subcellular cardiac structure and function.

Topics: Actins; Animals; Biomechanical Phenomena; Cells, Cultured; Heterocyclic Compounds, 4 or More Rings; Microscopy, Atomic Force; Models, Biological; Myocytes, Cardiac; Myofibrils; Myosins; Rats; Rats, Sprague-Dawley; Time Factors

Blebbistatin, an inhibitor of myosin-II-specific ATPase, has been used to inhibit contraction of invertebrate and mammalian muscle preparations containing non-muscle myosin. Activated hepatic stellate cells have contractile properties and play an important role in the pathophysiology of liver fibrosis and portal hypertension. Therefore, hepatic stellate cells are considered as therapeutic target cells. In the present study, we studied the effect of blebbistatin during the transition of mouse hepatic stellate cells into contractile myofibroblasts.. Effects of blebbistatin on cell morphology were evaluated by phase contrast microscopy. Cell stress fibres and focal adhesions were investigated by dual immunofluorescence staining and visualized using fluorescence microscopy. Contractile force generation was examined by silicone wrinkle formation assays and collagen gel contraction assays. Intracellular Ca(2+) release in response to endothelin-1 was measured by using Fluo-4. Cell migration was measured by wound healing experiments.. In culture-activated hepatic stellate cells, blebbistatin was found to change both cell morphology and function. In the presence of blebbistatin, stellate cells became smaller, acquired a dendritic morphology and had less myosin IIA-containing stress fibres and vinculin-containing focal adhesions. Moreover, blebbistatin impaired silicone wrinkle formation, reduced collagen gel contraction and blocked endothelin-1-induced intracellular Ca(2+) release. Finally, it promoted wound-induced cell migration.. By inhibiting myosin II ATPase, blebbistatin has profound effects on the morphology and function of activated hepatic stellate cells. Our data suggest that myosin II could be a therapeutic target in the treatment of liver fibrosis and portal hypertension.

Topics: Actins; Animals; Cell Movement; Cell Size; Cell Transdifferentiation; Cells, Cultured; Hepatic Stellate Cells; Heterocyclic Compounds, 4 or More Rings; Mice; Myosin Type II; Stress Fibers

The motor protein nonmuscle myosin II (NMII) through its interaction with the actin cytoskeleton constitutes the machinery of cell crawling and has an important role in driving locomotion and infiltration of immune competent cells during inflammatory response and immune reaction. Blebbistatin is a highly selective inhibitor of NMII adenosine triphosphatase. This study examined the effect of NMII inhibition by blebbistatin on inflammation. In vitro, blebbistatin markedly induced actinomyosin complex disassembly in various cultured immunocytes, and functionally impaired their motile activity and invasive capacity as assessed by the Boyden chamber motility assay and the matrigel invasion assay. In vivo, in a rat model of acute inflammation induced by tumor necrosis factor, blebbistatin obliterated renal sequestration of circulating fluorescence-labeled macrophages in a dose-dependent fashion. Moreover, in rats with progressive obstructive nephropathy, blebbistatin treatment exhibited a remarkable renoprotective effect, as evidenced by normalized kidney weight, improved gross morphology, and diminished histologic injury in the tubulointerstitium. This beneficial effect was associated with significant amelioration of renal inflammation, consistent with a primary anti-inflammatory action by blebbistatin. In addition, in rats with established obstructive nephropathy, blebbistatin pretreated macrophages showed obliterated recruitment into the inflamed renal parenchyma, denoting that blebbistatin directly impedes inflammatory infiltration by immunocytes. Collectively, our findings suggest that inhibition of NMII has a potent and direct anti-inflammatory effect on the basis of impairment of the actinomyosin powered locomotive machinery, which is essential for migration and infiltration of immune competent cells.

Topics: Actins; Animals; Cell Movement; Heterocyclic Compounds, 4 or More Rings; Humans; Jurkat Cells; Kidney; Macrophages; Male; Nephritis; Nonmuscle Myosin Type IIA; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha; Ureteral Obstruction

Maintaining a physical connection across cytoplasm is crucial for many biological processes such as matrix force generation, cell motility, cell shape and tissue development. However, in the absence of stress fibers, the coherent structure that transmits force across the cytoplasm is not understood. We find that nonmuscle myosin-II (NMII) contraction of cytoplasmic actin filaments establishes a coherent cytoskeletal network irrespective of the nature of adhesive contacts. When NMII activity is inhibited during cell spreading by Rho kinase inhibition, blebbistatin, caldesmon overexpression or NMIIA RNAi, the symmetric traction forces are lost and cell spreading persists, causing cytoplasm fragmentation by membrane tension that results in 'C' or dendritic shapes. Moreover, local inactivation of NMII by chromophore-assisted laser inactivation causes local loss of coherence. Actin filament polymerization is also required for cytoplasmic coherence, but microtubules and intermediate filaments are dispensable. Loss of cytoplasmic coherence is accompanied by loss of circumferential actin bundles. We suggest that NMIIA creates a coherent actin network through the formation of circumferential actin bundles that mechanically link elements of the peripheral actin cytoskeleton where much of the force is generated during spreading.

Topics: Actins; Animals; Blotting, Western; Cells, Cultured; Cytoskeleton; Fluorescent Antibody Technique; Heterocyclic Compounds, 4 or More Rings; Mice; NIH 3T3 Cells; Nonmuscle Myosin Type IIA; rho-Associated Kinases

The utilization of motor proteins for the movement and assembly of synthetic components is currently a goal of nanoengineering research. Application of the myosin actin motor system for nanotechnological uses has been hampered due to the low flexural rigidity of individual F-actin filaments. Here it is demonstrated how actin bundling can be used to affect the translational behavior of myosin-propelled filaments, transport molecules across a motor-patterned surface, and that the movement of bundled actin can be regulated photonically. These data suggest that actin bundling may significantly improve the applicability of the myosin motor for future nanotechnological applications.

Topics: Actin Cytoskeleton; Actins; Animals; Biological Transport; Chickens; Heterocyclic Compounds, 4 or More Rings; Humans; Light; Myosins; Nanoparticles; Rabbits

During cell migration, forces generated by the actin cytoskeleton are transmitted through adhesion complexes to the substrate. To investigate the mechanism of force generation and transmission, we analyzed the relationship between actin network velocity and traction forces at the substrate in a model system of persistently migrating fish epidermal keratocytes. Front and lateral sides of the cell exhibited much stronger coupling between actin motion and traction forces than the trailing cell body. Further analysis of the traction-velocity relationship suggested that the force transmission mechanisms were different in different cell regions: at the front, traction was generated by a gripping of the actin network to the substrate, whereas at the sides and back, it was produced by the network's slipping over the substrate. Treatment with inhibitors of the actin-myosin system demonstrated that the cell body translocation could be powered by either of the two different processes, actomyosin contraction or actin assembly, with the former associated with significantly larger traction forces than the latter.

Topics: Actin Cytoskeleton; Animals; Biomechanical Phenomena; Cell Adhesion; Cell Movement; Cell Polarity; Cell Shape; Cells, Cultured; Cytochalasin D; Cytoskeleton; Fishes; Heterocyclic Compounds, 4 or More Rings; Keratinocytes; Models, Biological; Myosins; Protein Synthesis Inhibitors; Stress, Mechanical; Tensile Strength

To explore the precise mechanisms of the inhibitory effects of blebbistatin, a potent inhibitor of myosin II, on smooth muscle contraction, we studied the blebbistatin effects on the mechanical properties and the structure of contractile filaments of skinned (cell membrane permeabilized) preparations from guinea pig taenia cecum. Blebbistatin at 10 microM or higher suppressed Ca(2+)-induced tension development at any given Ca(2+) concentration but had little effects on the Ca(2+)-induced myosin light chain phosphorylation. Blebbistatin also suppressed the 10 and 2.75 mM Mg(2+)-induced, "myosin light chain phosphorylation-independent" tension development at more than 10 microM. Furthermore, blebbistatin induced conformational change of smooth muscle myosin (SMM) and disrupted arrangement of SMM and thin filaments, resulting in inhibition of actin-SMM interaction irrespective of activation with Ca(2+). In addition, blebbistatin partially inhibited Mg(2+)-ATPase activity of native actomyosin from guinea pig taenia cecum at around 10 microM. These results suggested that blebbistatin suppressed skinned smooth muscle contraction through disruption of structure of SMM by the agent.

Topics: Actin Cytoskeleton; Actins; Animals; Calcium; Cecum; Fluorescence Resonance Energy Transfer; Guinea Pigs; Heterocyclic Compounds, 4 or More Rings; Magnesium; Male; Muscle Contraction; Muscle, Smooth; Myosin Type II; Protein Transport; Smooth Muscle Myosins

The cytoskeleton is an important factor in the functional and structural adaption of cells to mechanical forces. In this study we investigated the impact of microtubules and the acto-myosin machinery on the kinetics of force-induced reorientation of NIH3T3 fibroblasts. These cells were subjected to uniaxial stretching forces that are known to induce cellular reorientation perpendicular to the stretch direction. We found that disruption of filamentous actin using cytochalasin D and latrunculin B as well as an induction of a massive unpolarized actin polymerization by jasplakinolide, inhibited the stretch-induced reorientation. Similarly, blocking of myosin II activity abolished the stretch-induced reorientation of cells but, interestingly, increased their motility under stretching conditions in comparison to myosin-inhibited nonstretched cells. Investigating the contribution of microtubules to the cellular reorientation, we found that, although not playing a significant role in reorientation itself, microtubule stability had a significant impact on the kinetics of this event. Overall, we conclude that acto-myosin, together with microtubules, regulate the kinetics of force-induced cell reorientation.

Topics: Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Cell Movement; Cell Polarity; Cells, Cultured; Cytochalasin D; Depsipeptides; Heterocyclic Compounds, 4 or More Rings; Kinetics; Mice; Microtubules; NIH 3T3 Cells; Nocodazole; Paclitaxel; Structure-Activity Relationship; Thiazolidines

Plasma membrane depolarization activates the Rho/Rho kinase (ROK) pathway and thereby enhances myosin light chain (MLC) phosphorylation, which in turn is thought to be a key regulator of paracellular permeability. However, the upstream mechanisms that couple depolarization to Rho activation and permeability changes are unknown. Here we show that three different depolarizing stimuli (high extracellular K(+) concentration, the lipophilic cation tetraphenylphosphonium, or l-alanine, which is taken up by electrogenic Na(+) cotransport) all provoke robust phosphorylation of ERK in LLC-PK1 and Madin-Darby canine kidney (MDCK) cells. Importantly, inhibition of ERK prevented the depolarization-induced activation of Rho. Searching for the underlying mechanism, we have identified the GTP/GDP exchange factor GEF-H1 as the ERK-regulated critical exchange factor responsible for the depolarization-induced Rho activation. This conclusion is based on our findings that 1) depolarization activated GEF-H1 but not p115RhoGEF, 2) short interfering RNA-mediated GEF-H1 silencing eliminated the activation of the Rho pathway, and 3) ERK inhibition prevented the activation of GEF-H1. Moreover, we found that the Na(+)-K(+) pump inhibitor ouabain also caused ERK, GEF-H1, and Rho activation, partially due to its depolarizing effect. Regarding the functional consequences of this newly identified pathway, we found that depolarization increased paracellular permeability in LLC-PK1 and MDCK cells and that this effect was mitigated by inhibiting myosin using blebbistatin or a dominant negative (phosphorylation incompetent) MLC. Taken together, we propose that the ERK/GEF-H1/Rho/ROK/pMLC pathway could be a central mechanism whereby electrogenic transmembrane transport processes control myosin phosphorylation and regulate paracellular transport in the tubular epithelium.

Topics: Alanine; Animals; Butadienes; Calcium; Dogs; Enzyme Activation; Enzyme Inhibitors; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Guanine Nucleotide Exchange Factors; Heterocyclic Compounds, 4 or More Rings; Kidney Tubules; LLC-PK1 Cells; Membrane Potentials; Myosin Light Chains; Nitriles; Onium Compounds; Organophosphorus Compounds; Ouabain; Permeability; Phosphorylation; Potassium; Protein Kinase Inhibitors; ras Proteins; rho GTP-Binding Proteins; Sodium-Potassium-Exchanging ATPase; Swine; Time Factors; Transfection

Polymerization of microfilaments and their subsequent rearrangements under control of actin-myosin interactions are two main processes underlined morphogenetic reactions of cells. We studied their role during spreading of normal and transformed REF52tetRas fibroblasts with adjustable ras oncogene expression. Treatment with inhibitors of cell contractility (Y27532 or blebbistatin) led to disappearance of actin bundles and focal adhesions, but both normal and transformed cells preserved high pseudopodial activity. Spreading was considerably accelerated in normal cells and less accelerated in ras-transformed cells under these conditions. When actin polymerization was suppressed with low concentrations of latrunculin A, stress-fibrills and focal contacts were preserved, but lamellipodial activity was lost in normal cells, so spreading was dramatically inhibited. In the case of transformed fibroblasts, actin bundles and focal adhesions virtually disappeared, but pseudopofial activity was not lost and spreading was suppressed to a lesser extent. Therefore, the most essential process in regulation of cell spreading and polarization is microfilament polymerization at the leading edge. Incidentally, ras-transformed cells are less sensitive to inhibitors affecting cytoskeletal structure than non-transformed ones. Possible mechanisms underlying these diversities are discussed.

Topics: Actin Cytoskeleton; Actins; Animals; Cell Adhesion; Cell Line; Cell Line, Transformed; Enzyme Inhibitors; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Mice; Myosins; rho-Associated Kinases

Focal adhesions (FAs) are mechanosensitive adhesion and signaling complexes that grow and change composition in response to myosin II-mediated cytoskeletal tension in a process known as FA maturation. To understand tension-mediated FA maturation, we sought to identify proteins that are recruited to FAs in a myosin II-dependent manner and to examine the mechanism for their myosin II-sensitive FA association. We find that FA recruitment of both the cytoskeletal adapter protein vinculin and the tyrosine kinase FA kinase (FAK) are myosin II and extracellular matrix (ECM) stiffness dependent. Myosin II activity promotes FAK/Src-mediated phosphorylation of paxillin on tyrosines 31 and 118 and vinculin association with paxillin. We show that phosphomimic mutations of paxillin can specifically induce the recruitment of vinculin to adhesions independent of myosin II activity. These results reveal an important role for paxillin in adhesion mechanosensing via myosin II-mediated FAK phosphorylation of paxillin that promotes vinculin FA recruitment to reinforce the cytoskeletal ECM linkage and drive FA maturation.

Topics: Amides; Animals; Cells, Cultured; Extracellular Matrix; Fibroblasts; Fibronectins; Focal Adhesions; Heterocyclic Compounds, 4 or More Rings; Mice; Myosin Type II; Paxillin; Phosphorylation; Protein-Tyrosine Kinases; Pyridines; Structure-Activity Relationship; Vinculin

The zebrafish is an emerging model system for the study of cardiac electrophysiology and human arrhythmias. High resolution imaging techniques are powerful tools for the study of zebrafish cardiac electrophysiology, but these methods require the complete absence of cardiac contraction. Many pharmacological agents that uncouple cardiac contraction also markedly alter the cardiac action potential (AP). In this study, we compared the effects two uncoupling agents, 2,3-Butanedione monoxime (BDM) and blebbistatin, on contractility and AP parameters in embryonic zebrafish heart.. Zebrafish hearts were explanted (48 hpf) and superfused with either BDM (15 mM) or blebbistatin (1, 5 or 10 microM), while recording atrial or ventricular APs with the disrupted patch technique. Calcium transients were recorded with a high-speed confocal scanning microscope in hearts loaded intracellularly with 10 microM fluo-4 and superfused with 10 microM blebbistatin.. Despite abolishing cardiac contractility, BDM altered ventricular AP morphology and inhibited spontaneous APs. In contrast, blebbistatin (10 microM) abolished contractility without significantly altering AP morphology or generation of spontaneous APs. Blebbistatin allowed for high fidelity measurements of atrial and ventricular calcium transients.. Blebbistatin is a potent and effective excitation-contraction uncoupling agent in embryonic zebrafish heart.

Topics: Action Potentials; Animals; Diacetyl; Heart; Heterocyclic Compounds, 4 or More Rings; Myocardial Contraction; Zebrafish

The powerstroke of the myosin motor is the basis of cell division and bodily movement, but has eluded empirical description due to the short lifetime and low abundance of intermediates during force generation. To gain insight into this process, we used well-established single-tryptophan and pyrene fluorescent sensors and electron microscopy to characterize the structural and kinetic properties of myosin complexed with ADP and blebbistatin, a widely used inhibitor. We found that blebbistatin does not weaken the tight actin binding of myosin.ADP, but unexpectedly it induces lever priming, a process for which the gamma-phosphate of ATP (or its analog) had been thought necessary. The results indicate that a significant fraction of the myosin.ADP.blebbistatin complex populates a previously inaccessible conformation of myosin resembling the start of the powerstroke.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Binding Sites; Dictyostelium; Fluorescent Dyes; Heterocyclic Compounds, 4 or More Rings; Kinetics; Microscopy, Electron; Microscopy, Fluorescence; Models, Biological; Molecular Conformation; Myosins; Protein Conformation; Rabbits

Podosomes are spot-like actin-rich structures formed at the ventral surface of monocytic and haematopoietic cells. Podosomes degrade extracellular matrix and are proposed to be involved in cell migration. A key question is whether podosomes form protrusions similar to the invadopodia of cancer cells. We characterised podosomes of immature dendritic cells using electron microscopy combined with both conventional and novel high-resolution structured illumination light microscopy. Dendritic cell podosomes are composed of actin foci surrounded by a specialised ring region that is rich in material containing paxillin. We found that podosomes were preferential sites for protrusion into polycarbonate filters impregnated with crosslinked gelatin, degrading up to 2 micrometers of matrix in 24 hours. Podosome-associated uptake of colloidal gold-labelled gelatin matrix appeared to occur via large phagosome-like structures or narrow tubular invaginations. The motor protein myosin-II was excluded from ring or core regions but was concentrated around them and the myosin-II inhibitor Blebbistatin reduced the length of podosome protrusions. Finally, we found that degradation, protrusion and endocytosis in this system are dependent on the matrix metalloproteinase MMP-14. We propose that podosomes mediate migration of dendritic cells through tissues by means of myosin-II-dependent protrusion coupled to MMP-14-dependent degradation and endocytosis.

Topics: Animals; Cell Surface Extensions; Cross-Linking Reagents; Dendritic Cells; Endocytosis; Extracellular Matrix; Gelatin; Gene Knockout Techniques; Gold; Heterocyclic Compounds, 4 or More Rings; Matrix Metalloproteinase 14; Mice; Microscopy, Electron; Models, Biological; Myosin Type II; Polycarboxylate Cement; Porosity

Contraction of detrusor smooth muscle (DSM) at short muscle lengths generates a stiffness component we termed adjustable passive stiffness (APS) that is retained in tissues incubated in a Ca(2+)-free solution, shifts the DSM length-passive tension curve up and to the left, and is softened by muscle strain and release (strain softened). In the present study, we tested the hypothesis that APS is due to slowly cycling actomyosin cross bridges. APS and active tension produced by the stimulus, KCl, displayed similar length dependencies with identical optimum length values. The myosin II inhibitor blebbistatin relaxed active tension maintained during a KCl-induced contraction and the passive tension maintained during stress-relaxation induced by muscle stretch in a Ca(2+)-free solution. Passive tension was attributed to tension maintaining rather than tension developing cross bridges because tension did not recover after a rapid 10% stretch and release as it did during a KCl-induced contraction. APS generated by a KCl-induced contraction in intact tissues was preserved in tissues permeabilized with Triton X-100. Blebbistatin and the actin polymerization inhibitor latrunculin-B reduced the degree of APS generated by a KCl-induced contraction. The degree of APS generated by KCl was inhibited to a greater degree than was the peak KCl-induced tension by rhoA kinase and cyclooxygenase inhibitors. These data support the hypothesis that APS is due to slowly cycling actomyosin cross bridges and suggest that cross bridges may play a novel role in DSM that uniquely serves to ensure proper contractile function over an extreme working length range.

Topics: Actomyosin; Animals; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cyclooxygenase Inhibitors; Detergents; Excitation Contraction Coupling; Female; Heterocyclic Compounds, 4 or More Rings; In Vitro Techniques; Muscle Contraction; Muscle, Smooth; Myosin Type II; Myosins; Octoxynol; Potassium Chloride; Prostaglandin-Endoperoxide Synthases; Protein Kinase Inhibitors; Rabbits; rho-Associated Kinases; Thiazolidines; Time Factors; Urinary Bladder

Internalization of the obligate intracellular apicomplexan parasite, Cryptosporidium parvum, results in the formation of a unique intramembranous yet extracytoplasmic niche on the apical surfaces of host epithelial cells, a process that depends on host cell membrane extension. We previously demonstrated that efficient C. parvum invasion of biliary epithelial cells (cholangiocytes) requires host cell actin polymerization and localized membrane translocation/insertion of Na(+)/glucose cotransporter 1 (SGLT1) and of aquaporin 1 (Aqp1), a water channel, at the attachment site. The resultant localized water influx facilitates parasite cellular invasion by promoting host-cell membrane protrusion. However, the molecular mechanisms by which C. parvum induces membrane translocation/insertion of SGLT1/Aqp1 are obscure. We report here that cultured human cholangiocytes express several nonmuscle myosins, including myosins IIA and IIB. Moreover, C. parvum infection of cultured cholangiocytes results in the localized selective aggregation of myosin IIB but not myosin IIA at the region of parasite attachment, as assessed by dual-label immunofluorescence confocal microscopy. Concordantly, treatment of cells with the myosin light chain kinase inhibitor ML-7 or the myosin II-specific inhibitor blebbistatin or selective RNA-mediated repression of myosin IIB significantly inhibits (P < 0.05) C. parvum cellular invasion (by 60 to 80%). Furthermore ML-7 and blebbistatin significantly decrease (P < 0.02) C. parvum-induced accumulation of SGLT1 at infection sites (by approximately 80%). Thus, localized actomyosin-dependent membrane translocation of transporters/channels initiated by C. parvum is essential for membrane extension and parasite internalization, a phenomenon that may also be relevant to the mechanisms of cell membrane protrusion in general.

Topics: Aquaporin 1; Bile Ducts; Blotting, Western; Cell Line; Cryptosporidiosis; Cryptosporidium parvum; Fluorescent Antibody Technique; Heterocyclic Compounds, 4 or More Rings; Host-Parasite Interactions; Humans; Microscopy, Electron, Scanning; Nonmuscle Myosin Type IIA; Nonmuscle Myosin Type IIB; Reverse Transcriptase Polymerase Chain Reaction; Sodium-Glucose Transporter 1

Like other positive-strand RNA viruses, alphaviruses replicate their genomes in association with modified intracellular membranes. Alphavirus replication sites consist of numerous bulb-shaped membrane invaginations (spherules), which contain the double-stranded replication intermediates. Time course studies with Semliki Forest virus (SFV)-infected cells were combined with live-cell imaging and electron microscopy to reveal that the replication complex spherules of SFV undergo an unprecedented large-scale movement between cellular compartments. The spherules first accumulated at the plasma membrane and were then internalized using an endocytic process that required a functional actin-myosin network, as shown by blebbistatin treatment. Wortmannin and other inhibitors indicated that the internalization of spherules also required the activity of phosphatidylinositol 3-kinase. The spherules therefore represent an unusual type of endocytic cargo. After endocytosis, spherule-containing vesicles were highly dynamic and had a neutral pH. These primary carriers fused with acidic endosomes and moved long distances on microtubules, in a manner prevented by nocodazole. The result of the large-scale migration was the formation of a very stable compartment, where the spherules were accumulated on the outer surfaces of unusually large and static acidic vacuoles localized in the pericentriolar region. Our work highlights both fundamental similarities and important differences in the processes that lead to the modified membrane compartments in cells infected by distinct groups of positive-sense RNA viruses.

Topics: Actins; Androstadienes; Animals; Cell Line; Cell Membrane; Cricetinae; Endocytosis; Enzyme Inhibitors; Heterocyclic Compounds, 4 or More Rings; Lysosomes; Microtubules; Nocodazole; Phosphatidylinositol 3-Kinases; RNA, Viral; Semliki forest virus; Viral Proteins; Virus Replication; Wortmannin

Crawling locomotion of eukaryotic cells is achieved by a process dependent on the actin cytoskeleton: protrusion of the leading edge requires assembly of a network of actin filaments, which must be disassembled at the cell rear for sustained motility. Although ADF/cofilin proteins have been shown to contribute to actin disassembly, it is not clear how activity of these locally acting proteins could be coordinated over the distance scale of the whole cell. Here we show that non-muscle myosin II has a direct role in actin network disassembly in crawling cells. In fish keratocytes undergoing motility, myosin II is concentrated in regions at the rear with high rates of network disassembly. Activation of myosin II by ATP in detergent-extracted cytoskeletons results in rear-localized disassembly of the actin network. Inhibition of myosin II activity and stabilization of actin filaments synergistically impede cell motility, suggesting the existence of two disassembly pathways, one of which requires myosin II activity. Our results establish the importance of myosin II as an enzyme for actin network disassembly; we propose that gradual formation and reorganization of an actomyosin network provides an intrinsic destruction timer, enabling long-range coordination of actin network treadmilling in motile cells.

Topics: Actins; Adenosine Triphosphate; Animals; Cell Movement; Cichlids; Cytoskeleton; Depsipeptides; Detergents; Epithelial Cells; Heterocyclic Compounds, 4 or More Rings; Myosin Type II; Protein Binding; Protein Transport

Several myosin isotypes are discussed to be involved in the migration of various cells ranging from tumor cells to leukocytes. We investigated the involvement of myosins II and VI in the lymphoid leukemia cells lines Jurkat, NB4, Dohh-2, and Molt-4 by a three-dimensional, collagen-based migration assay. Down-regulation of myosin VI by siRNA significantly reduced the migratory activity of all cells, whereas the pharmacological inhibition of non-muscle myosin II using blebbistatin had only marginal influence. Therefore, in contrast to differentiated leukocytes and cells from solid tumors, myosin VI plays a crucial role in the migration of leukemic cells.

Topics: Cell Movement; Heterocyclic Compounds, 4 or More Rings; Humans; Jurkat Cells; K562 Cells; Leukemia, Lymphoid; Myosin Heavy Chains; Myosin Type II; Neoplasm Proteins; RNA, Small Interfering

S100A4, a member of the S100 family of Ca(2+)-binding proteins, is directly involved in tumor metastasis. In addition to its expression in tumor cells, S100A4 is expressed in normal cells and tissues, including fibroblasts and cells of the immune system. To examine the contribution of S100A4 to normal physiology, we established S100A4-deficient mice by gene targeting. Homozygous S100A4(-/-) mice are fertile, grow normally and exhibit no overt abnormalities; however, the loss of S100A4 results in impaired recruitment of macrophages to sites of inflammation in vivo. Consistent with these observations, primary bone marrow macrophages (BMMs) derived from S100A4(-/-) mice display defects in chemotactic motility in vitro. S100A4(-/-) BMMs form unstable protrusions, overassemble myosin-IIA, and exhibit altered colony-stimulating factor-1 receptor signaling. These studies establish S100A4 as a regulator of physiological macrophage motility and demonstrate that S100A4 mediates macrophage recruitment and chemotaxis in vivo.

Topics: Actomyosin; Animals; Bone Marrow Cells; Cell Count; Cell Surface Extensions; Chemotaxis; Cytoskeleton; Heterocyclic Compounds, 4 or More Rings; Humans; Inflammation; Macrophage Colony-Stimulating Factor; Macrophages; Mice; Mice, Knockout; Models, Biological; Receptor, Macrophage Colony-Stimulating Factor; S100 Calcium-Binding Protein A4; S100 Proteins; Signal Transduction

Arrhythmias are benign or lethal, depending on their sustainability and frequency. To determine why lethal arrhythmias are prone to occur in diseased hearts, usually characterized by nonuniform muscle contraction, we investigated the effect of nonuniformity on sustainability and frequency of triggered arrhythmias.. Force, membrane potential, and intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured in 51 rat ventricular trabeculae. Nonuniform contraction was produced by exposing a restricted region of muscle to a jet of 20 mmol/L 2,3-butanedione monoxime (BDM) or 20 mumol/L blebbistatin. Sustained arrhythmias (>10 seconds) could be induced by stimulus trains for 7.5 seconds only with the BDM or blebbistatin jet (100 nmol/L isoproterenol, 1.0 mmol/L [Ca(2+)](o), 24 degrees C). During sustained arrhythmias, Ca(2+) surges preceded synchronous increases in [Ca(2+)](i), whereas the stoppage of the BDM jet made the Ca(2+) surges unclear and arrested sustained arrhythmias (n=6). With 200 nmol/L isoproterenol, 2.5 mmol/L [Ca(2+)](o), and the BDM jet, lengthening or shortening of the muscle during sustained arrhythmias accelerated or decelerated their cycle in both the absence (n=10) and presence (n=10) of 100 mumol/L streptomycin, a stretch-activated channel blocker, respectively. The maximum rate of force relaxation correlated inversely with the change in cycle lengths (n=14; P<0.01). Sustained arrhythmias with the BDM jet were significantly accelerated by 30 mumol/L SCH00013, a Ca(2+) sensitizer of myofilaments (n=10).. These results suggest that nonuniformity of muscle contraction is an important determinant of the sustainability and frequency of triggered arrhythmias caused by the surge of Ca(2+) dissociated from myofilaments in cardiac muscle.

Topics: Actin Cytoskeleton; Animals; Arrhythmias, Cardiac; Calcium Signaling; Diacetyl; Heart; Heart Ventricles; Heterocyclic Compounds, 4 or More Rings; Membrane Potentials; Myocardial Contraction; Rats

Connexin-assembled gap junctions (GJs) and hemichannels coordinate intercellular signaling processes. Although the regulation of connexins in GJs has been well characterized, the molecular determinants controlling connexin-hemichannel activity are unresolved. Here we investigated the regulation of Cx43-hemichannel activity by actomyosin contractility and intracellular [Ca(2+)] ([Ca(2+)](i)) using plasma membrane-permeable TAT peptides (100 μM) designed to interfere with interactions between the cytoplasmic loop (CL) and carboxy-terminal (CT) in primary bovine corneal endothelial cells and HeLa, C6 glioma, and Xenopus oocytes ectopically expressing Cx43. Peptides corresponding to the last 10 CT aa (TAT-Cx43CT) prevented the inhibition of Cx43-hemichannel activity by contractility/high [Ca(2+)](i), whereas a reverse peptide (TAT-Cx43CTrev) did not. These effects were independent of zonula occludens-1, a cytoskeletal-associated Cx43-binding protein. In contrast, peptides corresponding to CL (TAT-L2) inhibited Cx43-hemichannel responses, whereas a mutant peptide (TAT-L2(H126K/I130N)) did not inhibit. In these assays, TAT-Cx43CT acted as a scaffold for TAT-L2 and vice versa, a finding supported by surface plasmon resonance measurements. Loop/tail interactions appeared essential for Cx43-hemichannel activity, because TAT-Cx43CT restored the activity of nonfunctional hemichannels, consisting of either Cx43 lacking the C-terminal tail (Cx43(M239)) or intact Cx43 ectopically expressed in Xenopus oocytes. We conclude that intramolecular loop/tail interactions control Cx43-hemichannel activity, laying the basis for developing hemichannel-specific blockers.

Topics: Adenosine Triphosphate; Animals; Calcium; Cattle; Cell Line; Cell Line, Tumor; Connexin 43; Cornea; Endothelial Cells; Gene Products, tat; HeLa Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Intracellular Space; Ion Channels; Oocytes; Protein Binding; Rats; Thrombin; Xenopus laevis

Enucleation of mammalian erythroblasts is a process whose mechanism is largely undefined. The prevailing model suggests that nuclear extrusion occurs via asymmetric cytokinesis. To test this hypothesis, we treated primary erythroblasts with inhibitors of cytokinesis, including blebbistatin, hesperadin, and nocodazole, and then assayed for enucleation. Although these agents inhibited cell-cycle progression and subsequent enucleation when added early in culture, they failed to block enucleation proper when added to postmitotic cells. These results suggest that contraction of the actomyosin ring is not essential for nuclear expulsion. Next, by ultrastructural examination of primary erythroblasts, we observed an accumulation of vacuoles in the cytoplasm proximal to the extruding nucleus. This finding led us to hypothesize that vesicle trafficking contributes to erythroblast enucleation. Here, we show that chemical inhibitors of vesicle trafficking block enucleation of primary erythroblasts without affecting differentiation, cell division, or apoptosis. Moreover, knock-down of clathrin inhibited the enucleation of late erythroblasts. In contrast, vacuolin-1, a small molecule that induces vacuole formation, increased the percentage of enucleated cells. Together, these results illustrate that vesicle trafficking, specifically the formation, movement, and subsequent coalescence of vacuoles at the junction of the nucleus and the cytoplasm, is a critical component of mammalian erythroblast enucleation.

Topics: Animals; Cells, Cultured; Clathrin; Cytokinesis; Endocytosis; Erythroblasts; Erythropoiesis; Gene Knockdown Techniques; Heterocyclic Compounds, 4 or More Rings; Humans; Liver; Mice; Monensin; Nocodazole; Spleen; Vacuoles

Human ESCs are the pluripotent precursor of the three embryonic germ layers. Human ESCs exhibit basal-apical polarity, junctional complexes, integrin-dependent matrix adhesion, and E-cadherin-dependent cell-cell adhesion, all characteristics shared by the epiblast epithelium of the intact mammalian embryo. After disruption of epithelial structures, programmed cell death is commonly observed. If individualized human ESCs are prevented from reattaching and forming colonies, their viability is significantly reduced. Here, we show that actin-myosin contraction is a critical effector of the cell death response to human ESC dissociation. Inhibition of myosin heavy chain ATPase, downregulation of myosin heavy chain, and downregulation of myosin light chain all increase survival and cloning efficiency of individualized human ESCs. ROCK inhibition decreases phosphorylation of myosin light chain, suggesting that inhibition of actin-myosin contraction is also the mechanism through which ROCK inhibitors increase cloning efficiency of human ESCs.

Topics: Actin Cytoskeleton; Actins; Cell Communication; Cell Death; Cell Surface Extensions; Cell Survival; Clone Cells; Embryonic Stem Cells; Gene Knockdown Techniques; Heterocyclic Compounds, 4 or More Rings; Humans; Molecular Motor Proteins; Myosin Heavy Chains; Myosin Light Chains; Myosins; Phosphorylation; rho-Associated Kinases

Epithelial invagination is a fundamental morphogenetic behavior that transforms a flat cell sheet into a pit or groove. Previous studies of invagination have focused on the role of actomyosin-dependent apical contraction; other mechanisms remain largely unexplored.. We combined experimental and computational approaches to identify a two-step mechanism for endoderm invagination during ascidian gastrulation. During Step 1, which immediately precedes invagination, endoderm cells constrict their apices because of Rho/Rho-kinase-dependent apical enrichment of 1P-myosin. Our data suggest that endoderm invagination itself occurs during Step 2, without further apical shrinkage, via a novel mechanism we call collared rounding: Rho/Rho-kinase-independent basolateral enrichment of 1P-myosin drives apico-basal shortening, whereas Rho/Rho-kinase-dependent enrichment of 1P and 2P myosin in circumapical collars is required to prevent apical expansion and for deep invagination. Simulations show that boundary-specific tension values consistent with these distributions of active myosin can explain the cell shape changes observed during invagination both in normal embryos and in embryos treated with pharmacological inhibitors of either Rho-kinase or Myosin II ATPase. Indeed, we find that the balance of strong circumapical and basolateral tension is the only mechanism based on differential cortical tension that can explain ascidian endoderm invagination. Finally, simulations suggest that mesectoderm cells resist endoderm shape changes during both steps, and we confirm this prediction experimentally.. Our findings suggest that early ascidian gastrulation is driven by the coordinated apposition of circumapical and lateral endoderm contraction, working against a resisting mesectoderm. We propose that similar mechanisms may operate during other invaginations.

Topics: Amides; Animals; Endoderm; Heterocyclic Compounds, 4 or More Rings; Models, Biological; Myosins; Pyridines; rhoA GTP-Binding Protein; Urochordata

 Inactivation of the mouse Myh9 gene (Myh9Δ) or its mutation in MYH9-related diseases leads to macrothrombocytopenia. Paradoxically, previous studies using in vitro differentiated megakaryocytes showed an increased capacity for proplatelet formation when myosin was absent or inhibited..  To explore the origin of the thrombocytopenia induced by myosin deficiency, we studied proplatelet formation using bone marrow explants of wild-type (WT) and Myh9Δ mouse where megakaryocytes have matured in their native environment..  A dramatic decrease in the number and complexity of proplatelets was observed in megakaryocytes from Myh9Δ mice, while inhibition of myosin activity by blebbistatin increased proplatelet formation from WT mature megakaryocytes. Moreover, Myh9Δ megakaryocytes had a smaller size than the WT cells. These data indicate that myosin deficiency acts negatively on proplatelet formation, probably by impairing in situ megakaryocyte maturation, while myosin activity is dispensable at the latest stage of proplatelet formation. In addition, ultrastructural examination of Myh9Δ bone marrow revealed an increased proportion of megakaryocytes exhibiting signs of non-apoptotic cell death as compared with the WT mice..  These data indicate that thrombocytopenia in Myh9Δ mice results from defective development of megakaryocyte size, impaired proplatelet formation and increased cell death.

Topics: Animals; Blood Platelets; Bone Marrow; Caspase 3; Cell Death; Cell Lineage; Cell Survival; Female; Heterocyclic Compounds, 4 or More Rings; In Situ Nick-End Labeling; Male; Megakaryocytes; Mice; Microscopy, Electron, Transmission; Mutation; Myosin Heavy Chains; Nonmuscle Myosin Type IIA; Thrombocytopenia

When activated muscle fibers are stretched at low speeds [≤ 2 optimal length (L(o))/s], force increases in two phases, marked by a change in slope [critical force (P(c))] that happens at a critical sarcomere length extension (L(c)). Some studies attribute P(c) to the number of attached cross bridges before stretch, while others attribute it to cross bridges in a pre-power-stroke state. In this study, we reinvestigated the mechanisms of forces produced during stretch by altering either the number of cross bridges attached to actin or the cross-bridge state before stretch. Two sets of experiments were performed: 1) activated fibers were stretched by 3% L(o) at speeds of 1.0, 2.0, and 3.0 L(o)/s in different pCa(2+) (4.5, 5.0, 5.5, 6.0), or 2) activated fibers were stretched by 3% L(o) at 2 L(o)/s in pCa(2+) 4.5 containing either 5 μM blebbistatin(+/-) or its inactive isomer (+/+). All stretches started at a sarcomere length (SL) of 2.5 μm. When fibers were activated at a pCa(2+) of 4.5, P(c) was 2.47 ± 0.11 maximal force developed before stretch (P(o)) and decreased with lower concentrations of Ca(2+). L(c) was not Ca(2+) dependent; the pooled experiments provided a L(c) of 14.34 ± 0.34 nm/half-sarcomere (HS). P(c) and L(c) did not change with velocities of stretch. Fibers activated in blebbistatin(+/-) showed a higher P(c) (2.94 ± 0.17 P(o)) and L(c) (16.30 ± 0.38 nm/HS) than control fibers (P(c) 2.31 ± 0.08 P(o); L(c) 14.05 ± 0.63 nm/HS). The results suggest that forces produced during stretch are caused by both the number of cross bridges attached to actin and the cross bridges in a pre-power-stroke state. Such cross bridges are stretched by large amplitudes before detaching from actin and contribute significantly to the force developed during stretch.

Topics: Animals; Biomechanical Phenomena; Calcium; Heterocyclic Compounds, 4 or More Rings; Muscle Contraction; Muscle Fibers, Skeletal; Muscle, Skeletal; Rabbits; Stress, Mechanical

Tendons attach muscles to bone and thereby transmit tensile forces during joint movement. However, a detailed understanding of the mechanisms that establish the mechanical properties of tendon has remained elusive because of the practical difficulties of studying tissue mechanics in vivo. Here we have performed a study of tendon-like constructs made by culturing embryonic tendon cells in fixed-length fibrin gels. The constructs display mechanical properties (toe-linear-fail stress-strain curve, stiffness, ultimate tensile strength, and failure strain) as well as collagen fibril volume fraction and extracellular matrix (ECM)/cell ratio that are statistically similar to those of embryonic chick metatarsal tendons. The development of mechanical properties during time in culture was abolished when the constructs were treated separately with Triton X-100 (to solubilise membranes), cytochalasin (to disassemble the actin cytoskeleton) and blebbistatin (a small molecule inhibitor of non-muscle myosin II). Importantly, these treatments had no effect on the mechanical properties of the constructs that existed prior to treatment. Live-cell imaging and (14)C-proline metabolic labeling showed that blebbistatin inhibited the contraction of the constructs without affecting cell viability, procollagen synthesis, or conversion of procollagen to collagen. In conclusion, the mechanical properties per se of the tendon constructs are attributable to the ECM generated by the cells but the improvement of mechanical properties during time in culture was dependent on non-muscle myosin II-derived forces.

Topics: Actin Cytoskeleton; Actins; Animals; Biomechanical Phenomena; Cell Count; Cell Movement; Cell Survival; Chick Embryo; Cytochalasin B; Elastic Modulus; Extracellular Matrix; Fibrillar Collagens; Heterocyclic Compounds, 4 or More Rings; Metatarsus; Myosins; Nonmuscle Myosin Type IIA; Nonmuscle Myosin Type IIB; Octoxynol; Procollagen; Tendons; Tensile Strength; Tissue Engineering

Human pluripotent stem (hPS) cells such as human embryonic stem (hES) and induced pluripotent stem (hiPS) cells are vulnerable under single cell conditions, which hampers practical applications; yet, the mechanisms underlying this cell death remain elusive. In this paper, we demonstrate that treatment with a specific inhibitor of non-muscle myosin II (NMII), blebbistatin, enhances the survival of hPS cells under clonal density and suspension conditions, and, in combination with a synthetic matrix, supports a fully defined environment for self-renewal. Consistent with this, genetically engineered mouse embryonic stem cells lacking an isoform of NMII heavy chain (NMHCII), or hES cells expressing a short hairpin RNA to knock down NMHCII, show greater viability than controls. Moreover, NMII inhibition increases the expression of self-renewal regulators Oct3/4 and Nanog, suggesting a mechanistic connection between NMII and self-renewal. These results underscore the importance of the molecular motor, NMII, as a novel target for chemically engineering the survival and self-renewal of hPS cells.

Topics: Animals; Cell Line; Cell Survival; Embryonic Stem Cells; Genetic Vectors; Heterocyclic Compounds, 4 or More Rings; Humans; In Situ Nick-End Labeling; Karyotyping; Lentivirus; Mice; Myosin Type II; Pluripotent Stem Cells; RNA, Small Interfering

Cardiomyocytes in vivo are continuously subjected to electrical signals that evoke contractions and instigate drastic changes in the cells' morphology and function. Studies on how electrical stimulation affects the cardiac transcriptome have remained limited to a small number of heart-specific genes. Furthermore, these studies have ignored the interplay between the electrical excitation and the subsequent contractions. We carried out a genomewide assessment of the effects of electrical signaling on gene expression, while distinguishing between the effects deriving from the electrical pulses themselves and the effects instigated by the evoked contractions. Changes in gene expression in primary cultures of neonatal ventricular cardiomyocytes from Lewis Rattus norvegicus were investigated with microarrays and RT-quantitative PCR (QPCR). A series of experiments was included in which the culture medium was supplemented with the contraction inhibitor blebbistatin to allow for electrical stimulation in the absence of contraction. Electrical stimulation was shown to directly enhance calcium handling and induce cardiomyocyte differentiation by arresting cell division and activating key cardiac transcription factors as well as additional differentiation mechanisms such as wnt signaling. Several genes involved in metabolism were also directly activated by electrical stimulation. Furthermore, our data suggest that contraction exerts negative feedback on the transcription of various genes. Together, these observations indicate that intercellular electric currents between adjacent cardiomyocytes have an important role in cardiomyocyte development. They act at least partially through a pulse-specific gene expression program that is activated independently from the evoked contractions.

Topics: Animals; Cell Separation; Electric Stimulation; Female; Gene Expression Profiling; Heterocyclic Compounds, 4 or More Rings; Male; Myocardial Contraction; Myocytes, Cardiac; Rats

Previous studies suggest that Dupuytren's disease is caused by fibroblast and myofibroblast contractility within Dupuytren's nodules; however, the stimulus for cell contractility is unknown. Sphingosine-1-phosphate (S1P) is a serum-derived lysophospholipid mediator that enhances cell contractility by activating the S1P receptor, S1P(2). It is hypothesized that S1P stimulates Dupuytren's fibroblast contractility through S1P(2) activation of non-muscle myosin II (NMMII). This investigation examined the role of S1P and NMMII activation in Dupuytren's disease progression and suggests potential targets for treatment.. We enmeshed Dupuytren's fibroblasts into fibroblast-populated collagen lattices (FPCLs) and assayed S1P-stimulated FPCL contraction in the presence of the S1P(2) receptor inhibitor JTE-013, the Rho kinase inhibitor Y-27632, the myosin light chain kinase inhibitor ML-7, and the NMMII inhibitor blebbistatin. Tissues from Dupuytren's fascia (n = 10) and normal palmar fascia (n = 10) were immunostained for NMMIIA and NMMIIB.. Sphingosine-1-phosphate stimulated FPCL contraction in a dose-dependent manner. Inhibition of S1P(2) and NMMII prevented S1P-stimulated FPCL contraction. Rho kinase and myosin light chain kinase inhibited both S1P and control FPCL contraction. Dupuytren's nodule fibroblasts robustly expressed NMMIIA and NMMIIB, compared with quiescent-appearing cords and normal palmar fascia.. Sphingosine-1-phosphate promotes Dupuytren's fibroblast contractility through S1P(2), which stimulates activation of NMMII. NMMII isoforms are ubiquitously expressed throughout Dupuytren's nodules, which suggests that nodule fibroblasts are primed to respond to S1P stimulation to cause contracture formation. S1P-promoted activation of NMMII may be a target for disease treatment.

Topics: Cell Line; Collagen; Disease Progression; Dupuytren Contracture; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Humans; Immunoenzyme Techniques; Lysophospholipids; Muscle Contraction; Muscle, Smooth; Myosin Type II; Sphingosine

Activation of thin filaments in striated muscle occurs when tropomyosin exposes myosin binding sites on actin either through calcium-troponin (Ca-Tn) binding or by actin-myosin (A-M) strong binding. However, the extent to which these binding events contributes to thin filament activation remains unclear. Here we propose a simple analytical model in which strong A-M binding and Ca-Tn binding independently activates the rate of A-M weak-to-strong binding. The model predicts how the level of activation varies with pCa as well as A-M attachment, N·k(att), and detachment, k(det), kinetics. To test the model, we use an in vitro motility assay to measure the myosin-based sliding velocities of thin filaments at different pCa, N·k(att), and k(det) values. We observe that the combined effects of varying pCa, N·k(att), and k(det) are accurately fit by the analytical model. The model and supporting data imply that changes in attachment and detachment kinetics predictably affect the calcium sensitivity of striated muscle mechanics, providing a novel A-M kinetic-based interpretation for perturbations (e.g. disease-related mutations) that alter calcium sensitivity.

Topics: Actin Cytoskeleton; Actins; Animals; Calcium; Heterocyclic Compounds, 4 or More Rings; Kinetics; Models, Theoretical; Muscle Contraction; Muscle, Skeletal; Mutation; Myosins; Rabbits; Tropomyosin; Troponin

Cancer deaths are primarily caused by metastases, not by the parent tumor. During metastasis, malignant cells detach from the parent tumor, and spread through the circulatory system to invade new tissues and organs. The physical-chemical mechanisms and parameters within the cellular microenvironment that initiate the onset of metastasis, however, are not understood. Here we show that human colon carcinoma (HCT-8) cells can exhibit a dissociative, metastasis-like phenotype (MLP) in vitro when cultured on substrates with appropriate mechanical stiffness. This rather remarkable phenotype is observed when HCT-8 cells are cultured on gels with intermediate-stiffness (physiologically relevant 21-47 kPa), but not on very soft (1 kPa) and very stiff (3.6 GPa) substrates. The cell-cell adhesion molecule E-Cadherin, a metastasis hallmark, decreases 4.73 ± 1.43 times on cell membranes in concert with disassociation. Both specific and nonspecific cell adhesion decrease once the cells have disassociated. After reculturing the disassociated cells on fresh substrates, they retain the disassociated phenotype regardless of substrate stiffness. Inducing E-Cadherin overexpression in MLP cells only partially reverses the MLP phenotype in a minority population of the dissociated cells. This important experiment reveals that E-Cadherin does not play a significant role in the upstream regulation of the mechanosensing cascade. Our results indicate, during culture on the appropriate mechanical microenvironment, HCT-8 cells undergo a stable cell-state transition with increased in vitro metastasis-like characteristics as compared to parent cells grown on standard, very stiff tissue culture dishes. Nuclear staining reveals that a large nuclear deformation (major/minor axis ratio, 2:5) occurs in HCT-8 cells when cells are cultured on polystyrene substrates, but it is markedly reduced (ratio, 1:3) in cells grown on 21 kPa substrates, suggesting the cells are experiencing different intracellular forces when grown on stiff as compared to soft substrates. Furthermore, MLP can be inhibited by blebbistatin, which inactivates myosin II activity and relaxes intracellular forces. This novel finding suggests that the onset of metastasis may, in part, be linked to the intracellular forces and the mechanical microenvironment of the tumor.

Topics: Actins; Biomechanical Phenomena; Cadherins; Cell Adhesion; Cell Line, Tumor; Cell Nucleus; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 4 or More Rings; Humans; Mechanical Phenomena; Neoplasm Metastasis; Phenotype

A new method for studying wound healing under realistic conditions in vitro was developed. The method involves creating defined patterns of damaged cell debris with poly(dimethyl)siloxane (PDMS) stamping. This novel assay permitted the quantification of wound healing rates in the presence of cell debris. Experimental results with this assay suggest that cell migration in the presence of cell debris is a two step process requiring (1) non-muscle myosin II-dependent cell clearance followed by (2) cell migration into newly cleared wound areas. The novel stamp wound assay allows the study of coupled cell migration and debris clearance and is a more realistic wound healing assay in vitro.

Topics: Cell Death; Cell Line; Cell Movement; Cells, Immobilized; Dimethylpolysiloxanes; Epithelial Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Myosin Type II; Time Factors; Wound Healing

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck (1-2). To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Topics: Actins; Cell Adhesion; Cytological Techniques; Cytoskeleton; Drug Evaluation, Preclinical; HeLa Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Myosins; Staining and Labeling

When cells cease migrating through the vasculature, adhere to extracellular matrix, and begin to spread, they exhibit rapid changes in contraction and relaxation at peripheral regions newly contacting the underlying substrata. We describe here a requirement in this process for myosin II disassembly at the cell cortex via the action of myosin phosphatase (MP), which in turn is regulated by a plasma membrane signaling lipid. Cells in suspension exhibit high levels of activity of the signaling enzyme phospholipase D2 (PLD2), elevating production of the lipid second messenger phosphatidic acid (PA) at the plasma membrane, which in turn recruits MP and stores it there in a presumed inactive state. On cell attachment, down-regulation of PLD2 activity decreases PA production, leading to MP release, myosin dephosphorylation, and actomyosin disassembly. This novel model for recruitment and restraint of MP provides a means to effect a rapid cytoskeletal reorganization at the cell cortex upon demand.

Topics: Amides; Animals; Cell Membrane; Cell Movement; CHO Cells; Cricetinae; Cricetulus; Enzyme Inhibitors; Heterocyclic Compounds, 4 or More Rings; Humans; Lipids; Myosin Type II; Myosin-Light-Chain Phosphatase; Phosphatidic Acids; Phospholipase D; Pyridines; rho-Associated Kinases; RNA Interference; Second Messenger Systems

Physiological processes, occurring as a result of specific receptor stimulation, are generally assessed via molecular biology techniques and microscopic approaches with the involvement of specific molecular markers. The recent progress in experimental approaches, allowing the mechanical characterization of individual biological entities, now makes it possible to address cellular processes occurring in individual cells as a result of their stimulation by hormones. Here, we demonstrate that the atomic force microscope (AFM) can be used to mechanically probe individual cells following the activation of the angiotensin-1 receptor, a receptor well known for its role in cell homeostasis regulation. Our goal is to demonstrate that the measurement of cantilever deflection can be used to quantify in real time the mechanical and morphological cell activity associated with the activation of the receptor. By combining the AFM with time-lapse sequences of phase-contrast and confocal micrographs, we show that the angiotensin-1 receptor stimulation with 100 nM angiotensin II produces an actin-dependent contractile response with an amplitude of 262 +/- 52 nm. We validated the mechanical origin of the responses by measuring the elastic modulus of the cell from indentation experiments performed at 30-s intervals. Additionally, nanoscaled height fluctuations of the cell membrane occurring after the initial contraction response could be attributed to an increased actin cytoskeleton activity and remodeling detected by confocal microscopy. Finally, by using inhibitors for specific elements of the angiotensin-1 receptor signaling pathways, we demonstrate that AFM real-time height monitoring allows a read out of the molecular processes responsible for the cell mechanical response.

Topics: Actins; Amides; Angiotensin II; Azepines; Cell Line; Cell Size; Cytological Techniques; Cytoskeleton; Elastic Modulus; Heterocyclic Compounds, 4 or More Rings; Humans; Kidney; Microscopy, Atomic Force; Microscopy, Confocal; Muscle Contraction; Pyridines; Receptor, Angiotensin, Type 1

Sudden cardiac death due to arrhythmia in the settings of chronic myocardial infarction (MI) is an important clinical problem. Arrhythmic risk post-MI continues indefinitely even if heart failure and acute ischemia are not present due to the anatomic substrate of the scar and border zone (BZ) tissue.. The purpose of this study was to determine mechanisms of arrhythmia initiation and termination in a rabbit model of chronic MI.. Ligation of the lateral division of the left circumflex artery was performed 72 +/- 29 days before acute experiments (n = 11). Flecainide (2.13 +/- 0.64 microM) was administered to promote sustained arrhythmias, which were induced with burst pacing or a multiple shock protocol (four pulses, 140-200 ms coupling interval).. Panoramic optical mapping with blebbistatin (5 microM) revealed monomorphic ventricular tachycardia (VT) maintained by a single mother rotor (cycle length [CL] = 174.7 +/- 38.4 ms) as the primary mechanism of arrhythmia. Mother rotors were anchored to the scar or BZ for 16 of the 19 rotor locations recorded. Cardioversion thresholds (CVTs) were determined at various phases throughout the VT CL from external shock electrodes. CVTs were found to be phase dependent, and the maximum versus minimum CVT was 7.8 +/- 1.9 vs. 4.1 +/- 1.6 V/cm, respectively (P = .005). Antitachycardia pacing was found to be effective in only 2.7% of cases in this model.. These results indicate that scar and BZ tissue heterogeneity provide the substrate for VT by attracting and stabilizing rotors. Additionally, a significant reduction in CVT may be achieved by appropriately timed shocks in which the shock-induced virtual electrode polarization interacts with the rotor to destabilize VT.

Topics: Animals; Chronic Disease; Diagnostic Imaging; Disease Models, Animal; Electric Countershock; Female; Heart Rate; Heterocyclic Compounds, 4 or More Rings; Imaging, Three-Dimensional; Male; Myocardial Infarction; Rabbits; Tachycardia, Ventricular

Multilayered fibroblast sheets have applications as cell transplants for tissue engineering. One way to increase their therapeutic efficacy is to increase cell numbers in a graft, but the factors influencing multilayered growth remain poorly understood. In this study, we investigated the roles of focal adhesion (FA) assembly and intercellular cohesion through fibronectin (FN) in the proliferation of normal human fibroblasts at confluence. Density-dependent growth-arrested fibroblasts resumed DNA synthesis when cultured in multilayer formation medium (MFM) containing transforming growth factor-beta1, ascorbic acid, and serum. This proliferation depended on alpha 5 beta 1-integrin-mediated cell-FN-cell interactions because blocking them with antibodies inhibited DNA synthesis. However, cell-FN-cell cohesion operated well regardless of exposure to MFM, judging from several parameters, including FN matrix deposition, activated beta1 integrin expression, and stress fiber development. Density-arrested cells formed few FAs at the cell center. Exposure of the cells to MFM induced the formation of vinculin-, paxillin-, and phosphotyrosine-containing FAs throughout the ventral cell-surface, indicating ROCK-mediated actomyosin contractile force generation. When the assembly of FAs was inhibited with either the ROCK inhibitor Y-27632 or the myosin II inhibitor blebbistatin, the up-regulation of DNA synthesis by MFM was suppressed. The drugs did not impair FN matrix deposition, activated beta1 integrin expression, and stress fiber development. Thus, these results indicate that the formation of FAs promotes the proliferation of confluent fibroblasts with the support of alpha 5 beta 1-integrin-mediated cell-FN-cell cohesion. The present findings provide insights into the rational design of high-density fibroblast transplants.

Topics: Adult; Amides; Animals; Antibodies, Monoclonal; Antioxidants; Ascorbic Acid; Cell Proliferation; Cells, Cultured; Enzyme Inhibitors; Extracellular Matrix; Fibroblasts; Fibronectins; Focal Adhesions; Heterocyclic Compounds, 4 or More Rings; Humans; Integrin alpha5beta1; Middle Aged; Myosin Type II; Paxillin; Pyridines; rho-Associated Kinases; Stress Fibers; Transforming Growth Factor beta1; Vinculin

Mertk is a key phagocytic receptor in the immune, male reproductive, and visual systems. In the retinal pigment epithelium, Mertk is required for the daily ingestion of photoreceptor outer segment (OS) tips. Loss of Mertk function causes retinal degeneration in rats, mice, and humans; however, little is known about the mechanism by which Mertk regulates the ingestion phase of retinal pigment epithelial (RPE) phagocytosis. To address this, the authors sought proteins that associated with Mertk during OS phagocytosis.. Lysates of RPE-J cells challenged with OS for various times were immunoprecipitated with Mertk antibody. Potential interacting proteins were identified by mass spectrometry and characterized with confocal microscopy, pharmacologic inhibition, and siRNA knockdown coupled with an in vitro phagocytic assay in primary RPE cells.. Myh9, the non-muscle myosin II-A heavy chain, was enriched in immunoprecipitates from OS-treated samples. Myosin II-A and II-B isoforms exhibited a striking redistribution in wild-type rat primary RPE cells challenged with OS, moving from the cell periphery to colocalize with ingested OS over time. In contrast, myosin II-A redistribution in response to OS was blunted in primary RPE cells from RCS rats, which lack functional Mertk. Wild-type rat primary RPE cells treated with the myosin II-specific inhibitor blebbistatin or myosin II siRNAs exhibited a significant phagocytic defect.. Mertk mobilizes myosin II from the RPE cell periphery to sites of OS engulfment, where myosin II function is essential for the normal phagocytic ingestion of OS.

Topics: Actins; Animals; c-Mer Tyrosine Kinase; Cattle; Heterocyclic Compounds, 4 or More Rings; Mass Spectrometry; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Nonmuscle Myosin Type IIA; Nonmuscle Myosin Type IIB; Phagocytosis; Proto-Oncogene Proteins; Rats; Rats, Long-Evans; Rats, Mutant Strains; Rats, Sprague-Dawley; Receptor Protein-Tyrosine Kinases; Retinal Pigment Epithelium; RNA, Small Interfering; Rod Cell Outer Segment; Skeletal Muscle Myosins; Transfection

Phosphorylation of skeletal myosin regulatory light chain (RLC) occurs in fatigue and may play a role in the inhibition of shortening velocities observed in vivo. Forces and shortening velocities were measured in permeabilized rabbit psoas fibers with either phosphorylated or dephosphorylated RLCs and in the presence or absence of the myosin inhibitor blebbistatin. Addition of 20 microM blebbistatin decreased tensions by approximately 80% in fibers, independent of phosphorylation. In blebbistatin maximal shortening velocities (V(max)) at 30 degrees C, were decreased by 45% (3.2 +/- 0.34 vs. 5.8 +/- 0.18 lengths/s) in phosphorylated fibers but were not inhibited in dephosphorylated fibers (6.0 +/- 0.30 vs. 5.4 +/- 0.30). In the presence of 20 microM blebbistatin, K(m) for V(max) as a function of [ATP] was lower for phosphorylated fibers than for dephosphorylated fibers (50 +/- 20 vs. 330 +/- 84 microM) indicating that the apparent binding of ATP is stronger in these fibers. Phosphorylation of RLC in situ during fiber preparation or by addition of myosin light chain kinase yielded similar data. RLC phosphorylation inhibited velocity in blebbistatin at both 30 and 10 degrees C, unlike previous reports where RLC phosphorylation only affected shortening velocities at higher temperatures.

Topics: Animals; Heterocyclic Compounds, 4 or More Rings; Muscle Contraction; Muscle Fibers, Skeletal; Myosin Light Chains; Phosphorylation; Rabbits

The roles of nonmuscle myosin II and cortical actin filaments in chromaffin granule exocytosis were studied by confocal fluorescence microscopy, amperometry, and cell-attached capacitance measurements. Fluorescence imaging indicated decreased mobility of granules near the plasma membrane following inhibition of myosin II function with blebbistatin. Slower fusion pore expansion rates and longer fusion pore lifetimes were observed after inhibition of actin polymerization using cytochalasin D. Amperometric recordings revealed increased amperometric spike half-widths without change in quantal size after either myosin II inhibition or actin disruption. These results suggest that actin and myosin II facilitate release from individual chromaffin granules by accelerating dissociation of catecholamines from the intragranular matrix possibly through generation of mechanical forces.

Topics: Actins; Animals; Azepines; Catecholamines; Cattle; Cells, Cultured; Chromaffin Cells; Chromaffin Granules; Cytochalasin D; Electric Capacitance; Exocytosis; Heterocyclic Compounds, 4 or More Rings; Microscopy, Confocal; Myosin Type II; Naphthalenes; Nucleic Acid Synthesis Inhibitors

Interactions between cells and the surrounding matrix are critical to the development and engineering of tissues. We have investigated the role of cell-derived traction forces in the assembly of extracellular matrix using what we believe is a novel assay that allows for simultaneous measurement of traction forces and fibronectin fibril growth at discrete cell-matrix attachment sites. NIH3T3 cells were plated onto arrays of deformable cantilever posts for 2-24 h. Data indicate that developing fibril orientation is guided by the direction of the traction force applied to that fibril. In addition, cells initially establish a spatial distribution of traction forces that is largest at the cell edge and decreases toward the cell center. This distribution progressively shifts from a predominantly peripheral pattern to a more uniform pattern as compressive strain at the cell perimeter decreases with time. The impact of these changes on fibrillogenesis was tested by treating cells with blebbistatin or calyculin A to tonically block or augment, respectively, myosin II activity. Both treatments blocked the inward translation of traction forces, the dissipation of compressive strain, and fibronectin fibrillogenesis over time. These data indicate that dynamic spatial and temporal changes in traction force and local strain may contribute to successful matrix assembly.

Topics: Algorithms; Animals; Cell Adhesion; Extracellular Matrix; Fibronectins; Fluorescent Antibody Technique; Heterocyclic Compounds, 4 or More Rings; Image Processing, Computer-Assisted; Marine Toxins; Mice; Myosin Type II; NIH 3T3 Cells; Oxazoles

Cell detachment is central to a broad range of physiopathological changes, but there are no quantitative methods to study this process. Here we report programmed subcellular release, a method for spatially and temporally controlled cellular detachment, and present quantitative results of the detachment dynamics of 3T3 fibroblasts at the subcellular level.

Topics: 3T3 Cells; Actin Cytoskeleton; Actomyosin; ADP Ribose Transferases; Algorithms; Animals; Azepines; Botulinum Toxins; Bridged Bicyclo Compounds, Heterocyclic; Cell Adhesion; Cell Movement; Cell Shape; Cytological Techniques; Electric Stimulation; Electrodes; Enzyme Inhibitors; Fibroblasts; Focal Adhesions; Glass; Gold; Heterocyclic Compounds, 4 or More Rings; Kinetics; Mice; Naphthalenes; Oligopeptides; Stress Fibers; Thiazolidines

The earliest stage in the development of neuronal polarity is characterized by extension of undifferentiated "minor processes" (MPs), which subsequently differentiate into the axon and dendrites. We investigated the role of the myosin II motor protein in MP extension using forebrain and hippocampal neuron cultures. Chronic treatment of neurons with the myosin II ATPase inhibitor blebbistatin increased MP length, which was also seen in myosin IIB knockouts. Through live-cell imaging, we demonstrate that myosin II inhibition triggers rapid minor process extension to a maximum length range. Myosin II activity is determined by phosphorylation of its regulatory light chains (rMLC) and mediated by myosin light chain kinase (MLCK) or RhoA-kinase (ROCK). Pharmacological inhibition of MLCK or ROCK increased MP length moderately, with combined inhibition of these kinases resulting in an additive increase in MP length similar to the effect of direct inhibition of myosin II. Selective inhibition of RhoA signaling upstream of ROCK, with cell-permeable C3 transferase, increased both the length and number of MPs. To determine whether myosin II affected development of neuronal polarity, MP differentiation was examined in cultures treated with direct or indirect myosin II inhibitors. Significantly, inhibition of myosin II, MLCK, or ROCK accelerated the development of neuronal polarity. Increased myosin II activity, through constitutively active MLCK or RhoA, decreased both the length and number of MPs and, consequently, delayed or abolished the development of neuronal polarity. Together, these data indicate that myosin II negatively regulates MP extension, and the developmental time course for axonogenesis.

Topics: Animals; Cell Growth Processes; Cell Polarity; Cells, Cultured; Chick Embryo; Enzyme Inhibitors; Gene Expression Regulation, Developmental; Heterocyclic Compounds, 4 or More Rings; Hippocampus; Isoenzymes; Metalloendopeptidases; Myosin Light Chains; Myosin Type II; Myosin-Light-Chain Kinase; Myosins; Neurogenesis; Neuronal Plasticity; Neurons; Phosphorylation; Prosencephalon; rho-Associated Kinases; Time Factors; Transfection

Megalin plays a critical role in the endocytosis of albumin and other filtered low-molecular-weight proteins. Here we studied the interaction between megalin and Disabled-2 (Dab2), an adaptor protein that binds to the cytoplasmic domain of megalin and appears to control its trafficking. We co-immunoprecipitated megalin and Dab2 from cultured proximal tubule cells and identified the proteins by liquid chromatography and tandem mass spectrometry. We found two proteins associated with the megalin/Dab2 complex, nonmuscle myosin heavy chain IIA (NMHC-IIA) and beta-actin. Subcellular fractionation followed by sucrose velocity gradient separation showed that megalin, Dab2, and NMHC-IIA existed as a complex in the same endosomal fractions. In vitro pull-down assays demonstrated that NMHC-IIA was bound to the carboxyl-terminal region of Dab2, but not to megalin's cytoplasmic domain. We then transfected COS-7 cells with plasmids that induced the expression of Dab2, NMHC-IIA, and the megalin minireceptor, a truncated form of megalin. Co-immunoprecipitation studies showed that the minireceptor and NMHC-IIA co-immunoprecipitated only with Dab2. Furthermore, the uptake of (125)I-lactoferrin, an endocytic ligand of megalin, by rat yolk sac-derived megalin-expressing L2 cells was inhibited by blebbistatin, a specific inhibitor of nonmuscle myosin II. Our study shows that NMHC-IIA is functionally linked to megalin by interaction with Dab2 and is likely involved in megalin-mediated endocytosis in proximal tubule cells.

Topics: Adaptor Proteins, Vesicular Transport; Animals; Cells, Cultured; Endocytosis; Heterocyclic Compounds, 4 or More Rings; Kidney Tubules, Proximal; Lactoferrin; Ligands; Low Density Lipoprotein Receptor-Related Protein-2; Molecular Motor Proteins; Multiprotein Complexes; Myosin Heavy Chains; Nonmuscle Myosin Type IIA; Protein Interaction Domains and Motifs; Rats

During new blood vessel formation, the cessation of angiogenic sprouting is necessary for the generation of functional vasculature. How sprouting is halted is not known, but it is contemporaneous with the development of stable intercellular junctions [1]. We report that VE-cadherin, which is responsible for endothelial adherens junction organization [2, 3], plays a crucial role in the cessation of sprouting. Abrogating VE-cadherin function in an organotypic angiogenesis assay and in zebrafish embryos stimulates sprouting. We show that VE-cadherin signals to Rho-kinase-dependent myosin light-chain 2 phosphorylation, leading to actomyosin contractility [4], which regulates the distribution of VE-cadherin at cell-cell junctions. VE-cadherin antagonizes VEGFR2 signaling, and consequently, inhibition of VE-cadherin, Rho-kinase, or actomyosin contractility leads to VEGF-driven, Rac1-dependent sprouting. These findings suggest a novel mechanism by which cell-cell adhesion suppresses Rac1-dependent migration and sprouting by increasing actomyosin contractility at cell junctions.

Topics: Actins; Animals; Antigens, CD; Cadherins; Cardiac Myosins; Cell Communication; Cell Line; Endothelial Cells; Gene Knockdown Techniques; Heterocyclic Compounds, 4 or More Rings; Humans; Intercellular Junctions; Myosin Light Chains; Myosins; Neovascularization, Physiologic; Phosphorylation; rac1 GTP-Binding Protein; rho-Associated Kinases; Signal Transduction; Vascular Endothelial Growth Factor Receptor-2; Zebrafish

The spreading and migration of cells on adhesive substrates is regulated by the counterbalance of contractile and protrusive forces. Non-muscle myosin IIA, an ubiquitously expressed contractile protein and enzyme, is implicated in the regulation of cell spreading and directional migration in response to various stimuli. Here we show that discoidin domain receptor 1 (DDR1), a tyrosine kinase receptor activated by type I collagen, associates with the non-muscle myosin IIA heavy chain (NMHC-IIA) upon ligand stimulation. An association was also indicated by coimmunoprecipitation of NMHC-IIA with full-length DDR1, but not with the truncated DDR1d-isoform lacking the kinase domain. DDR1 was important for assembly of NMHC-IIA into filaments on cells plated on collagen. DDR1 expression inhibited cell spreading over collagen but promoted cell migration. By contrast, blockade of non-muscle myosin II activity by blebbistatin enhanced cell spreading but inhibited migration over collagen. We propose that myosin and DDR1 impact cell spreading and migration by regulating adhesive contacts with collagen.

Topics: Actin Cytoskeleton; Animals; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Shape; Collagen Type I; Discoidin Domain Receptor 1; Heterocyclic Compounds, 4 or More Rings; Humans; Mice; Mice, Knockout; Molecular Motor Proteins; Mutation; Myosin Heavy Chains; NIH 3T3 Cells; Nonmuscle Myosin Type IIA; Protein Binding; Protein Transport; Receptor Protein-Tyrosine Kinases; Time Factors; Transfection

The mechanics of the actin cytoskeleton have a central role in the regulation of cells and tissues, but the details of how molecular sensors recognize deformations and forces are elusive. By performing cytoskeleton laser nanosurgery in cultured epithelial cells and fibroblasts, we show that the retraction of stress fibers (SFs) is restricted to the proximity of the cut and that new adhesions form at the retracting end. This suggests that SFs are attached to the substrate. A new computational model for SFs confirms this hypothesis and predicts the distribution and propagation of contractile forces along the SF. We then analyzed the dynamics of zyxin, a focal adhesion protein present in SFs. Fluorescent redistribution after laser nanosurgery and drug treatment shows a high correlation between the experimentally measured localization of zyxin and the computed localization of forces along SFs. Correlative electron microscopy reveals that zyxin is recruited very fast to intermediate substrate anchor points that are highly tensed upon SF release. A similar acute localization response is found if SFs are mechanically perturbed with the cantilever of an atomic force microscope. If actin bundles are cut by nanosurgery in living Drosophila egg chambers, we also find that zyxin redistribution dynamics correlate to force propagation and that zyxin relocates at tensed SF anchor points, demonstrating that these processes also occur in living organisms. In summary, our quantitative analysis shows that force and protein localization are closely correlated in stress fibers, suggesting a very direct force-sensing mechanism along actin bundles.

Topics: Actins; Animals; Computer Simulation; Drosophila; Drosophila Proteins; Elasticity; Epithelial Cells; Fibroblasts; Focal Adhesions; Green Fluorescent Proteins; Heterocyclic Compounds, 4 or More Rings; Homeodomain Proteins; Laser Therapy; Mechanotransduction, Cellular; Mice; Microscopy, Atomic Force; Microscopy, Electron, Transmission; Models, Biological; Nonmuscle Myosin Type IIA; Potoroidae; Recombinant Fusion Proteins; Stress Fibers; Stress, Mechanical; Swiss 3T3 Cells; Time Factors; Transfection; Zyxin

The degree of helical order of the thick filament of mammalian skeletal muscle is highly dependent on temperature and the nature of the ligand. Previously, we showed that there was a close correlation between the conformation of the myosin heads on the surface of the thick filaments and the extent of their helical order. Helical order required the heads to be in the closed conformation. In addition, we showed that, with the same ligand bound at the active site, three conformations of myosin coexisted in equilibrium. Hitherto, however, there was no detectable helical order as measured by x-ray diffraction under the temperatures studied for myosin with MgADP and the nucleotide-free myosin, raising the possibility that the concept of multiple conformations has limited validity. In this study, blebbistatin was used to stabilize the closed conformation of myosin. The degree of helical order is substantially improved with MgATP at low temperature or with MgADP or in the absence of nucleotide. The thermodynamic parameters of the disorder<-->order transition and the characteristics of the ordered array were not significantly altered by binding blebbistatin. The simplest explanation is that the binding of blebbistatin increases the proportion of myosin in the closed conformation from being negligible to substantial. These results provide further evidence for the coexistence of multiple conformations of myosin under a wide range of conditions and for the closed conformation being directly coupled to helical order.

Topics: Actins; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Heterocyclic Compounds, 4 or More Rings; Myosins; Protein Conformation; Psoas Muscles; Rabbits; Temperature; Thermodynamics; X-Ray Diffraction

Adrenal medullary chromaffin cells are innervated by the sympathetic splanchnic nerve and translate graded sympathetic firing into a differential hormonal exocytosis. Basal sympathetic firing elicits a transient kiss-and-run mode of exocytosis and modest catecholamine release, whereas elevated firing under the sympathetic stress response results in full granule collapse to release catecholamine and peptide transmitters into the circulation. Previous studies have shown that rearrangement of the cell actin cortex regulates the mode of exocytosis. An intact cortex favors kiss-and-run exocytosis, whereas disrupting the cortex favors the full granule collapse mode. Here, we investigate the specific roles of two actin-associated proteins, myosin II and myristoylated alanine-rich C-kinase substrate (MARCKS) in this process. Our data demonstrate that MARCKS phosphorylation under elevated cell firing is required for cortical actin disruption but is not sufficient to elicit peptide transmitter exocytosis. Our data also demonstrate that myosin II is phospho-activated under high stimulation conditions. Inhibiting myosin II activity prevented disruption of the actin cortex, full granule collapse, and peptide transmitter release. These results suggest that phosphorylation of both MARCKS and myosin II lead to disruption of the actin cortex. However, myosin II, but not MARCKS, is required for the activity-dependent exocytosis of the peptide transmitters.

Topics: Actins; Action Potentials; Animals; Azepines; Carbazoles; Catecholamines; Cells, Cultured; Chromaffin Cells; Chromogranins; Enzyme Inhibitors; Exocytosis; Heterocyclic Compounds, 4 or More Rings; Immunohistochemistry; Indoles; Intracellular Signaling Peptides and Proteins; Maleimides; Membrane Proteins; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Myosin Type II; Myosin-Light-Chain Kinase; Myristoylated Alanine-Rich C Kinase Substrate; Naphthalenes; Neuropeptides; Patch-Clamp Techniques; Phosphorylation; Protein Kinase C; Reverse Transcriptase Polymerase Chain Reaction

The material properties of a cell determine how mechanical forces are transmitted through and sensed by that cell. Some types of cells stiffen passively under large external forces, but they can also alter their own stiffness in response to the local mechanical environment or biochemical cues. Here we show that the actin-binding protein filamin A is essential for the active stiffening of cells plated on collagen-coated substrates. This appears to be due to a diminished capability to build up large internal contractile stresses in the absence of filamin A. To show this, we compare the material properties and contractility of two human melanoma cell lines that differ in filamin A expression. The filamin A-deficient M2 cells are softer than the filamin A-replete A7 cells, and exert much smaller contractile stresses on the substratum, even though the M2 cells have similar levels of phosphorylated myosin II light chain and only somewhat diminished adhesion strength. In contrast to A7 cells, the stiffness and contractility of M2 cells are insensitive to either myosin-inhibiting drugs or the stiffness of the substratum. Surprisingly, however, filamin A is not required for passive stiffening under large external forces.

Topics: Actins; Cell Adhesion; Cell Line, Tumor; Contractile Proteins; Cytoskeleton; Elasticity; Filamins; Heterocyclic Compounds, 4 or More Rings; Humans; Microfilament Proteins; Myosin Light Chains; Myosin Type II; Phosphorylation; Stress, Mechanical

Yeast is a powerful genetic model system, but its rigid cell wall has prohibited microinjection. Using microfabricated channels to constrain the fission yeast Schizosaccharomyces pombe, we sheared local regions of individual cells with a piezoelectric unit. The cells remained viable, we detected actin patches in the cell after introduction of fluorescent phalloidin into the medium, and the cytokinetic ring was disrupted after injection of the myosin II inhibitor blebbistatin.

Topics: Actins; Fluorescent Dyes; Heterocyclic Compounds, 4 or More Rings; Microinjections; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Phalloidine; Schizosaccharomyces; Schizosaccharomyces pombe Proteins

The dynamics of astral and midzone microtubules (MTs) must be separately regulated during cell division, but the mechanism of selective stabilization of midzone MTs is poorly understood. Here we show that, in HT1080 cells, activation of Rho is required to stabilize midzone MTs, and to maintain the midzone structures after anaphase onset or during cytokinesis. Ect2-depleted cells undergoing conventional cytokinesis (cytokinesis A) or contractile ring-independent cytokinesis (cytokinesis B) formed abnormally thin bundles of midzone MTs. C3-loaded mitotic cells with inactivated Rho showed similar but more severe disorganization of midzone MTs. In addition, the bundles of astral MTs were abnormally abundant along the cell periphery in both Ect2-depleted and C3-loaded mitotic cells. Mitotic kinesin-like protein 1 (MKLP1), a component of the spindle midzone required for bundling of MTs, was localized only in the narrower equatorial regions in Ect2-depleted cells, and disappeared from the midzone accompanying the progression of the mitotic phase in C3-loaded cells. Stabilization of MTs by taxol was sufficient to maintain the midzone structures in C3-loaded mitotic cells. These results, when combined with a preceding analysis on another, microtubule-associated Rho GEF (C.J. Bakal, D. Finan, J. LaRose, C.D. Wells, G. Gish, S. Kulkarni, P. DeSepulveda, A. Wilde, R. Rottapel, The Rho GTP exchange factor Lfc promotes spindle assembly in early mitosis, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 9529-9534), suggest that mammalian cells have two potential steps that require active Rho for the stabilization of midzone MTs during mitosis and cytokinesis.

Topics: Anaphase; Animals; Cell Line, Tumor; Cytokinesis; Enzyme Activation; Fibrosarcoma; Heterocyclic Compounds, 4 or More Rings; Humans; Microtubule-Associated Proteins; Microtubules; Nocodazole; Paclitaxel; Proto-Oncogene Proteins; rho GTP-Binding Proteins; RNA Interference; Spindle Apparatus; Tubulin Modulators

ZO-1 binds numerous transmembrane and cytoplasmic proteins and is required for assembly of both adherens and tight junctions, but its role in defining barrier properties of an established tight junction is unknown. We depleted ZO-1 in MDCK cells using siRNA methods and observed specific defects in the barrier for large solutes, even though flux through the small claudin pores was unaffected. This permeability increase was accompanied by morphological alterations and reorganization of apical actin and myosin. The permeability defect, and to a lesser extent morphological changes, could be rescued by reexpression of either full-length ZO-1 or an N-terminal construct containing the PDZ, SH3, and GUK domains. ZO-2 knockdown did not replicate either the permeability or morphological phenotypes seen in the ZO-1 knockdown, suggesting that ZO-1 and -2 are not functionally redundant for these functions. Wild-type and knockdown MDCK cells had differing physiological and morphological responses to pharmacologic interventions targeting myosin activity. Use of the ROCK inhibitor Y27632 or myosin inhibitor blebbistatin increased TER in wild-type cells, whereas ZO-1 knockdown monolayers were either unaffected or changed in the opposite direction; paracellular flux and myosin localization were also differentially affected. These studies are the first direct evidence that ZO-1 limits solute permeability in established tight junctions, perhaps by forming a stabilizing link between the barrier and perijunctional actomyosin.

Topics: Animals; Calcium; Cell Line; Cell Membrane Permeability; Cell Shape; Cytochalasin D; Cytoskeleton; Dogs; Gene Knockdown Techniques; Heterocyclic Compounds, 4 or More Rings; Humans; Membrane Proteins; Nucleic Acid Synthesis Inhibitors; Phenotype; Phosphoproteins; rho-Associated Kinases; RNA, Small Interfering; Tight Junctions; Zonula Occludens-1 Protein; Zonula Occludens-2 Protein

Migration is a dynamic process in which a cell searches the environment and translates acquired information into somal advancement. In particular, interneuron migration during development is accomplished by two distinct processes: the extension of neurites tipped with growth cones; and nucleus translocation, termed nucleokinesis. The primary purpose of our study is to investigate neurite branching and nucleokinesis using high-resolution time-lapse confocal microscopy and computational modeling. We demonstrate that nucleokinesis is accurately modeled by a spring-dashpot system and that neurite branching is independent of the nucleokinesis event, and displays the dynamics of a stochastic birth-death process. This is in contrast to traditional biological descriptions, which suggest a closer relationship between the two migratory mechanisms. Our models are validated on independent data sets acquired using two different imaging protocols, and are shown to be robust to alterations in guidance cues and cellular migratory mechanisms, through treatment with brain-derived neurotrophic factor, neurotrophin-4, and blebbistatin. We postulate that the stochastic branch dynamics exhibited by interneurons undergoing guidance-directed migration permit efficient exploration of the environment.

Topics: Algorithms; Animals; Brain; Brain-Derived Neurotrophic Factor; Cell Movement; Computer Simulation; Databases, Factual; Green Fluorescent Proteins; Heterocyclic Compounds, 4 or More Rings; Humans; Interneurons; Mice; Mice, Transgenic; Microscopy, Confocal; Models, Neurological; Nerve Growth Factors; Neurites; Stochastic Processes; Time Factors; Tissue Culture Techniques

Blebbistatin (BLEB) is a small cell permeable molecule originally reported as a selective inhibitor of myosin II isoforms expressed by striated muscle and non-muscle cells (IC(50) = 0.5-5 microM) with poor inhibition of turkey gizzard smooth muscle (SM) myosin II (IC(50) approximately 80 microM). However, recently it was found that BLEB can potently inhibit mammalian arterial SM (IC(50) approximately 5 microM).. To investigate the effect of BLEB on corpus cavernosum SM (CCSM) tone and erectile function (EF).. CC tissue obtained from penile implant patients along with CC, aorta and bladder from adult male rats were used for BLEB organ bath studies. Intracavernosal BLEB was administered to rats and EF was assessed via intracavernous pressure (ICP).. Effects of BLEB on agonist-induced CCSM, aorta and bladder contraction in vitro and ICP in vivo.. BLEB completely relaxed human CCSM pre-contracted with phenylephrine (PE) in a dose-dependent manner decreasing tension by 76.5% at 10 microM. BLEB pre-incubation attenuated PE-induced contraction of human CC by approximately 85%. Human CC strips pre-contracted with endothelin-1 or KCl were almost completely relaxed by BLEB. Rat CCSM pre-contracted with PE showed BLEB relaxation comparable to human CCSM. BLEB inhibition was similar for rat aorta but slower for bladder. Both maximal ICP and ICP/mean arterial pressure were dose-dependently increased by BLEB intracavernous injections with full erection at 1 micromole.. Our novel data reveals that BLEB nearly completely relaxes rat and human CCSM pre-contracted with a variety of potent agonists and exhibits tissue selectivity. Coupled with our in vivo data in which nanomole doses of BLEB significantly increase ICP, our data substantiates an important role for the SM contractile apparatus in the molecular mechanism for EF and suggests the possibility of BLEB binding at myosin II as a therapeutic treatment for ED by targeting SM contractile pathways.

Topics: Adult; Aged; Animals; Aorta; Dose-Response Relationship, Drug; Heterocyclic Compounds, 4 or More Rings; Humans; In Vitro Techniques; Male; Middle Aged; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Myosin Type II; Penis; Phenylephrine; Rats; Rats, Sprague-Dawley; Urinary Bladder

Cytosolic fluid dynamics have been implicated in cell motility because of the hydrodynamic forces they induce and because of their influence on transport of components of the actin machinery to the leading edge. To investigate the existence and the direction of fluid flow in rapidly moving cells, we introduced inert quantum dots into the lamellipodia of fish epithelial keratocytes and analysed their distribution and motion. Our results indicate that fluid flow is directed from the cell body towards the leading edge in the cell frame of reference, at about 40% of cell speed. We propose that this forward-directed flow is driven by increased hydrostatic pressure generated at the rear of the cell by myosin contraction, and show that inhibition of myosin II activity by blebbistatin reverses the direction of fluid flow and leads to a decrease in keratocyte speed. We present a physical model for fluid pressure and flow in moving cells that quantitatively accounts for our experimental data.

Topics: Actins; Animals; Cell Movement; Cells; Chromogenic Compounds; Cytoplasm; Epithelial Cells; Fishes; Heterocyclic Compounds, 4 or More Rings; Intracellular Fluid; Kinetics; Models, Biological; Myosin Type II; Myosins; Pseudopodia; Quantum Dots

Living cells sense the rigidity of their environment and adapt their activity to it. In particular, cells cultured on elastic substrates align their shape and their traction forces along the direction of highest stiffness and preferably migrate towards stiffer regions. Although numerous studies investigated the role of adhesion complexes in rigidity sensing, less is known about the specific contribution of acto-myosin based contractility. Here we used a custom-made single-cell technique to measure the traction force as well as the speed of shortening of isolated myoblasts deflecting microplates of variable stiffness. The rate of force generation increased with increasing stiffness and followed a Hill force-velocity relationship. Hence, cell response to stiffness was similar to muscle adaptation to load, reflecting the force-dependent kinetics of myosin binding to actin. These results reveal an unexpected mechanism of rigidity sensing, whereby the contractile acto-myosin units themselves can act as sensors. This mechanism may translate anisotropy in substrate rigidity into anisotropy in cytoskeletal tension, and could thus coordinate local activity of adhesion complexes and guide cell migration along rigidity gradients.

Topics: Animals; Cell Line; Cell Shape; Heterocyclic Compounds, 4 or More Rings; Mice; Muscles; Myoblasts; Stress, Mechanical

Entry of vaccinia virus (VACV) into cells occurs by fusion with the plasma membrane and via a low pH-dependent endosomal pathway, presumably involving unidentified cellular receptors. In addition to approximately 25 viral proteins, the membrane of VACV mature virions contains several phospholipids including phosphatidylserine (PS). A recent model posits that PS flags virions as apoptotic debris to activate a common cellular uptake pathway to gain cell entry, perhaps through an interaction with a PS-specific cell surface receptor. To evaluate the apoptotic mimicry model, we reconstituted the membrane of detergent-extracted virions with several different phospholipids. Although the ability of the L-stereoisomer of PS to reconstitute infectivity was confirmed, the nonbiologically relevant D-stereoisomer of PS, and phosphatidylglycerol, which are not normally present in the virion membrane, functioned as well. Regardless of which phospholipid reconstituted infectivity, virus entry was inhibited by a neutralizing monoclonal antibody to a virion surface protein and by the drugs blebbistatin and bafilomycin A1, suggesting that in each case virus uptake was specific and occurred by a similar mechanism involving macropinocytosis and a low-pH endocytic pathway. Lipid-reconstituted and nonreconstituted, membrane-extracted virions were equally capable of binding to cells. However, the physical association of phospholipids with virus particles during membrane reconstitution correlated directly with rescue of particle infectivity and cell entry capability. Our results support a role for PS in poxvirus entry, but demonstrate that other phospholipids, not known to signal uptake of apoptotic debris, can function similarly.

Topics: Animals; Detergents; Endosomes; Fireflies; Flow Cytometry; Heterocyclic Compounds, 4 or More Rings; Hydrogen-Ion Concentration; Luciferases; Molecular Mimicry; Octoxynol; Phospholipids; Polyethylene Glycols; Vaccinia virus; Virion

Interkinetic nuclear migration (INM) is a hallmark of the polarized stem and progenitor cells in the ventricular zone (VZ) of the developing vertebrate CNS. INM is responsible for the pseudostratification of the VZ, a crucial aspect of brain evolution. The nuclear migration toward the apical centrosomes in G2 is thought to be a dynein-microtubule-based process. By contrast, the cytoskeletal machinery involved in the basally directed nuclear translocation away from the centrosome in G1 has been enigmatic. Studying the latter aspect of INM requires manipulation of the cytoskeleton without impairing mitosis and cytokinesis. To this end, we have established a culture system of mouse embryonic telencephalon that reproduces cortical development, and have applied it to explore a role of actomyosin in INM. Using the nonmuscle myosin II inhibitor blebbistatin at a low concentration at which neither cell cycle progression nor cytokinesis is impaired, we show that myosin II is required for the apical-to-basal (ap-->bl), ab-centrosomal INM. Myosin II activity is also necessary for the nuclear translocation during delamination of subventricular zone (SVZ) cells, a second, telencephalon-specific type of neural progenitor. Moreover, the inhibition of ab-centrosomal INM changes the balance between VZ and SVZ progenitor cell fate. Our data suggest a unifying concept in which the actomyosin contraction underlying ab-centrosomal INM sets the stage for the evolutionary increase in VZ pseudostratification and for SVZ progenitor delamination, a key process in cortical expansion.

Topics: Animals; Bromodeoxyuridine; Cell Culture Techniques; Cell Cycle; Cell Nucleus; Cell Proliferation; Cells, Cultured; Centrosome; Cerebral Ventricles; Fluorescent Antibody Technique; Green Fluorescent Proteins; Heterocyclic Compounds, 4 or More Rings; Immediate-Early Proteins; Mice; Mice, Inbred C57BL; Microscopy, Electron, Transmission; Myosin Type II; Neurons; Recombinant Fusion Proteins; Stem Cells; Time Factors; Tumor Suppressor Proteins

Mast cells stimulated with antigen undergo extensive changes in their cytoskeleton. In the present study, we assess the impact of actin-modifying drugs and report that, in the presence of cytochalasin D, mast cells stop membrane ruffling, but instead bleb. Bleb formation is reversible following washout of cytochalasin D and occurs in an actin-polymerization-dependent manner. Bleb formation is inhibited by expression of constitutively active ezrin-T567D. Blebbing is also inhibited by blebbistatin, a myosin II inhibitor, implying myosin II activation in the process. We used a selection of inhibitors and observed that myosin II activation is dependent mainly on Ca2+-calmodulin, with only a small contribution from Rho kinase. The signalling pathways stimulated by antigen include PLC (phospholipase C) and PLD (phospholipase D). Bleb formation was dependent on activation of PLC, but not PLD. Primary alcohols, used previously as a means to reduce PLD-derived phosphatidic acid, were potent inhibitors of membrane blebbing, but a more selective inhibitor of PLD, FIPI (5-fluoro-2-indolyl des-chlorohalopemide), was without effect. FIPI also did not inhibit membrane ruffling or degranulation of mast cells, indicating that inhibition by primary alcohols works through an unidentified mechanism rather than via diversion of PLD activity as assumed. We also examined the requirement for ARF6 (ADP-ribosylation factor 6) and observed that its expression led to an increase in bleb size and a further increase was observed with the dominant-active mutant, ARF6-Q67L. Since ARF6-T27N had no effect on bleb size, we conclude that ARF6 needs to be active to regulate the size of the blebs.

Topics: 1-Butanol; Actins; ADP-Ribosylation Factor 6; ADP-Ribosylation Factors; Animals; Antigens; Calcium; Calmodulin; Cell Line, Tumor; Cell Membrane; Cytochalasin D; Dinitrophenols; Domperidone; Enzyme Inhibitors; Green Fluorescent Proteins; Heterocyclic Compounds, 4 or More Rings; Immunoglobulin E; Indoles; Mast Cells; Microscopy, Fluorescence; Mutation; Phosphatidic Acids; Phospholipase D; Rats; Transfection; Type C Phospholipases

Endogenous granules (EGs) that consist of lipid droplets and mitochondria have been commonly used to assess intracellular mechanical properties via multiple particle tracking microrheology (MPTM). Despite their widespread use, the nature of interaction of EGs with the cytoskeletal network and the type of forces driving their dynamics--both of which are crucial for the interpretation of the results from MPTM technique--are yet to be resolved. In this report, we study the dynamics of endogenous granules in mammalian cells using particle tracking methods. We find that the ensemble dynamics of EGs is diffusive in three types of mammalian cells (endothelial cells, smooth muscle cells and fibroblasts), thereby suggesting an apparent universality in their dynamical behavior. Moreover, in a given cell, the amplitude of the mean-squared displacement for EGs is an order of magnitude larger than that of injected particles. This observation along with results from ATP depletion and temperature intervention studies suggests that cytoskeletal active forces drive the dynamics of EGs. To elucidate the dynamical origin of the diffusive-like nonthermal motion, we consider three active force generation mechanisms--molecular motor transport, actomyosin contractility and microtubule polymerization forces. We test these mechanisms using pharmacological interventions. Experimental evidence and model calculations suggest that EGs are intimately linked to microtubules and that microtubule polymerization forces drive their dynamics. Thus, endogenous granules could serve as non-invasive probes for microtubule network dynamics in mammalian cells.

Topics: Actomyosin; Adenosine Triphosphate; Animals; Biological Transport; Biomechanical Phenomena; Bridged Bicyclo Compounds, Heterocyclic; Cells, Cultured; Chlorocebus aethiops; COS Cells; Cytoplasmic Granules; Cytoskeleton; Depsipeptides; Endothelial Cells; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Humans; Kinetics; Microscopy, Confocal; Microtubules; Models, Biological; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nocodazole; Taxoids; Thermodynamics; Thiazolidines

We developed a novel atomic force microscope (AFM) indentation technique for mapping spatiotemporal stiffness of spontaneously beating neonatal rat cardiac myocytes. Cells were indented at a rate close but unequal to their contractile frequency. Resultant apparent elastic modulus cycled at a predictable envelope frequency between a systolic value of 26.2 +/- 5.1 kPa and a diastolic value of 7.8 +/- 4.1 kPa. In cells probed along their axis, spatial heterogeneity of systolic stiffness correlated with the sarcomeric structure of underlying myofibrils. Treatment with blebbistatin eliminated contractile activity and resulted in a uniform modulus of 6.5 +/- 4.8 kPa. The technique provides a unique means of probing the mechanical effects of disease processes and pharmacological treatments on beating cardiomyocytes at the subcellular level, providing new insights relating myocardial structure and function.

Topics: Animals; Biomechanical Phenomena; Biomedical Engineering; Cells, Cultured; Elastic Modulus; Elasticity Imaging Techniques; Heterocyclic Compounds, 4 or More Rings; Microscopy, Atomic Force; Myocardial Contraction; Myocytes, Cardiac; Rats

We measure the temporal evolution of three-dimensional membrane topography on living fibroblasts and characterize the propagation of membrane waves using a wide-field optical profiling technique. The measured membrane profiles are compared with the numerical results calculated by the active membrane model recently proposed by Shlomovitz and Gov. After the treatments of blebbistatin and latrunculin A separately, the membrane waves disappear and the membrane surfaces are flattened, verifying that the membrane waves are driven by the interactions between myosin II and actin polymerization.

Topics: Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Cell Membrane; Cell Survival; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Microscopy; Thiazolidines; Time Factors

The small molecule blebbistatin is now a front line tool in the study of myosin function. Chemical modification of the tricyclic core of blebbistatin could deliver the next generation of myosin inhibitors and to help address this we report here on the impact of structural changes in the methyl-substituted aromatic ring of blebbistatin on its biological activity. Chemical methods for the preparation of isomeric methyl-containing analogues are reported and a series of co-crystal structures are used to rationalise the observed variations in their biological activity. These studies further support the view that the previously identified binding mode of blebbistatin to Dictyostelium discoideum myosin II is of relevance to its mode of action. A discussion of the role that these observations have on planning the synthesis of focused libraries of blebbistatin analogues is also provided including an assessment of possibilities by computational methods. These studies are ultimately directed at the development of novel myosin inhibitors with improved affinity and different selectivity profiles from blebbistatin itself.

Topics: Crystallography; Heterocyclic Compounds, 4 or More Rings; Mass Spectrometry; Models, Molecular; Myosins; Nuclear Magnetic Resonance, Biomolecular; Stereoisomerism

Metalloproteinase-dependent tissue invasion requires the formation of podosomes and invadopodia for localized matrix degradation. Actin cytoskeleton remodeling via Arp2/3-mediated actin polymerization is essential for podosome formation, and dynamic microtubules have an important role in maintaining podosome turnover in macrophages and osteoclasts. Little is known, however, about the involvement of the intermediate filament cytoskeleton in formation, stabilization, and turnover of podosomes. Here we show that vimentin intermediate filaments colocalize with the early sites of podosome formation at the stress fiber - focal adhesion interface in cultured vascular smooth muscle cells, but do not directly contribute to podosome formation, or stabilization. In unstimulated A7r5 cells the cytolinker protein plectin poorly colocalized with vimentin and the microdomains, but following induction by phorbol ester accumulated in the rings that surround the podosomes. In plectin-deficient A7r5 cells actin stress fiber remodelling is reduced in response to PDBu, and small podosomes remain localized at stable actin stress fibres. Pharmacological inhibition of actomyosin contractility by blebbistatin leads to an aberrant localization of podosomes away from the cell periphery and induces failure of plectin to surround the outer perimeter of these invasive adhesions. Taken together, we conclude that plectin is involved in growth and maturation of podosomes by reducing focal adhesion and stress fiber turnover, and that actomyosin-dependent contractility is required for the peripheral localization and specific deposition of plectin at the podosome rings.

Topics: Actins; Animals; Blotting, Western; Cell Line; Cell Movement; Cytoskeleton; Focal Adhesions; Heterocyclic Compounds, 4 or More Rings; Intermediate Filaments; Microscopy, Fluorescence; Muscle, Smooth; Myosins; Phorbol 12,13-Dibutyrate; Plectin; Pseudopodia; Rats; RNA Interference; Vimentin

The mechanism by which anaesthetic agents produce general anaesthesia is not yet fully understood. Retraction of neurites is an important function of individual neurones and neural plexuses during normal and pathological conditions, and it has been shown that such a retraction pathway exists in developing and mature neurones. We hypothesized that propofol decreases neuronal activity by causing retraction of neuronal neurites.. Primary cultures of rat cortical neurones were exposed in concentration- and time-response experiments to 0.02, 0.2, 2, and 20 microM propofol or lipid vehicle. Neurones were pretreated with the GABA(A) receptor (GABA(A)R) antagonist, bicuculline, the myosin II ATPase activity inhibitor, blebbistatin, and the F-actin stabilizing agent, phalloidin, followed by administration of propofol (20 microM). Changes in neurite retraction were evaluated using time-lapse light microscopy.. Propofol caused a concentration- and time-dependent reversible retraction of cultured cortical neurone neurites. Bicuculline, blebbistatin, and phalloidin completely inhibited propofol-induced neurite retraction. Images of retracted neurites were characterized by a retraction bulb and a thin trailing membrane remnant.. Cultured cortical rat neurones retract their neurites after exposure to propofol in a concentration- and time-dependent manner. This retraction is GABA(A)R mediated, reversible, and dependent on actin and myosin II. Furthermore, the concentrations and times to full retraction and recovery correspond to those observed during propofol anaesthesia.

Topics: Anesthetics, Intravenous; Animals; Bicuculline; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; GABA Antagonists; Heterocyclic Compounds, 4 or More Rings; Neurites; Phalloidine; Propofol; Rats; Rats, Sprague-Dawley

Blebbistatin is a small-molecule, high-affinity, noncompetitive inhibitor of myosin II. We have used negative staining electron microscopy to study the effects of blebbistatin on the organization of the myosin heads on muscle thick filaments. Loss of ADP and Pi from the heads causes thick filaments to lose their helical ordering. In the presence of 100 microM blebbistatin, disordering was at least 10 times slower. In the M.ADP state, myosin heads are also disordered. When blebbistatin was added to M.ADP thick filaments, helical ordering was restored. However, blebbistatin did not improve the order of thick filaments lacking bound nucleotide. Addition of calcium to relaxed muscle homogenates induced thick-thin filament interaction and filament sliding. In the presence of blebbistatin, filament interaction was inhibited. These structural observations support the conclusion, based on biochemical studies, that blebbistatin inhibits myosin ATPase and actin interaction by stabilizing the closed switch 2 structure of the myosin head. These properties make blebbistatin a useful tool in structural and functional studies of cell motility and muscle contraction.

Topics: Actins; Adenosine Diphosphate; Animals; Apyrase; Arachnida; Heterocyclic Compounds, 4 or More Rings; Muscles; Myosins; Phosphates; Protein Binding

For both cells and tissues, shape is closely correlated with function presumably via geometry-dependent distribution of tension. In this study, we identify common shape determinants spanning cell and tissue scales. For cells whose sites of adhesion are restricted to small adhesive islands on a micropatterned substrate, shape resembles a sequence of inward-curved circular arcs. The same shape is observed for fibroblast-populated collagen gels that are pinned to a flat substrate. Quantitative image analysis reveals that, in both cases, arc radii increase with the spanning distance between the pinning points. Although the Laplace law for interfaces under tension predicts circular arcs, it cannot explain the observed dependence on the spanning distance. Computer simulations and theoretical modeling demonstrate that filamentous network mechanics and contractility give rise to a modified Laplace law that quantitatively explains our experimental findings on both cell and tissue scales. Our model in conjunction with actomyosin inhibition experiments further suggests that cell shape is regulated by two different control modes related to motor contractility and structural changes in the actin cytoskeleton.

Topics: Amides; Animals; Biomechanical Phenomena; Cattle; Cell Adhesion; Cell Line, Tumor; Cell Shape; Computer Simulation; Cytoskeleton; Elasticity; Heterocyclic Compounds, 4 or More Rings; Mice; Models, Biological; Myosin Type II; Pyridines; Rats; rho-Associated Kinases; Tissue Engineering

The cell biological processes underlying axon growth and guidance are still not well understood. An outstanding question is how a new segment of the axon shaft is formed in the wake of neuronal growth cone advance. For this to occur, the highly dynamic, splayed-out microtubule (MT) arrays characteristic of the growth cone must be consolidated (bundled together) to form the core of the axon shaft. MT-associated proteins stabilize bundled MTs, but how individual MTs are brought together for initial bundling is unknown. Here, we show that laterally moving actin arcs, which are myosin II-driven contractile structures, interact with growing MTs and transport them from the sides of the growth cone into the central domain. Upon Myosin II inhibition, the movement of actin filaments and MTs immediately stopped and MTs unbundled. Thus, Myosin II-dependent compressive force is necessary for normal MT bundling in the growth cone neck.

Topics: Actins; Animals; Aplysia; Cell Culture Techniques; Cells, Cultured; Growth Cones; Heterocyclic Compounds, 4 or More Rings; Immunohistochemistry; Kymography; Microtubules; Myosin Type II; Neurons; Time Factors

Thrombin inhibits intercellular Ca(2+) wave propagation in bovine corneal endothelial cells (BCECs) through a mechanism dependent on myosin light chain (MLC) phosphorylation. In this study, blebbistatin, a selective myosin II ATPase inhibitor, was used to investigate whether the effect of thrombin is mediated by enhanced actomyosin contractility.. BCECs were exposed to thrombin (2 U/mL) for 5 minutes. MLC phosphorylation was assayed by immunocytochemistry. Ca(2+) waves were visualized by confocal microscopy with Fluo-4AM. Fluorescence recovery after photobleaching (FRAP) was used to investigate intercellular communication (IC) via gap junctions. ATP release was measured by luciferin-luciferase assay. Lucifer yellow (LY) uptake was used to investigate hemichannel activity, and Fura-2 was used to assay thrombin- and ATP-mediated Ca(2+) responses.. Pretreatment with blebbistatin (5 microM for 20 minutes) or its nitro derivative prevented the thrombin-induced inhibition of the Ca(2+) wave. Neither photo-inactivated blebbistatin nor the inactive enantiomers prevented the thrombin effect. Blebbistatin also prevented thrombin-induced inhibition of LY uptake, ATP release and FRAP, indicating that it prevented the thrombin effect on paracrine and gap junctional IC. In the absence of thrombin, blebbistatin had no significant effect on paracrine or gap junctional IC. The drug had no influence on MLC phosphorylation or on [Ca(2+)](i) transients in response to thrombin or ATP.. Blebbistatin prevents the inhibitory effects of thrombin on intercellular Ca(2+) wave propagation. The findings demonstrate that myosin II-mediated actomyosin contractility plays a central role in thrombin-induced inhibition of gap junctional IC and of hemichannel-mediated paracrine IC.

Topics: Adenosine Triphosphatases; Animals; Blotting, Western; Calcium; Calcium Channels; Cattle; Endothelium, Corneal; Fluorescent Dyes; Gap Junctions; Gene Expression; Hemostatics; Heterocyclic Compounds, 4 or More Rings; Immunohistochemistry; Intracellular Fluid; Isoquinolines; Myosin Type II; Phosphorylation; Physical Stimulation; Reverse Transcriptase Polymerase Chain Reaction; RNA; Thrombin

Interplay of two cytoskeletal systems--microfilaments and microtubules is essential for directional cell movement. To better understand the role of those cytoskeletal systems in polarization of cells, rat fibroblasts were incubated with drugs inhibiting activity of myosin II: blebbistatin and Y-27632. Both drugs led to disappearance of actin-myosin bundles and mature focal cell-matrix adhesions but did not affect polarization and directional motility. The rate of motility even increased after inhibitor treatment. The characteristic feature of inhibitor-treated fibroblasts was collapse of the cytoplasm accompanied by bundling of microtubules that led to transformation of lamellae into long immobile tails. The only exception was the leading anterior lamella which was not transformed into the tail and supported directional movement of the cell. The tail at the cell rear determined the position of anterior lamella and direction of locomotion. Depolymerization of microtubules by colcemid stopped directional locomotion of inhibitor-treated cells. These data show that integrity of the microtubular system provides the basic mechanism of polarization and orientation which is only modified by interactions with actin-myosin system and cell-substrate adhesions. We suggest that the position of bundled tail microtubules and dispersed microtubules in leading lamella determine polarization in cells lacking stress fibers and focal adhesions. Thus, polarization is based on microtubule-dependent mechanisms both in non-contractile and contractile cells. These mechanisms could switch dependent on circumstances as fibroblasts may acquire non-contractile phenotype, not only after direct inhibition of myosin II but also in certain conditions of microenvironment.

Topics: Actins; Amides; Animals; Cell Line; Cell Movement; Cell Polarity; Cell Shape; Demecolcine; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Microtubules; Models, Biological; Myosin Type II; Pyridines; Rats; Wound Healing

In vertebrate rod photoreceptor cells, arrestin and the visual G-protein transducin move between the inner segment and outer segment in response to changes in light. This stimulus dependent translocation of signalling molecules is assumed to participate in long term light adaptation of photoreceptors. So far the cellular basis for the transport mechanisms underlying these intracellular movements remains largely elusive. Here we investigated the dependency of these movements on actin filaments and the microtubule cytoskeleton of photoreceptor cells. Co-cultures of mouse retina and retinal pigment epithelium were incubated with drugs stabilizing and destabilizing the cytoskeleton. The actin and microtubule cytoskeleton and the light dependent distribution of signaling molecules were subsequently analyzed by light and electron microscopy. The application of cytoskeletal drugs differentially affected the cytoskeleton in photoreceptor compartments. During dark adaptation the depolymerization of microtubules as well as actin filaments disrupted the translocation of arrestin and transducin in rod photoreceptor cells. During light adaptation only the delivery of arrestin within the outer segment was impaired after destabilization of microtubules. Movements of transducin and arrestin required intact cytoskeletal elements in dark adapting cells. However, diffusion might be sufficient for the fast molecular movements observed as cells adapt to light. These findings indicate that different molecular translocation mechanisms are responsible for the dark and light associated translocations of arrestin and transducin in rod photoreceptor cells.

Topics: Actin Cytoskeleton; Actins; Animals; Arrestin; Cell Migration Assays; Cytochalasin D; Cytoskeleton; Dark Adaptation; Darkness; Heterocyclic Compounds, 4 or More Rings; Homozygote; Light; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Fluorescence; Microscopy, Immunoelectron; Microtubules; Paclitaxel; Phalloidine; Protein Transport; Retina; Retinal Rod Photoreceptor Cells; Rod Cell Outer Segment; Signal Transduction; Thiabendazole; Transducin; Vision, Ocular

Cultured confluent endothelial cells exhibit stable basal isometric tone associated with constitutive myosin II regulatory light chain (RLC) phosphorylation. Thrombin treatment causes a rapid increase in isometric tension concomitant with myosin II RLC phosphorylation, actin polymerization, and stress fiber reorganization while inhibitors of myosin light chain kinase (MLCK) and Rho-kinase prevent these responses. These findings suggest a central role for myosin II in the regulation of endothelial cell tension. The present studies examine the effects of blebbistatin, a specific inhibitor of myosin II activity, on basal tone and thrombin-induced tension development. Although blebbistatin treatment abolished basal tension, this was accompanied by an increase in myosin II RLC phosphorylation. The increase in RLC phosphorylation was Ca(2+) dependent and mediated by MLCK. Similarly, blebbistatin inhibited thrombin-induced tension without interfering with the increase in RLC phosphorylation or in F-actin polymerization. Blebbistatin did prevent myosin II filament incorporation and association with polymerizing or reorganized actin filaments leading to the disappearance of stress fibers. Thus the inhibitory effects of blebbistatin on basal tone and induced tension are consistent with a requirement for myosin II activity to maintain stress fiber integrity.

Topics: Actins; Animals; Biomechanical Phenomena; Cattle; Cell Adhesion; Cell Line; Endothelial Cells; Endothelium, Vascular; Heterocyclic Compounds, 4 or More Rings; Myosin Type II; Stress Fibers

Invadopodia are actin-rich subcellular protrusions with associated proteases used by cancer cells to degrade extracellular matrix (ECM) [1]. Molecular components of invadopodia include branched actin-assembly proteins, membrane trafficking proteins, signaling proteins, and transmembrane proteinases [1]. Similar structures exist in nontransformed cells, such as osteoclasts and dendritic cells, but are generally called podosomes and are thought to be more involved in cell-matrix adhesion than invadopodia [2-4]. Despite intimate contact with their ECM substrates, it is unknown whether physical or chemical ECM signals regulate invadopodia function. Here, we report that ECM rigidity directly increases both the number and activity of invadopodia. Transduction of ECM-rigidity signals depends on the cellular contractile apparatus [5-7], given that inhibition of nonmuscle myosin II, myosin light chain kinase, and Rho kinase all abrogate invadopodia-associated ECM degradation. Whereas myosin IIA, IIB, and phosphorylated myosin light chain do not localize to invadopodia puncta, active phosphorylated forms of the mechanosensing proteins p130Cas (Cas) and focal adhesion kinase (FAK) are present in actively degrading invadopodia, and the levels of phospho-Cas and phospho-FAK in invadopodia are sensitive to myosin inhibitors. Overexpression of Cas or FAK further enhances invadopodia activity in cells plated on rigid polyacrylamide substrates. Thus, in invasive cells, ECM-rigidity signals lead to increased matrix-degrading activity at invadopodia, via a myosin II-FAK/Cas pathway. These data suggest a potential mechanism, via invadopodia, for the reported correlation of tissue density with cancer aggressiveness.

Topics: Actin Cytoskeleton; Azepines; Cell Line, Tumor; Cell Surface Extensions; Crk-Associated Substrate Protein; Enzyme Inhibitors; Extracellular Matrix; Focal Adhesion Kinase 1; Gelatin; Heterocyclic Compounds, 4 or More Rings; Humans; Integrins; Myosin Type II; Myosin-Light-Chain Kinase; Naphthalenes; Signal Transduction

Podosomes are self-organized, dynamic, actin-containing structures that adhere to the extracellular matrix via integrins [1-5]. Yet, it is not clear what regulates podosome dynamics and whether podosomes can function as direct mechanosensors, like focal adhesions [6-9]. We show here that myosin-II proteins form circular structures outside and at the podosome actin ring to regulate podosome dynamics. Inhibiting myosin-II-dependent tension dissipated podosome actin rings before dissipating the myosin-ring structure. As podosome rings changed size or shape, tractions underneath the podosomes were exerted onto the substrate and were abolished when myosin-light-chain activity was inhibited. The magnitudes of tractions were comparable to those generated underneath focal adhesions, and they increased with substrate stiffness. The dynamics of podosomes and of focal adhesions were different. Torsional tractions underneath the podosome rings were generated with rotations of podosome rings in a nonmotile, nonrotating cell, suggesting a unique feature of these circular structures. Stresses applied via integrins at the apical surface directly displaced podosomes near the basal surface. Stress-induced podosome displacements increased nonlinearly with applied stresses. Our results suggest that podosomes are dynamic mechanosensors in which interactions of myosin tension and actin dynamics are crucial for regulating these self-organized structures in living cells.

Topics: Actin Cytoskeleton; Actins; Azepines; Cells, Cultured; Depsipeptides; Enzyme Inhibitors; Extracellular Matrix; Focal Adhesions; Heterocyclic Compounds, 4 or More Rings; Integrins; Mechanotransduction, Cellular; Myosin Type II; Myosin-Light-Chain Kinase; Naphthalenes

Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles to the plasma membrane. Our previous studies demonstrated that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. The experiments described in this report are the first to show a dual role for the myosin IIA isoform specifically in regulating insulin-stimulated glucose uptake in adipocytes. We demonstrate that inhibition of MLCK but not RhoK results in impaired insulin-stimulated glucose uptake. Furthermore, our studies show that insulin specifically stimulates the phosphorylation of the RLC associated with the myosin IIA isoform via MLCK. In time course experiments, we determined that GLUT4 translocates to the plasma membrane prior to myosin IIA recruitment. We further show that recruitment of myosin IIA to the plasma membrane requires that myosin IIA be activated via phosphorylation of the RLC by MLCK. Our findings also reveal that myosin II is required for proper GLUT4-vesicle fusion at the plasma membrane. We show that once at the plasma membrane, myosin II is involved in regulating the intrinsic activity of GLUT4 after insulin stimulation. Collectively, our results are the first to reveal that myosin IIA plays a critical role in mediating insulin-stimulated glucose uptake in 3T3-LI adipocytes, via both GLUT4 vesicle fusion at the plasma membrane and GLUT4 activity.

Topics: 3T3-L1 Cells; Adipocytes; Animals; Azepines; Cell Membrane; Enzyme Inhibitors; Glucose; Glucose Transporter Type 4; Heterocyclic Compounds, 4 or More Rings; Insulin; Membrane Fusion; Mice; Myosin Type II; Myosin-Light-Chain Kinase; Naphthalenes; Protein Isoforms; Signal Transduction

The myelin sheath forms by the spiral wrapping of a glial membrane around the axon. The mechanisms responsible for this process are unknown but are likely to involve coordinated changes in the glial cell cytoskeleton. We have found that inhibition of myosin II, a key regulator of actin cytoskeleton dynamics, has remarkably opposite effects on myelin formation by Schwann cells (SC) and oligodendrocytes (OL). Myosin II is necessary for initial interactions between SC and axons, and its inhibition or down-regulation impairs their ability to segregate axons and elongate along them, preventing the formation of a 1:1 relationship, which is critical for peripheral nervous system myelination. In contrast, OL branching, differentiation, and myelin formation are potentiated by inhibition of myosin II. Thus, by controlling the spatial and localized activation of actin polymerization, myosin II regulates SC polarization and OL branching, and by extension their ability to form myelin. Our data indicate that the mechanisms regulating myelination in the peripheral and central nervous systems are distinct.

Topics: Actins; Animals; Axons; Biomarkers; Cadherins; Cell Differentiation; Cell Proliferation; Cell Survival; Cells, Cultured; Central Nervous System; Coculture Techniques; Cytoskeleton; Ganglia, Spinal; Heterocyclic Compounds, 4 or More Rings; Myelin Basic Protein; Myelin Sheath; Myosin Type II; Oligodendroglia; Peripheral Nervous System; Rats; RNA Interference; Schwann Cells

Rap1 enhances integrin-mediated adhesion but the link between Rap1 activation and integrin function in collagen phagocytosis is not defined. Mass spectrometry of Rap1 immunoprecipitates showed that the association of Rap1 with nonmuscle myosin heavy-chain II-A (NMHC II-A) was enhanced by cell attachment to collagen beads. Rap1 colocalized with NM II-A at collagen bead-binding sites. There was a transient increase in myosin light-chain phosphorylation after collagen-bead binding that was dependent on myosin light-chain kinase but not Rho kinase. Inhibition of myosin light-chain phosphorylation, but not myosin II-A motor activity inhibited collagen-bead binding and Rap activation. In vitro binding assays demonstrated binding of Rap1A to filamentous myosin rods, and in situ staining of permeabilized cells showed that NM II-A filaments colocalized with F-actin at collagen bead sites. Knockdown of NM II-A did not affect talin, actin, or beta1-integrin targeting to collagen beads but targeting of Rap1 and vinculin to collagen was inhibited. Conversely, knockdown of Rap1 did not affect localization of NM II-A to beads. We conclude that MLC phosphorylation in response to initial collagen-bead binding promotes NM II-A filament assembly; binding of Rap1 to myosin filaments enables Rap1-dependent integrin activation and enhanced collagen phagocytosis.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Collagen; Enzyme Activation; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Humans; Integrin beta1; Mice; Molecular Sequence Data; Myosin-Light-Chain Kinase; Nonmuscle Myosin Type IIA; Peptides; Phagocytosis; rap1 GTP-Binding Proteins; Recombinant Fusion Proteins; RNA, Small Interfering; Sequence Alignment; Talin

Nonmuscle myosin II isoforms A and B (hereafter, IIA and IIB) perform unique roles in cell migration, even though both isoforms share the same basic molecular functions. That IIA and IIB assume distinct subcellular distribution in migrating cells suggests that discrete spatiotemporal regulation of each isoform's activity may provide a basis for its unique migratory functions. Here, we make the surprising finding that swapping a small C-terminal portion of the tail between IIA and IIB inverts the distinct distribution of these isoforms in migrating cells. Moreover, swapping this region between isoforms also inverts their specific turnover properties, as assessed by fluorescence recovery after photobleaching and Triton solubility. These data, acquired through the use of chimeras of IIA and IIB, suggest that the C-terminal region of the myosin heavy chain supersedes the distinct motor properties of the two isoforms as the predominant factor directing isoform-specific distribution. Furthermore, our results reveal a correlation between isoform solubility and distribution, leading to the proposal that the C-terminal region regulates isoform distribution by tightly controlling the amount of each isoform that is soluble and therefore available for redistribution into new protrusions.

Topics: Amides; Animals; Cell Line; Cell Movement; Enzyme Inhibitors; Fibroblasts; Fluorescence Recovery After Photobleaching; Heterocyclic Compounds, 4 or More Rings; Humans; Myosin Heavy Chains; Nonmuscle Myosin Type IIA; Nonmuscle Myosin Type IIB; Protein Isoforms; Pyridines; Recombinant Fusion Proteins

Mutations in contractile proteins in heart muscle can cause anatomical changes that result in cardiac arrhythmias and sudden cardiac death. However, a conundrum has existed because mutations in one such contractile protein, a so-called Ca2+ sensor troponin T (TnT), can promote ventricular rhythm disturbances even in the absence of hypertrophy or fibrosis. Thus, these mutations must enhance abnormal electrophysiological events via alternative means. In this issue of the JCI, Baudenbacher et al. report a novel mechanism to explain this puzzle (see the related article beginning on page 3893). They show that a selected TnT mutation in the adult mouse heart can markedly increase the sensitivity of cardiac muscle myofilaments to Ca2+ and enhance the susceptibility to arrhythmia, even in the absence of anatomical deformities. As these same mutations can cause some forms of arrhythmias in humans, these findings are of both basic and translational significance.

Topics: Actin Cytoskeleton; Action Potentials; Animals; Calcium; Cardiomyopathy, Hypertrophic; Cardiotonic Agents; Cats; Death, Sudden, Cardiac; Disease Models, Animal; Disease Susceptibility; Female; Fibrosis; Heterocyclic Compounds, 4 or More Rings; Humans; Male; Mice; Mice, Mutant Strains; Quinolines; Risk Factors; Tachycardia, Ventricular; Thiadiazines; Troponin T

In human cardiomyopathy, anatomical abnormalities such as hypertrophy and fibrosis contribute to the risk of ventricular arrhythmias and sudden death. Here we have shown that increased myofilament Ca2+ sensitivity, also a common feature in both inherited and acquired human cardiomyopathies, created arrhythmia susceptibility in mice, even in the absence of anatomical abnormalities. In mice expressing troponin T mutants that cause hypertrophic cardiomyopathy in humans, the risk of developing ventricular tachycardia was directly proportional to the degree of Ca2+ sensitization caused by the troponin T mutation. Arrhythmia susceptibility was reproduced with the Ca2+-sensitizing agent EMD 57033 and prevented by myofilament Ca2+ desensitization with blebbistatin. Ca2+ sensitization markedly changed the shape of ventricular action potentials, resulting in shorter effective refractory periods, greater beat-to-beat variability of action potential durations, and increased dispersion of ventricular conduction velocities at fast heart rates. Together these effects created an arrhythmogenic substrate. Thus, myofilament Ca2+ sensitization represents a heretofore unrecognized arrhythmia mechanism. The protective effect of blebbistatin provides what we believe to be the first direct evidence that reduction of Ca2+ sensitivity in myofilaments is antiarrhythmic and might be beneficial to individuals with hypertrophic cardiomyopathy.

Topics: Actin Cytoskeleton; Action Potentials; Animals; Calcium; Cardiomyopathy, Hypertrophic; Cardiotonic Agents; Cats; Death, Sudden, Cardiac; Disease Models, Animal; Disease Susceptibility; Female; Fibrosis; Heterocyclic Compounds, 4 or More Rings; Humans; Male; Mice; Mice, Mutant Strains; Quinolines; Risk Factors; Tachycardia, Ventricular; Thiadiazines; Troponin T

Blebbistatin (BLEB) is a recently discovered compound that inhibits myosin-II ATPase activity. In this study, we tested BLEB in intact and skinned isolated rat cardiac trabeculae, rat intact myocytes, and single rabbit psoas myofibrils. BLEB (10 muM) reduced twitch force and cell shortening that was reversed by exposure to light. BLEB treatment of skinned trabeculae in the dark (1 hr) reduced Ca(2+)-activated force (EC(50) = 0.38 +/- 0.03 muM). Rapid (<5 ms) BLEB application in Ca(2+)-activated rabbit myofibrils reduced force proportional to [BLEB]. Two-photon Indo1-AM ratio-metric confocal line-scan microscopy revealed no impact of BLEB on the cytosolic Ca(2+) transient. BLEB reduced contractile force in skinned trabeculae without affecting tension-dependent myofilament ATPase activity. We conclude that BLEB specifically uncouples cardiac myofilament activation from Ca(2+) activation without affecting EC coupling or cross-bridge cycling parameters. This agent could be useful to uncouple myofilament contractility from electrical events that lead to sarcoplasmic reticulum Ca(2+) release in the cardiac myocyte (uncoupling agent) However, the compound is very sensitive to light, a property that limits its application to mechanistic physiological studies.

Topics: Actin Cytoskeleton; Adenosine Triphosphatases; Aniline Compounds; Animals; Calcium Signaling; Cytosol; Data Interpretation, Statistical; Diacetyl; Dose-Response Relationship, Drug; Electric Stimulation; Enzyme Inhibitors; Fluorescent Dyes; Heterocyclic Compounds, 4 or More Rings; In Vitro Techniques; Male; Microscopy, Confocal; Muscle Contraction; Muscle Fibers, Skeletal; Muscle, Skeletal; Rats; Xanthenes

Although myelin-associated neurite outgrowth inhibitors express their effects through RhoA/Rho-kinase, the downstream targets of Rho-kinase remain unknown. We examined the involvement of myosin II, which is one of the downstream targets of Rho-kinase, by using blebbistatin - a specific myosin II inhibitor - and small interfering RNA targeting two myosin II isoforms, namely, MIIA and MIIB. We found that neurite outgrowth inhibition by repulsive guidance molecule (RGMa) was mediated via myosin II, particularly MIIA, in cerebellar granule neurons. RGMa induced myosin light chain (MLC) phosphorylation by a Rho-kinase-dependent mechanism. After spinal cord injury in rats, phosphorylated MLC in axons around the lesion site was up-regulated, and this effect depends on Rho-kinase activity. Further, RGMa-induced F-actin reduction in growth cones and growth cone collapse were mediated by MIIA. We conclude that Rho-kinase-dependent activation of MIIA via MLC phosphorylation induces F-actin reduction and growth cone collapse and the subsequent neurite retraction/outgrowth inhibition triggered by RGMa.

Topics: Actins; Animals; Animals, Newborn; Cells, Cultured; Cerebellum; Female; GPI-Linked Proteins; Heterocyclic Compounds, 4 or More Rings; Immunoprecipitation; In Situ Nick-End Labeling; Laminectomy; Membrane Glycoproteins; Myosin Light Chains; Myosin Type II; Nerve Tissue Proteins; Neural Inhibition; Neurites; Neurons; Phosphorylation; Rats; Rats, Wistar; rho-Associated Kinases; RNA, Small Interfering; Transfection

Myosin II motors play several important roles in a variety of cellular processes, some of which involve active assembly/disassembly of cytoskeletal substructures. Myosin II motors have been shown to function in actin bundle turnover in neuronal growth cones and in the recycling of actin filaments during cytokinesis. Close examination had shown an intimate relationship between myosin II motor adenosine triphosphatase activity and actin turnover rate. However, the direct implication of myosin II in actin turnover is still not understood. Herein, we show, using high-resolution cryo-transmission electron microscopy, that myosin II motors control the turnover of actin bundles in a concentration-dependent manner in vitro. We demonstrate that disassembly of actin bundles occurs through two main stages: the first stage involves unbundling into individual filaments, and the second involves their subsequent depolymerization. These evidence suggest that, in addition to their "classical" contractile abilities, myosin II motors may be directly implicated in active actin depolymerization. We believe that myosin II motors may function similarly in vivo (e.g., in the disassembly of the contractile ring by fine tuning the local concentration/activity of myosin II motors).

Topics: Actin Cytoskeleton; Actin Depolymerizing Factors; Actins; Adenosine Triphosphatases; Animals; Carrier Proteins; Cytoskeleton; Ethylmaleimide; Fluorescent Dyes; Glutathione Transferase; Heterocyclic Compounds, 4 or More Rings; Microfilament Proteins; Microscopy, Fluorescence; Microscopy, Video; Models, Biological; Molecular Motor Proteins; Myosin Type II; Rabbits; Recombinant Fusion Proteins

Amoeboid movement is believed to involve a pressure gradient along the cell length, with contractions in the posterior region driving cytoplasmic streaming forward. However, a parallel mechanism has yet to be demonstrated in migrating adhesive cells. To probe the distribution of intracellular forces, we microinjected high molecular weight linear polyacrylamide (PAA) as a passive force sensor into migrating NIH3T3 fibroblasts. Injected PAA appeared as amorphous aggregates that underwent shape change and directional movement in response to differential forces exerted by the surrounding environment. PAA injected into the posterior region moved toward the front, whereas PAA in the anterior region never moved to the posterior region. This preferential forward movement was observed only in migrating cells with a defined polarity. Disruption of myosin II activity by blebbistatin inhibited the forward translocation of PAA while cell migration persisted in a disorganized fashion. These results suggest a myosin II-dependent force gradient in migrating cells, possibly as a result of differential cortical contractions between the anterior and posterior regions. This gradient may be responsible for the forward transport of cellular components and for maintaining the directionality during cell migration.

Topics: Acrylic Resins; Animals; Cell Adhesion; Cell Movement; Cytoplasm; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Mice; Motor Cortex; Muscle Contraction; Myosin Type II; NIH 3T3 Cells

We have investigated the mechanical properties of fibroblast cells after adding the myosin inhibitor blebbistatin and the Rho-kinase inhibitor Y-27632 by atomic force microscopy (AFM). We have observed a decrease in the elastic modulus from a value of around 20 kPa down to a value around 8 kPa on a time scale of around 30-60 min when applying the myosin inhibitor blebbistatin, whereas the Y-27632 did not show any prominent mechanical effects. From topographic images, we can conclude that, after adding blebbistatin, actin filaments are not visible any more, whereas Y-27632 did not show any prominent effects in cell morphology. This study shows that tension generated by myosin contributes to the cellular stiffness and thus can be observed by measuring the elastic modulus of cells.

Topics: Actins; Amides; Animals; Biomechanical Phenomena; Cell Line; Cell Shape; Cytoskeleton; Elasticity; Enzyme Inhibitors; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Microscopy, Atomic Force; Myosin Type II; Pyridines; Rats; rho-Associated Kinases

mDia proteins are mammalian homologues of Drosophila diaphanous and belong to the formin family proteins that catalyze actin nucleation and polymerization. Although formin family proteins of nonmammalian species such as Drosophila diaphanous are essential in cytokinesis, whether and how mDia proteins function in cytokinesis remain unknown. Here we depleted each of the three mDia isoforms in NIH 3T3 cells by RNA interference and examined this issue. Depletion of mDia2 selectively increased the number of binucleate cells, which was corrected by coexpression of RNAi-resistant full-length mDia2. mDia2 accumulates in the cleavage furrow during anaphase to telophase, and concentrates in the midbody at the end of cytokinesis. Depletion of mDia2 induced contraction at aberrant sites of dividing cells, where contractile ring components such as RhoA, myosin, anillin, and phosphorylated ERM accumulated. Treatment with blebbistatin suppressed abnormal contraction, corrected localization of the above components, and revealed that the amount of F-actin at the equatorial region during anaphase/telophase was significantly decreased with mDia2 RNAi. These results demonstrate that mDia2 is essential in mammalian cell cytokinesis and that mDia2-induced F-actin forms a scaffold for the contractile ring and maintains its position in the middle of a dividing cell.

Topics: Actins; Anaphase; Animals; Cytokinesis; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Mice; Microtubule-Associated Proteins; NADPH Dehydrogenase; NIH 3T3 Cells; Protein Isoforms; Protein Transport; rho GTP-Binding Proteins; RNA Interference; Telophase

The characterization of cellular phenotypes of heart disorders can be achieved by isolating cardiac myocytes from mouse models or genetically modifying wild-type cells in culture. However, adult mouse cardiac myocytes show extremely low tolerance to isolation and primary culture conditions. Previous studies indicate that 2,3-butanedione monoximine (BDM), a nonspecific excitation-contraction coupling inhibitor, can improve the viability of isolated adult mouse cardiac myocytes. The mechanisms of the beneficial and unwanted nonspecific actions of BDM on cardiac myocytes are not understood. To understand what contributes to murine adult cardiac myocyte stability in primary culture and improve this model system for experimental use, the specific myosin II inhibitor blebbistatin was explored as a media supplement to inhibit mouse myocyte contraction. Enzymatically isolated adult mouse cardiac myocytes were cultured with blebbistatin or BDM as a media supplement. Micromolar concentrations of blebbistatin significantly increased the viability, membrane integrity, and morphology of adult cardiac myocytes compared with cells treated with previously described 10 mM BDM. Cells treated with blebbistatin also showed efficient adenovirus gene transfer and stable transgene expression, and unlike BDM, blebbistatin does not appear to interfere with cell adhesion. Higher concentrations of BDM actually worsened myocyte membrane integrity and transgene expression. Therefore, the specific inhibition of myosin II activity by blebbistatin has significant beneficial effects on the long-term viability of adult mouse cardiac myocytes. Furthermore, the unwanted effects of BDM on adult mouse cardiac myocytes, perhaps due to its nonspecific activities or action as a chemical phosphatase, can be avoided by using blebbistatin.

Topics: Adenoviridae; Animals; Cell Adhesion; Cell Culture Techniques; Cell Membrane; Cell Shape; Cell Survival; Cells, Cultured; Diacetyl; Dose-Response Relationship, Drug; Enzyme Inhibitors; Genetic Vectors; Green Fluorescent Proteins; Heterocyclic Compounds, 4 or More Rings; Mice; Mice, Inbred C57BL; Myocardial Contraction; Myocytes, Cardiac; Myosin Type II; Time Factors; Transfection; Transgenes

Blebbistatin is a myosin II-specific inhibitor. However, the mechanism and tissue specificity of the drug are not well understood. Blebbistatin blocked the chemotaxis of vascular smooth muscle cells (VSMCs) toward sphingosylphosphorylcholine (IC(50) = 26.1 +/- 0.2 and 27.5 +/- 0.5 microM for GbaSM-4 and A7r5 cells, respectively) and platelet-derived growth factor BB (IC(50) = 32.3 +/- 0.9 and 31.6 +/- 1.3 muM for GbaSM-4 and A7r5 cells, respectively) at similar concentrations. Immunofluorescence and fluorescent resonance energy transfer analysis indicated a blebbistatin-induced disruption of the actin-myosin interaction in VSMCs. Subsequent experiments indicated that blebbistatin inhibited the Mg(2+)-ATPase activity of the unphosphorylated (IC(50) = 12.6 +/- 1.6 and 4.3 +/- 0.5 microM for gizzard and bovine stomach, respectively) and phosphorylated (IC(50) = 15.0 +/- 0.6 microM for gizzard) forms of purified smooth muscle myosin II, suggesting a direct effect on myosin II motor activity. It was further observed that the Mg(2+)-ATPase activities of gizzard myosin II fragments, heavy meromyosin (IC(50) = 14.4 +/- 1.6 microM) and subfragment 1 (IC(50) = 5.5 +/- 0.4 microM), were also inhibited by blebbistatin. Assay by in vitro motility indicated that the inhibitory effect of blebbistatin was reversible. Electron-microscopic evaluation showed that blebbistatin induced a distinct conformational change (i.e., swelling) of the myosin II head. The results suggest that the site of blebbistatin action is within the S1 portion of smooth muscle myosin II.

Topics: Actins; Animals; Becaplermin; Cattle; Cell Line; Chemotaxis; Chickens; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fluorescence Resonance Energy Transfer; Fluorescent Antibody Technique; Guinea Pigs; Heterocyclic Compounds, 4 or More Rings; Microscopy, Confocal; Microscopy, Electron; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Myosin Subfragments; Myosin Type II; Phosphorylation; Phosphorylcholine; Platelet-Derived Growth Factor; Protein Conformation; Proto-Oncogene Proteins c-sis; Rats; Sphingosine

A fundamental property of platelets is their ability to transmit cytoskeletal contractile forces to extracellular matrices. While the importance of the platelet contractile mechanism in regulating fibrin clot retraction is well established, its role in regulating the primary hemostatic response, independent of blood coagulation, remains ill defined. Real-time analysis of platelet adhesion and aggregation on a collagen substrate revealed a prominent contractile phase during thrombus development, associated with a 30% to 40% reduction in thrombus volume. Thrombus contraction developed independent of thrombin and fibrin and resulted in the tight packing of aggregated platelets. Inhibition of the platelet contractile mechanism, with the myosin IIA inhibitor blebbistatin or through Rho kinase antagonism, markedly inhibited thrombus contraction, preventing the tight packing of aggregated platelets and undermining thrombus stability in vitro. Using a new intravital hemostatic model, we demonstrate that the platelet contractile mechanism is critical for maintaining the integrity of the primary hemostatic plug, independent of thrombin and fibrin generation. These studies demonstrate an important role for the platelet contractile mechanism in regulating primary hemostasis and thrombus growth. Furthermore, they provide new insight into the underlying bleeding diathesis associated with platelet contractility defects.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Blood Platelets; Clot Retraction; Fibrin; Hemostasis; Heterocyclic Compounds, 4 or More Rings; Humans; In Vitro Techniques; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Motor Proteins; Nonmuscle Myosin Type IIA; Platelet Adhesiveness; Protein Kinase Inhibitors; Receptors, Proteinase-Activated; rho-Associated Kinases; Thrombosis

Topics: Action Potentials; Atrioventricular Block; Atrioventricular Node; Bundle of His; Cardiac Pacing, Artificial; Electrophysiology; Fluorescent Dyes; Heterocyclic Compounds, 4 or More Rings; Humans; Image Processing, Computer-Assisted; In Vitro Techniques; Light; Pyridinium Compounds; Tachycardia, Atrioventricular Nodal Reentry

Salmonella enterica serovar Typhimurium grows within host cells in a permissive compartment termed the Salmonella-containing vacuole (SCV). These bacteria use two distinct type III secretion systems (T3SS) to deliver virulence proteins (effectors) into cells. Effectors secreted by the Salmonella pathogenicity island 1 (SPI-1)-encoded T3SS mediate invasion and early SCV maturation steps, while those secreted by the SPI-2 T3SS affect the SCV at later stages postinfection. Some SPI-2 effectors modulate microtubule motor activity on the SCV. Here, we show that the actin-based motor myosin II also affects SCV dynamics during infection. Following invasion, myosin II is required for SCV positioning near the nucleus of host cells. Later, myosin II counteracts the activities of the SPI-2 effectors PipB2 and SseJ to maintain SCV positioning and stability, respectively. Myosin II activity was required for maximal bacterial growth in macrophages. Rho kinase activity was required for SCV positioning. The effector SopB, a known activator of Rho GTPases, was found to be required for SCV positioning, and transfection of cells with SopB was sufficient to induce myosin II phosphorylation. These studies reveal a novel role for myosin II in controlling SCV dynamics during infection and suggest that SopB activates myosin II.

Topics: Animals; Bacterial Proteins; Cell Division; Cell Nucleus; Gene Expression Regulation; HeLa Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Macrophages; Mice; Myosin Type II; rho-Associated Kinases; Salmonella typhimurium; Vacuoles

Collecting duct cells swell when exposed to arginine vasopressin (AVP) in the presence of a transepithelial osmolality gradient. We investigated the mechanisms of AVP-induced cell swelling in isolated, perfused rat inner medullary collecting ducts (IMCDs) using quantitative video microscopy and fluorescence-based measurements of transepithelial water transport. We tested the roles of transepithelial water flow, basolateral solute entry, and the cytoskeleton (actomyosin). When a transepithelial osmolality gradient was imposed by addition of NaCl to the bath, AVP significantly increased both water flux and cell height. When the osmolality gradient was imposed by addition of mannitol, AVP increased water flux but not cell height, suggesting that AVP-induced cell swelling requires a NaCl gradient and is not merely dependent on the associated water flux. Bumetanide (Na-K-2Cl cotransporter inhibitor) added to the bath markedly diminished the AVP-induced cell height increase. AVP-induced cell swelling was absent in IMCDs from NKCC1-knockout mice. In rat IMCDs, replacement of Na, K, or Cl in the peritubular bath caused significant cell shrinkage, consistent with a basolateral solute transport pathway dependent on all three ions. Immunocytochemistry using an antibody to NKCC1 confirmed basolateral expression in IMCD cells. The conventional nonmuscle myosin II inhibitor blebbistatin also diminished the AVP-induced cell height increase and cell shape change, consistent with a role for the actin cytoskeleton and myosin II. We conclude that the AVP-induced cell height increase is dependent on basolateral solute uptake via NKCC1 and changes in actin organization via myosin II, but is not dependent specifically on increased apical water entry.

Topics: Animals; Arginine Vasopressin; Bumetanide; Cell Size; Heterocyclic Compounds, 4 or More Rings; In Vitro Techniques; Kidney Tubules, Collecting; Male; Mice; Mice, Knockout; Myosin Type II; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sodium-Potassium-Chloride Symporters; Solute Carrier Family 12, Member 2

The development of luminal organs begins with the formation of spherical cysts composed of a single layer of epithelial cells. Using a model three-dimensional cell culture, this study examines the role of a cytoskeletal motor, myosin II, in cyst formation. Caco-2 and SK-CO15 intestinal epithelial cells were embedded into Matrigel, and myosin II was inhibited by blebbistatin or siRNA-mediated knockdown. Whereas control cells formed spherical cysts with a smooth surface, inhibition of myosin II induced the outgrowth of F-actin-rich surface protrusions. The development of these protrusions was abrogated after inhibition of F-actin polymerization or of phospholipase C (PLC) activity, as well as after overexpression of a dominant-negative ADF/cofilin. Surface protrusions were enriched in microtubules and their formation was prevented by microtubule depolymerization. Myosin II inhibition caused a loss of peripheral F-actin bundles and a submembranous extension of cortical microtubules. Our findings suggest that inhibition of myosin II eliminates the cortical F-actin barrier, allowing microtubules to reach and activate PLC at the plasma membrane. PLC-dependent stimulation of ADF/cofilin creates actin-filament barbed ends and promotes the outgrowth of F-actin-rich protrusions. We conclude that myosin II regulates the spherical shape of epithelial cysts by controlling actin polymerization at the cyst surface.

Topics: Actin Cytoskeleton; Actins; Caco-2 Cells; Cell Polarity; Cell Shape; Cofilin 1; Down-Regulation; Epithelial Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Intestinal Mucosa; Myosin Type II; Nocodazole; Organ Culture Techniques; Polymers; RNA Stability; Type C Phospholipases

During anaphase, the nonkinetochore microtubules in the spindle midzone become compacted into the central spindle, a structure which is required to both initiate and complete cytokinesis. We show that Tektin 2 (Tek2) associates with the spindle poles throughout mitosis, organizes the spindle midzone microtubules during anaphase, and assembles into the midbody matrix surrounding the compacted midzone microtubules during cytokinesis. Tek2 small interfering RNA (siRNA) disrupts central spindle organization and proper localization of MKLP1, PRC1, and Aurora B to the midzone and prevents the formation of a midbody matrix. Video microscopy revealed that loss of Tek2 results in binucleate cell formation by aberrant fusion of daughter cells after cytokinesis. Although a myosin II inhibitor, blebbistatin, prevents actin-myosin contractility, the microtubules of the central spindle are compacted. Strikingly, Tek2 siRNA abolishes this actin-myosin-independent midzone microtubule compaction. Thus, Tek2-dependent organization of the central spindle during anaphase is essential for proper midbody formation and the segregation of daughter cells after cytokinesis.

Topics: Actins; Animals; Aurora Kinase B; Aurora Kinases; Cell Cycle Proteins; Centrosome; CHO Cells; Cricetinae; Cricetulus; Cytokinesis; Heterocyclic Compounds, 4 or More Rings; Mice; Microtubule Proteins; Microtubule-Associated Proteins; Microtubules; Myosins; Protein Serine-Threonine Kinases; Protein Transport; RNA, Small Interfering; Spindle Apparatus

Blebbistatin is reported to be a selective and specific small molecule inhibitor of the myosin II isoforms expressed by striated muscles and nonmuscle (IC(50) = 0.5-5 microM) but is a poor inhibitor of purified turkey smooth muscle myosin II (IC(50) approximately 80 microM). We found that blebbistatin potently (IC(50) approximately 3 microM) inhibited the actomyosin ATPase activities of expressed "slow" [smooth muscle myosin IIA (SMA)] and "fast" [smooth muscle myosin IIB (SMB)] smooth muscle myosin II heavy-chain isoforms. Blebbistatin also inhibited the KCl-induced tonic contractions produced by rabbit femoral and renal arteries that express primarily SMA and the weaker tonic contraction produced by the saphenous artery that expresses primarily SMB, with an equivalent potency comparable with that identified for nonmuscle myosin IIA (IC(50) approximately 5 microM). In femoral and saphenous arteries, blebbistatin had no effect on unloaded shortening velocity or the tonic increase in myosin light-chain phosphorylation produced by KCl but potently inhibited beta-escin permeabilized artery contracted with calcium at pCa 5, suggesting that cell signaling events upstream from KCl-induced activation of cross-bridges were unaffected by blebbistatin. It is noteworthy that KCl-induced contractions of chicken gizzard were less potently inhibited (IC(50) approximately 20 microM). Adult femoral, renal, and saphenous arteries did not express significant levels of nonmuscle myosin. These data together indicate that blebbistatin is a potent inhibitor of smooth muscle myosin II, supporting the hypothesis that the force-bearing structure responsible for tonic force maintenance in adult mammalian vascular smooth muscle is the cross-bridge formed from the blebbistatin-dependent interaction between actin and smooth muscle myosin II.

Topics: Animals; Chickens; Female; Heterocyclic Compounds, 4 or More Rings; Muscle, Smooth, Vascular; Myosin Type II; Potassium Chloride; Rabbits; Vasoconstriction

Activation of CD38 in lymphokine-activated killer (LAK) cells involves interleukin-8 (IL8)-mediated protein kinase G (PKG) activation and results in an increase in the sustained intracellular Ca(2+) concentration ([Ca(2+)](i)), cADP-ribose, and LAK cell migration. However, direct phosphorylation or activation of CD38 by PKG has not been observed in vitro. In this study, we examined the molecular mechanism of PKG-mediated activation of CD38. Nonmuscle myosin heavy chain IIA (MHCIIA) was identified as a CD38-associated protein upon IL8 stimulation. The IL8-induced association of MHCIIA with CD38 was dependent on PKG-mediated phosphorylation of MHCIIA. Supporting these observations, IL8- or cell-permeable cGMP analog-induced formation of cADP-ribose, increase in [Ca(2+)](i), and migration of LAK cells were inhibited by treatment with the MHCIIA inhibitor blebbistatin. Binding studies using purified proteins revealed that the association of MHCIIA with CD38 occurred through Lck, a tyrosine kinase. Moreover, these three molecules co-immunoprecipitated upon IL8 stimulation of LAK cells. IL8 treatment of LAK cells resulted in internalization of CD38, which co-localized with MHCIIA and Lck, and blebbistatin blocked internalization of CD38. These findings demonstrate that the association of phospho-MHCIIA with Lck and CD38 is a critical step in the internalization and activation of CD38.

Topics: ADP-ribosyl Cyclase 1; Calcium; Calcium Signaling; Cyclic ADP-Ribose; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Heterocyclic Compounds, 4 or More Rings; Humans; Interleukin-8; Jurkat Cells; Killer Cells, Lymphokine-Activated; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Nonmuscle Myosin Type IIA; Phosphorylation; Protein Binding; Protein Processing, Post-Translational

The main role of the chromosomal passenger complex is to ensure that Aurora B kinase is properly localized and activated before and during mitosis. Borealin, a member of the chromosomal passenger complex, shows increased expression during G2/M phases and is involved in targeting the complex to the centromere and the spindle midzone, where it ensures proper chromosome segregation and cytokinesis. Borealin has a consensus CDK1 phosphorylation site, threonine 106 and can be phosphorylated by Aurora B Kinase at serine 165 in vitro.. Here, we show that Borealin is phosphorylated during mitosis in human cells. Dephosphorylation of Borealin occurs as cells exit mitosis. The phosphorylated form of Borealin is found in an INCENP-containing complex in mitosis. INCENP-containing complexes from cells in S phase are enriched in the phosphorylated form suggesting that phosphorylation may encourage entry of Borealin into the chromosomal passenger complex. Although Aurora B Kinase is found in complexes that contain Borealin, it is not required for the mitotic phosphorylation of Borealin. Mutation of T106 or S165 of Borealin to alanine does not alter the electrophoretic mobility shift of Borealin. Experiments with cyclohexamide and the phosphatase inhibitor sodium fluoride suggest that Borealin is phosphorylated by a protein kinase that can be active in interphase and mitosis and that the phosphorylation may be regulated by a short-lived phosphatase that is active in interphase but not mitosis.. Borealin is phosphorylated during mitosis. Neither residue S165, T106 nor phosphorylation of Borealin by Aurora B Kinase is required to generate the mitotic, shifted form of Borealin. Suppression of phosphorylation during interphase is ensured by a labile protein, possibly a cell cycle regulated phosphatase.

Topics: Amino Acid Substitution; Aurora Kinase B; Aurora Kinases; Benzamides; CDC2 Protein Kinase; Cell Cycle Proteins; Centromere; Chromosomal Proteins, Non-Histone; Chromosome Segregation; Consensus Sequence; Cycloheximide; G2 Phase; HeLa Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Inhibitor of Apoptosis Proteins; Interphase; Metaphase; Microtubule-Associated Proteins; Mitosis; Multiprotein Complexes; Mutagenesis, Site-Directed; Mutation, Missense; Neoplasm Proteins; Nocodazole; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Phosphoserine; Piperazines; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein Synthesis Inhibitors; Quinazolines; Recombinant Fusion Proteins; Sodium Fluoride; Spindle Apparatus; Substrate Specificity; Survivin; Transfection

The molecular basis for asymmetric meiotic divisions in mammalian oocytes that give rise to mature eggs and polar bodies remains poorly understood. Previous studies demonstrated that the asymmetrically positioned meiotic chromosomes provide the cue for cortical polarity in mouse oocytes. Here we show that the chromatin-induced cortical response can be fully reconstituted by injecting DNA-coated beads into metaphase II-arrested eggs. The injected DNA beads induce a cortical actin cap, surrounded by a myosin II ring, in a manner that depends on the number of beads and their distance from the cortex. The Ran GTPase plays a critical role in this process, because dominant-negative and constitutively active Ran mutants disrupt DNA-induced cortical polarization. The Ran-mediated signaling to the cortex is independent of the spindle but requires cortical myosin II assembly. We hypothesize that a Ran(GTP) gradient serves as a molecular ruler to interpret the asymmetric position of the meiotic chromatin.

Topics: Animals; Bridged Bicyclo Compounds, Heterocyclic; Cell Polarity; Chromatin; DNA; Enzyme Activation; Female; Heterocyclic Compounds, 4 or More Rings; Mice; Microinjections; Microspheres; Microtubules; Mitogen-Activated Protein Kinases; Myosin Type II; Oocytes; ran GTP-Binding Protein; Signal Transduction; Thiazolidines

Cell repulsion responses to Eph receptor activation are linked to rapid actin cytoskeletal reorganizations, which in turn are partially mediated by Rho-ROCK (Rho kinase) signalling, driving actomyosin contractility. In the present study, we show that Rho alone is not sufficient for this repulsion response. Rather, Cdc42 (cell division cycle 42) and its effector MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) are also critical for ephrinB-induced cell retraction. Stimulation of endothelial cells with ephrinB2 triggers rapid, but transient, cell retraction. We show that, although membrane retraction is fully blocked by blebbistatin (a myosin-II ATPase inhibitor), it is only partially blocked by inhibiting Rho-ROCK signalling, suggesting that there is ROCK-independent signalling to actomyosin contractility downstream of EphBs. We find that a combination of either Cdc42 or MRCK inhibition with ROCK inhibition completely abolishes the repulsion response. Additionally, endocytosis of ephrin-Eph complexes is not required for initial cell retraction, but is essential for subsequent Rac-mediated re-spreading of cells. Our data reveal a complex interplay of Rho, Rac and Cdc42 in the process of EphB-mediated cell retraction-recovery responses.

Topics: Biotinylation; cdc42 GTP-Binding Protein; Cells, Cultured; Endothelium, Vascular; GTPase-Activating Proteins; Heterocyclic Compounds, 4 or More Rings; Humans; Immunohistochemistry; Receptors, Eph Family; Rho Factor; Signal Transduction; Umbilical Veins

Non-muscle myosin II has diverse functions in cell contractility, cytokinesis and locomotion, but the specific contributions of its different isoforms have yet to be clarified. Here, we report that ablation of the myosin IIA isoform results in pronounced defects in cellular contractility, focal adhesions, actin stress fibre organization and tail retraction. Nevertheless, myosin IIA-deficient cells display substantially increased cell migration and exaggerated membrane ruffling, which was dependent on the small G-protein Rac1, its activator Tiam1 and the microtubule moter kinesin Eg5. Myosin IIA deficiency stabilized microtubules, shifting the balance between actomyosin and microtubules with increased microtubules in active membrane ruffles. When microtubule polymerization was suppressed, myosin IIB could partially compensate for the absence of the IIA isoform in cellular contractility, but not in cell migration. We conclude that myosin IIA negatively regulates cell migration and suggest that it maintains a balance between the actomyosin and microtubule systems by regulating microtubule dynamics.

Topics: Actomyosin; Aminoquinolines; Animals; Azepines; Cell Adhesion; Cell Movement; Chlorocebus aethiops; COS Cells; Embryonic Stem Cells; Enzyme Inhibitors; Fibroblasts; Guanine Nucleotide Exchange Factors; Heterocyclic Compounds, 4 or More Rings; Humans; Kinesins; Mice; Microtubules; Naphthalenes; Nocodazole; Nonmuscle Myosin Type IIA; Nonmuscle Myosin Type IIB; Pyrimidines; rac1 GTP-Binding Protein; RNA, Small Interfering; T-Lymphoma Invasion and Metastasis-inducing Protein 1; Thiones; Transfection; Vinblastine

Endocytosis of chemokine receptors regulates signal transduction initiated by chemokines, but the molecular mechanisms underlying this process are not fully defined. In this work, we assessed the involvement of the motor protein nonmuscle myosin heavy chain IIA (MIIA) in the endocytosis of CXCR4 induced by SDF-1alpha (also known as CXCL12) in T lymphocytes. Overexpression of the C-terminal half of MIIA inhibited the ligand-induced endocytosis of CXCR4, but not that of transferrin receptor. Targeting MIIA either by silencing its expression with small interfering RNA (siRNA) or by blebbistatin treatment also inhibited endocytosis of CXCR4. Inhibition of endocytosis of CXCR4 by targeting endogenous MIIA resulted in an increased migration of T cells induced by SDF-1alpha, and in the inhibition of the HIV-1-Env antifusogenic activity of this chemokine. Coimmunoprecipitation and protein-protein binding studies demonstrated that MIIA interacts with both the cytoplasmic tail of CXCR4 and beta-arrestin. Moreover, SDF-1alpha promotes a rapid MIIA-beta-arrestin dissociation. Our data reveal a novel role for MIIA in CXCR4 endocytosis, which involves its dynamic association with beta-arrestin and highlights the role of endogenous MIIA as a regulator of CXCR4 internalization and, therefore, the onset of SDF-1alpha signaling.

Topics: Arrestins; beta-Arrestins; Cell Line, Tumor; Cell Movement; Chemokine CXCL12; Endocytosis; Heterocyclic Compounds, 4 or More Rings; Humans; Nonmuscle Myosin Type IIA; Protein Binding; Receptors, CXCR4; Receptors, Transferrin; Recombinant Proteins; Signal Transduction; T-Lymphocytes

Kidney-derived Madin Darby canine kidney (MDCK) cells form lumina at their apices, and target luminal proteins to an intracellular vacuolar apical compartment (VAC) when prevented from polarizing. Hepatocytes, by contrast, organize their luminal surfaces (the bile canaliculi; BC) between their lateral membranes, and, when nonpolarized, they display an intracellular luminal compartment that is distinct from the VACs of MDCK cells. Overexpression of the serine/threonine kinase Par1b/EMK1/MARK2 induces BC-like lateral lumina and a hepatic-type intracellular luminal compartment in MDCK cells, suggesting a role for Par1b in the branching decision between kidney- and hepatic-type epithelial phenotypes. Here, we report that Par1b promotes lateral lumen polarity in MDCK cells independently of Ca(2+)-mediated cell-cell adhesion by inhibiting myosin II in a rho kinase-dependent manner. Polarization was inhibited by E-cadherin depletion but promoted by an adhesion-defective E-cadherin mutant. By contrast, apical surface formation in control MDCK cells required Ca(2+)-dependent cell-cell adhesion, but it occurred in the absence of E-cadherin. We propose that E-cadherin, when in an adhesion-incompetent state at the lateral domain, serves as targeting patch for the establishment of lateral luminal surfaces. E-cadherin depletion also reverted the hepatic-type intracellular luminal compartment in Par1b-MDCK cells to VACs characteristic of control MDCK cells, indicating a novel link between E-cadherin and luminal protein targeting.

Topics: alpha Catenin; Animals; beta Catenin; Cadherins; Cell Adhesion; Cell Line; Cell Polarity; Dogs; Heterocyclic Compounds, 4 or More Rings; Intracellular Signaling Peptides and Proteins; Liver; Myosin Type II; Protein Serine-Threonine Kinases; rho-Associated Kinases; RNA Interference; Signal Transduction

Hepatic stellate cells play an essential role in the liver's injury response. Although stellate cells are defined by the presence of cytoplasmic protrusions, the function of these characteristic structures has been obscure. We hypothesized that stellate cell protrusions act by coupling injury-associated stimuli to chemotaxis. To test this hypothesis, we developed an assay for directly visualizing the response of living stellate cells in early primary culture to local stimulation of the tips of protrusions with platelet-derived growth factor-BB (PDGF). Stellate cells exhibited elongate protrusions containing actin, myosin, and tubulin. PDGF, but not cytochrome C, localized at a protrusion tip induced a coordinated series of morphological events--cell spreading at the tip, movement of the cell body toward the PDGF, and retraction of trailing protrusions--that resulted in chemotaxis. Soluble PDGF and AG 1296, a receptor tyrosine kinase inhibitor, both reduced stellate cell chemotaxis. PDGF-induced chemotaxis was associated with an early and transient increase in myosin phosphorylation within the spreading lamella. We observed that blebbistatin, a myosin II inhibitor, completely and reversibly blocked protrusion-mediated lamella formation and chemotaxis. Moreover, blockade of MRLC phosphorylation with the myosin light chain kinase inhibitor, ML-7, or the rho kinase inhibitor, Y-27632, blocked lamella formation, myosin phosphorylation within the protrusion, and chemotaxis.. These results support a model in which protrusions permit stellate cells to promptly detect PDGF distant from their cell bodies and transduce this signal into mechanical forces that propel the cell toward the site of injury.

Topics: Angiogenesis Inducing Agents; Animals; Becaplermin; Cells, Cultured; Chemotaxis; Hepatocytes; Heterocyclic Compounds, 4 or More Rings; Myosin Type II; Phosphorylation; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Pseudopodia; Rats; Rats, Sprague-Dawley; Signal Transduction; Tyrphostins

Application of fluorescence imaging of cardiac electrical activity is limited by motion artifacts and/or side effects of currently available pharmacologic excitation-contraction uncoupling agents.. The purpose of this study was to test whether blebbistatin, a recently discovered inhibitor of myosin II isoforms, can be used as an excitation-contraction uncoupler.. The specificity and potency of blebbistatin were examined by assaying the effects of blebbistatin on the contraction and basic cardiac electrophysiologic parameters of Langendorff-perfused rabbit hearts, isolated rabbit right ventricle and right atrium, and single rat ventricular myocytes using conventional ECG, surface electrograms, microelectrode recordings, and optical imaging with voltage-sensitive and Ca(2+)-sensitive dyes. Action potential morphology, ECG parameters, cardiac conduction, and refractoriness were determined after perfusion with 0.1-10 microM blebbistatin.. Blebbistatin 5-10 microM completely eliminated contraction in all cardiac preparations but did not have any effect on electrical activity, including ECG parameters, atrial and ventricular effective refractory periods, and atrial and ventricular activation patterns. Blebbistatin 10 microM had no effects on action potential morphology in rabbit cardiac tissue. Blebbistatin inhibited single cellular contraction in a dose-dependent manner with half-maximal inhibitory concentration (IC(50)) = 0.43 microM, without altering the morphologies of intracellular calcium transients. The blebbistatin effect was completely reversible by simultaneous washout and photobleaching by ultraviolet light. Blebbistatin is a promising novel selective excitation-contraction uncoupler that can be used for optical imaging of cardiac tissues.

Topics: Action Potentials; Animals; Dose-Response Relationship, Drug; Electrocardiography; Electrophysiologic Techniques, Cardiac; Heart Atria; Heart Ventricles; Heterocyclic Compounds, 4 or More Rings; Inhibitory Concentration 50; Isoenzymes; Male; Microelectrodes; Models, Animal; Myocardial Contraction; Myocytes, Cardiac; Myosin Type II; Rabbits; Rats; Rats, Sprague-Dawley; Refractory Period, Electrophysiological; Research Design; Sinoatrial Node; Uncoupling Agents; Ventricular Function

Topics: Action Potentials; Animals; Artifacts; Electrocardiography; Electrophysiologic Techniques, Cardiac; Heart Atria; Heart Ventricles; Heterocyclic Compounds, 4 or More Rings; Humans; Motion; Myocardial Contraction; Sinoatrial Node; Uncoupling Agents; Ventricular Function

Shape change and centralization of granules surrounded by a microtubular coil (internal contraction) are among the earliest morphologic changes observed following platelet activation. Myosin IIA contributes to initiation of platelet shape change, but its role in internal contraction has not been defined.. To define the contribution of myosin IIA to platelet internal contraction.. Aspirin-treated platelets suspended in calcium-free buffer were activated with a low concentration (25 nm) of the thromboxane A(2) analog U46619 which initiated shape change and internal contraction via a Rho kinase pathway. Shape change and internal contraction were assessed by aggregometry and transmission electron microscopy (TEM), and Rho activation and myosin regulatory light chain (MRLC) phosphorylation were studied concurrently.. Low-concentration blebbistatin (10 microm) inhibited internal contraction in the majority of platelets with minimal inhibition of shape change without significant suppression of MRLC phosphorylation. Higher blebbistatin concentrations (25-100 microm) produced concentration-dependent inhibition of aggregation, shape change, Rho activation, and MRLC phosphorylation. These data demonstrate: (i) direct platelet myosin IIA participation in internal contraction; and (ii) inhibition of Rho activation and MRLC phosphorylation by >10 microm blebbistatin.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Actins; Adult; Amides; Blood Platelets; Cell Shape; Heterocyclic Compounds, 4 or More Rings; Humans; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Microscopy, Electron, Transmission; Myosin Light Chains; Nonmuscle Myosin Type IIA; Phosphorylation; Platelet Aggregation; Protein Serine-Threonine Kinases; Pyridines; rho-Associated Kinases

Blebbistatin is a powerful inhibitor of actin-myosin interaction in isolated contractile proteins. To examine whether blebbistatin acts in a similar manner in the organized contractile system of striated muscle, the effects of blebbistatin on contraction of cardiac tissue from mouse were studied. The contraction of paced intact papillary muscle preparations and shortening of isolated cardiomyocytes were inhibited by blebbistatin with inhibitory constants in the micromolar range (1.3-2.8 muM). The inhibition constants are similar to those previously reported for isolated cardiac myosin subfragments showing that blebbistatin action is similar in filamentous myosin of the cardiac contractile apparatus and isolated proteins. The inhibition was not associated with alterations in action potential duration or decreased influx through L-type Ca(2+) channels. Experiments on permeabilized cardiac muscle preparations showed that the inhibition was not due to alterations in Ca(2+) sensitivity of the contractile filaments. The maximal shortening velocity was not affected by 1 muM blebbistatin. In conclusion, we show that blebbistatin is an inhibitor of the actin-myosin interaction in the organized contractile system of cardiac muscle and that its action is not due to effects on the Ca(2+) influx and activation systems.

Topics: Actins; Action Potentials; Animals; Calcium; Female; Heterocyclic Compounds, 4 or More Rings; In Vitro Techniques; Mice; Mice, Inbred C57BL; Myocardial Contraction; Myocytes, Cardiac; Myosins; Papillary Muscles; Patch-Clamp Techniques

An essential feature of dendritic cell immune surveillance is endocytic sampling of the environment for non-self antigens primarily via macropinocytosis and phagocytosis. The role of several members of the myosin family of actin based molecular motors in dendritic cell endocytosis and endocytic vesicle movement was assessed through analysis of dendritic cells derived from mice with functionally null myosin mutations. These include the dilute (myosin Va), Snell's waltzer (myosin VI) and shaker-1 (myosin VIIa) mouse lines. Non muscle myosin II function was assessed by treatment with the inhibitor, blebbistatin. Flow cytometric analysis of dextran uptake by dendritic cells revealed that macropinocytosis was enhanced in Snell's waltzer dendritic cells while shaker-1 and blebbistatin-treated cells were comparable to controls. Comparison of fluid phase uptake using pH insensitive versus pH sensitive fluorescent dextrans revealed that in dilute cells rates of uptake were normal but endosomal acidification was accelerated. Phagocytosis, as quantified by uptake of E. coli, was normal in dilute while dendritic cells from Snell's waltzer, shaker-1 and blebbistatin treated cells exhibited decreased uptake. Microtubule mediated movements of dextran-or transferrin-tagged endocytic vesicles were significantly faster in dendritic cells lacking myosin Va. Loss of myosin II, VI or VIIa function had no significant effects on rates of endocytic vesicle movement.

Topics: Animals; Bone Marrow Cells; Cells, Cultured; Cytoskeleton; Dendritic Cells; Endocytosis; Heterocyclic Compounds, 4 or More Rings; Mice; Mice, Mutant Strains; Myosin Type V; Myosins; Nonmuscle Myosin Type IIA; Phagocytosis; Pinocytosis; Transport Vesicles

During brain development, neural precursor cells migrate along radial glial fibers to populate the neocortex. RNA interference (RNAi) of the lissencephaly gene LIS1 (also known as PAFAH1b1) inhibits somal movement but not process extension of neural precursors in live brain slices. Here we report imaging of the subcellular events accompanying neural precursor migration and the effects of LIS1, cytoplasmic dynein and myosin II inhibition. Centrosomes move continuously and often far in advance of nuclei, which show extreme saltatory behavior. LIS1 and dynein RNAi inhibit centrosomal and nuclear movement independently, whereas myosin II inhibition blocks only nuclear translocation. Imaging of the microtubule end-binding protein 3 (EB3) reveals a centrosome-centered array of microtubules in live neural precursors under all conditions examined. Dynein is concentrated both at a swelling in the leading process reported to initiate each migratory cycle and in the soma. Thus, dynein pulls on the microtubule network from the swelling. The nucleus is transported along the trailing microtubules by dynein assisted by myosin II.

Topics: Animals; Cell Movement; Cerebral Cortex; Dyneins; Electroporation; Embryo, Mammalian; Heterocyclic Compounds, 4 or More Rings; Humans; Intracellular Space; Luminescent Proteins; Mice; Microscopy, Confocal; Microtubule-Associated Proteins; Myosin Type II; Nerve Tissue Proteins; Neurons; Oligonucleotides, Antisense; Organ Culture Techniques; Rats; Stem Cells; Time Factors

Nonmuscle myosin II (Myo2) has been shown to associate with membranes of the trans-Golgi network and to be involved in Golgi to ER retrograde protein transport. Here, we provide evidence that Myo2 not only associates with membranes but functions to transport vesicles on actin filaments (AFs). We used extracts from unactivated clam oocytes for these studies. AFs assembled spontaneously in these extracts and myosin-dependent vesicle transport was observed upon activation. In addition, actin bundles formed and moved relative to each other at an average speed of 0.30 microm/s. Motion analysis revealed that vesicles moved on the spontaneously assembled AFs at speeds greater than 1 microm/s. The motor on these vesicles was identified as a member of the nonmuscle Myo2 family based on sequence determination by Edman chemistry. Vesicles in these extracts were purified by sucrose gradient centrifugation and movement was reconstituted in vitro using skeletal muscle actin coated coverslips. When peripheral membrane proteins of vesicles including Myo2 were removed by salt stripping or when extracts were treated with an antibody specific to clam oocyte nonmuscle Myo2, vesicle movement was inhibited. Blebbistatin, a Myo2 specific inhibitor, also blocked vesicle movement. Myo2 light chain kinase activity was found to be essential for vesicle movement and sliding of actin bundles. Together, our data provide direct evidence that nonmuscle Myo2 is involved in actin-dependent vesicle transport in clam oocytes.

Topics: Actins; Amino Acid Sequence; Animals; Biological Transport; Cytoskeleton; Heterocyclic Compounds, 4 or More Rings; Molecular Sequence Data; Myosin Type II; Oocytes; Secretory Vesicles; Sequence Homology, Amino Acid; Spisula

To understand the mechanism of cell migration, one needs to know how the parts of the motile machinery of the cell are assembled and how they move with respect to each other. Actin and myosin II are thought to be the major structural and force-generating components of this machinery (Mitchison and Cramer, 1996; Parent, 2004). The movement of myosin II along actin filaments is thought to generate contractile force contributing to cell translocation, but the relative motion of the two proteins has not been investigated. We use fluorescence speckle and conventional fluorescence microscopy, image analysis, and computer tracking techniques to generate comparative velocity and assembly maps of actin and myosin II over the entire cell in a simple model system of persistently migrating fish epidermal keratocytes. The results demonstrate contrasting polarized assembly patterns of the two components, indicate force generation at the lamellipodium-cell body transition zone, and suggest a mechanism of anisotropic network contraction via sliding of myosin II assemblies along divergent actin filaments.

Topics: Actin Cytoskeleton; Animals; Biomechanical Phenomena; Cell Movement; Cell Polarity; Cytoskeleton; Fishes; Heterocyclic Compounds, 4 or More Rings; Keratinocytes; Models, Biological; Motion; Myosin Type II; Pseudopodia

MyosinIIs are adenosine triphosphate-driven molecular motors that form part of a cell's contractile machinery. They are activated by phosphorylation of their light chains, by either activation of myosin light chain (MLC) kinase or inhibition of MLC phosphatase via Rho kinase (ROCK). MyosinIIa phosphorylation underlies platelet rounding and stress fiber formation.. To identify the functional significance of myosinIIa in platelet spreading and thrombus formation on collagen using inhibitors of ROCK (Y27632) and myosinII (blebbistatin).. Stress fiber formation on collagen is inhibited by both Y27632 and blebbistatin. A substantial proportion of spread platelets generate internal holes or splits on collagen, presumably because of a reduction in contractile strength. Platelet integrity, however, is maintained. In an in vitro model, thrombus embolization on collagen is increased in the presence of Y27632 and blebbistatin at intermediate shear, leading to a reduction in platelet aggregate growth. Moreover, Y27632 causes a marked reduction in thrombus formation in an in vivo laser-injury model.. MyosinIIa contractility is required for maintenance of platelet structure during spreading on collagen and contributes to thrombus stability.

Topics: Actins; Amides; Blood Platelets; Cell Movement; Collagen; Cytoskeleton; Heterocyclic Compounds, 4 or More Rings; Humans; Models, Biological; Myosin Light Chains; Nonmuscle Myosin Type IIA; Phosphorylation; Platelet Aggregation; Pyridines; rho-Associated Kinases; Thrombosis

The integrity and function of the epithelial barrier is dependent on the apical junctional complex (AJC) composed of tight and adherens junctions and regulated by the underlying actin filaments. A major F-actin motor, myosin II, was previously implicated in regulation of the AJC, however direct evidence of the involvement of myosin II in AJC dynamics are lacking and the molecular identity of the myosin II motor that regulates formation and disassembly of apical junctions in mammalian epithelia is unknown. We investigated the role of nonmuscle myosin II (NMMII) heavy chain isoforms, A, B, and C in regulation of epithelial AJC dynamics and function. Expression of the three NMMII isoforms was observed in model intestinal epithelial cell lines, where all isoforms accumulated within the perijunctional F-actin belt. siRNA-mediated downregulation of NMMIIA, but not NMMIIB or NMMIIC expression in SK-CO15 colonic epithelial cells resulted in profound changes of cell morphology and cell-cell adhesions. These changes included acquisition of a fibroblast-like cell shape, defective paracellular barrier, and substantial attenuation of the assembly and disassembly of both adherens and tight junctions. Impaired assembly of the AJC observed after NMMIIA knock-down involved dramatic disorganization of perijunctional actin filaments. These findings provide the first direct non-pharmacological evidence of myosin II-dependent regulation of AJC dynamics in mammalian epithelia and highlight a unique role of NMMIIA in junctional biogenesis.

Topics: Cell Adhesion; Cell Line, Tumor; Cell Polarity; DNA Primers; Down-Regulation; Epithelial Cells; Gene Silencing; Heterocyclic Compounds, 4 or More Rings; Humans; Intercellular Junctions; Intestinal Mucosa; Myosin Heavy Chains; Polymerase Chain Reaction; Protein Isoforms; RNA, Small Interfering

To identify cytoskeletal proteins that change conformation or assembly within stressed cells, in situ labeling of sterically shielded cysteines with fluorophores was analyzed by fluorescence imaging, quantitative mass spectrometry, and sequential two-dye labeling. Within red blood cells, shotgun labeling showed that shielded cysteines in the two isoforms of the cytoskeletal protein spectrin were increasingly labeled as a function of shear stress and time, indicative of forced unfolding of specific domains. Within mesenchymal stem cells-as a prototypical adherent cell-nonmuscle myosin IIA and vimentin are just two of the cytoskeletal proteins identified that show differential labeling in tensed versus drug-relaxed cells. Cysteine labeling of proteins within live cells can thus be used to fluorescently map out sites of molecular-scale deformation, and the results also suggest means to colocalize signaling events such as phosphorylation with forced unfolding.

Topics: Chromatography, Liquid; Cysteine; Cytoskeletal Proteins; Erythrocytes; Fluorescence; Fluorescent Antibody Technique; Fluorescent Dyes; Heterocyclic Compounds, 4 or More Rings; Humans; Mesenchymal Stem Cells; Naphthalenesulfonates; Nonmuscle Myosin Type IIA; Protein Conformation; Protein Folding; Protein Structure, Quaternary; Protein Structure, Tertiary; Spectrin; Stress, Mechanical; Tandem Mass Spectrometry; Temperature; Vimentin

In the retinal pigment epithelium (RPE) of fish, melanosomes (pigment granules) migrate long distances through the cell body into apical projections in the light, and aggregate back into the cell body in the dark. RPE cells can be isolated from the eye, dissociated, and cultured as single cells in vitro. Treatment of isolated RPE cells with cAMP or the phosphatase inhibitor, okadaic acid (OA), stimulates melanosome aggregation, while cAMP or OA washout in the presence of dopamine triggers dispersion. Previous studies have shown that actin filaments are both necessary and sufficient for aggregation and dispersion of melanosomes within apical projections of isolated RPE. The role of myosin II in melanosome motility was investigated using the myosin II inhibitor, blebbistatin, and a specific rho kinase (ROCK) inhibitor, H-1152. Blebbistatin and H-1152 partially blocked melanosome aggregation triggered by cAMP in dissociated, isolated RPE cells and isolated sheets of RPE. In contrast, neither drug affected melanosome dispersion. In cells exposed to either blebbistatin or H-1152, then triggered to aggregate using OA, melanosome aggregation was completely inhibited. These results demonstrate that (1) melanosome aggregation and dispersion occur through different, actin-dependent mechanisms; (2) myosin II and ROCK activity are required for full melanosome aggregation, but not dispersion; (3) partial aggregation that occurred despite myosin II or ROCK inhibition suggests a second component of aggregation that is dependent on cAMP signaling, but independent of ROCK and myosin II.

Topics: Actins; Animals; Cell Aggregation; Cell Movement; Fishes; Heterocyclic Compounds, 4 or More Rings; Melanosomes; Microscopy, Video; Microtubules; Myosin Type II; Okadaic Acid; Pigment Epithelium of Eye; Retina; rho-Associated Kinases

Atrial fibrillation (AF) causes tachycardia-induced atrial electrical remodeling, contributing to the progressive nature of the arrhythmia. Ventricular dysfunction due to a rapid response to AF can cause structural remodeling, but whether AF itself directly promotes atrial fibrosis is controversial. This study investigated the hypothesis that rapid atrial cardiomyocyte activation produces factors that influence atrial fibroblast proliferation and secretory functions.. Cultured canine atrial fibroblasts were treated with medium from rapidly-paced atrial cardiomyocytes, non-paced cardiomyocytes and cardiomyocyte-pacing medium only, and analyzed by [(3)H]thymidine incorporation, Western blot and real-time RT-PCR.. Rapidly-paced cardiomyocyte-conditioned medium reduced [(3)H]thymidine uptake compared to non-paced cardiomyocyte-conditioned medium and medium alone (approximately 85%, P<0.01). Rapidly-paced cardiomyocyte medium increased alpha SMA protein (approximately 55%, p<0.001), collagen-1 (approximately 85%, P<0.05) and fibronectin-1 (approximately 205%, P<0.05) mRNA expression vs. controls. The angiotensin-1 receptor blocker valsartan attenuated pacing-induced alpha SMA changes but did not affect fibroblast proliferation. Suppression of contraction with blebbistatin did not prevent tachypacing-induced changes in [(3)H]thymidine uptake or alpha SMA upregulation, pointing to a primary role of electrical over mechanical cardiomyocyte activity. Atrial tissue from 1-week atrial-tachypaced dogs with ventricular rate control similarly showed upregulation of alpha SMA protein (approximately 40%, P<0.05), collagen-1 (approximately 380%, P<0.01) and fibronectin-1 (approximately 430%, P<0.001) mRNA versus shams.. Rapidly-paced cardiomyocytes release substances that profoundly alter cardiac fibroblast function, inducing an activated myofibroblast phenotype that is reflected by increased ECM-gene expression in vivo. These findings are consistent with recent observations that AF per se may cause ECM remodeling, and have potentially important consequences for understanding and preventing the mechanisms underlying AF progression.

Topics: Actins; Angiotensin II Type 1 Receptor Blockers; Animals; Atrial Fibrillation; Cell Communication; Cell Proliferation; Cells, Cultured; Collagen Type I; Culture Media; Disease Models, Animal; Dogs; Extracellular Matrix; Fibroblasts; Fibronectins; Heterocyclic Compounds, 4 or More Rings; Muscle Contraction; Myocytes, Cardiac; Tachycardia; Tetrazoles; Valine; Valsartan; Ventricular Remodeling

The myosinII-specific inhibitor blebbistatin was used to attenuate actinomyosinII contractility in E7-chicken retina explant, medulla and spinal cord neuronal cell cultures. Addition of 20-100 microM blebbistatin, a concentration range that reversibly disrupts actin stress fibers, led to a reduction of growth cone lamellipodial areas and to an elongation of filopodia within 5 to 10 min. These morphological changes were completely reversed after removing the inhibitor. In the continued presence of blebbistatin for several hours, a dose-dependent acceleration (up to 6-fold) of neurite outgrowth was observed. The rapidly elongating neuritic processes displayed narrowed growth cones with one to three long filopodia at the leading edge. At the same time, thin neuritic branches emerged in a "push"-like fashion guided by filopodial extensions. Immunocytochemical characterization of these thin sprouts revealed that they contained actin filaments, myosinIIA, phosphorylated neurofilament/tau epitopes, MAP2, NCAM-PSA, and microtubules, demonstrating that these processes presented neurites and not filopodia. The crucial involvement of microtubules in blebbistatin-induced accelerated neurite extension was confirmed by its inhibition in the presence of nocodazole or taxol. The promotion by blebbistatin of neurite outgrowth occurred on polylysine, laminin, as well as on fibronectin as substrate. The presence of the Rho/ROCK-inhibitor Y-27632 also caused a dose-dependent promotion of neurite growth which was, however, 3-fold less pronounced as compared to blebbistatin. In contrast to blebbistatin, Y-27632 led to the enlargement of growth cone lamellipodial extensions. Our data demonstrate that neurite outgrowth and branching are inversely correlated with the degree of actinomyosinII contractility which determines the speed of retrograde flow and turnover of actin filaments and, by this, microtubule extension.

Topics: Actomyosin; Amides; Animals; Central Nervous System; Chick Embryo; Contractile Proteins; Cytoskeletal Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibronectins; Growth Cones; Heterocyclic Compounds, 4 or More Rings; Laminin; Microtubules; Neural Pathways; Neurites; Neurogenesis; Nocodazole; Paclitaxel; Pseudopodia; Pyridines; rho GTP-Binding Proteins; Tubulin Modulators

Although recent studies have demonstrated the importance of calcium/calmodulin (Ca(2+)/CAM) signaling in mammalian fertilization, many targets of Ca(2+)/CAM have not been investigated and represent potentially important regulatory pathways to transduce the Ca2+ signal that is responsible for most events of egg activation. A well-established Ca(2+)/CAM-dependent enzyme is myosin light chain kinase (MYLK2), the downstream target of which is myosin II, an isoform of myosin known to be important in cytokinesis. In fertilized mouse eggs, established inhibitors of MYLK2 and myosin II were investigated for their effects on events of egg activation. The MYLK2 antagonist, ML-7, did not decrease the activity of Ca(2+)/CAM protein kinase II or the elevation of intracellular Ca2+, and it did not delay the onset of Ca2+ oscillations. In contrast, ML-7 inhibited second polar body (PB) formation in a dose-dependent manner and reduced cortical granule (CG) exocytosis by a mean of approximately 50%. The myosin II isoform-specific inhibitor, blebbistatin, had similar inhibitory effects. Although both antagonists had no effect on anaphase onset, they inhibited second PB formation by preventing spindle rotation before telophase II and normal contractile ring constriction. To our knowledge, this is the first report that MYLK2 and myosin II are involved in regulating the position of the meiotic spindle, formation of the second PB, and CG exocytosis. The present results suggest that MYLK2 is one of a family of CAM-dependent proteins that act as multifunctional regulators and transduce the Ca2+ signal at fertilization.

Topics: Animals; Azepines; Calcium Signaling; Cell Cycle; Cell Degranulation; Female; Heterocyclic Compounds, 4 or More Rings; Male; Mice; Myosin Type II; Myosin-Light-Chain Kinase; Naphthalenes; Zygote

During the execution phase of apoptosis, a cell undergoes cytoplasmic and nuclear changes that prepare it for death and phagocytosis. The end-point of the execution phase is condensation into a single apoptotic body or fragmentation into multiple apoptotic bodies. Fragmentation is thought to facilitate phagocytosis; however, mechanisms regulating fragmentation are unknown. An isoform of Rho kinase, ROCK-I, drives membrane blebbing through its activation of actin-myosin contraction; this raises the possibility that ROCK-I may regulate other execution phase events, such as cellular fragmentation. Here, we show that COS-7 cells fragment into a number of small apoptotic bodies during apoptosis; treating with ROCK inhibitors (Y-27632 or H-1152) prevents fragmentation. Latrunculin B and blebbistatin, drugs that interfere with actin-myosin contraction, also inhibit fragmentation. During apoptosis, ROCK-I is cleaved and activated by caspases, while ROCK-II is not activated, but rather translocates to a cytoskeletal fraction. siRNA knock-down of ROCK-I but not ROCK-II inhibits fragmentation of dying cells, consistent with ROCK-I being required for apoptotic fragmentation. Finally, cells dying in the presence of the ROCK inhibitor Y-27632 are not efficiently phagocytized. These data show that ROCK plays an essential role in fragmentation and phagocytosis of apoptotic cells.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Actins; Amides; Animals; Apoptosis; Bridged Bicyclo Compounds, Heterocyclic; Caspase Inhibitors; Caspases; Cell Membrane; Chlorocebus aethiops; COS Cells; DNA Fragmentation; Enzyme Activation; Enzyme Inhibitors; Heterocyclic Compounds, 4 or More Rings; Intracellular Signaling Peptides and Proteins; Marine Toxins; Muscle Contraction; Myosins; PC12 Cells; Phagocytosis; Protein Serine-Threonine Kinases; Protein Transport; Pyridines; Rats; rho-Associated Kinases; RNA, Small Interfering; Thiazoles; Thiazolidines

Dendritic spines show rapid motility and plastic morphology, which may mediate information storage in the brain. It is presently believed that polymerization/depolymerization of actin is the primary determinant of spine motility and morphogenesis. Here, we show that myosin IIB, a molecular motor that binds and contracts actin filaments, is essential for normal spine morphology and dynamics and represents a distinct biophysical pathway to control spine size and shape. Myosin IIB is enriched in the postsynaptic density (PSD) of neurons. Pharmacologic or genetic inhibition of myosin IIB alters protrusive motility of spines, destabilizes their classical mushroom-head morphology, and impairs excitatory synaptic transmission. Thus, the structure and function of spines is regulated by an actin-based motor in addition to the polymerization state of actin.

Topics: Animals; Cell Size; Cells, Cultured; Dendrites; DNA; Electrophysiology; Excitatory Postsynaptic Potentials; Heterocyclic Compounds, 4 or More Rings; Immunohistochemistry; Microscopy, Confocal; Nonmuscle Myosin Type IIB; Rats; Rats, Sprague-Dawley; RNA Interference; Subcellular Fractions; Synapses; Synaptic Transmission

The fluorescent probe 3-[4-(3-phenyl-2-pyrazolin-1-yl)benzene-1-sulfonyl amido]phenylboronic acid (PPBA) acts as a fluorescent inhibitor for the ATPases of skeletal [Hiratsuka (1994) J. Biol. Chem. 269, 27251-27257] and Dictyostelium discoideum [Bobkov et al. (1997) J. Muscle Res. Cell Motil. 18, 563-571] myosins. The former paper suggested that, upon addition of excess nucleotides to the binary complex of subfragment-1 from skeletal myosin (S1) with PPBA, a stable ternary complex of S1 with PPBA and nucleotide is formed. Useful fluorescence properties of PPBA enable us to distinguish the conformation of the myosin ATPase at the ATP state from that at the ADP state. In the present paper, to determine the PPBA-binding site in the complexes, enzymatic and fluorescence properties of the S1.PPBA.nucleotide complexes were investigated. Upon formation of the ternary complex with ATP, a new peak appeared at 398 nm in the PPBA fluorescence spectrum. Experiments using model compounds of aromatic amino acid suggested that this fluorescence peak at 398 nm is originated from PPBA interacting with Phe residue(s). Taking into account differences in fluorescence spectra between complexes of S1 and those of subfragment-1 from D. discoideum myosin (S1dC), in the ternary complex of S1 formed with ATP, PPBA was suggested to interact with Phe residue(s) that is absent in S1dC. Docking simulation of PPBA on the S1.nucleotide complex revealed that Phe472 interacts with PPBA. Binding sites of PPBA and blebbistatin, an inhibitor showing high affinity and selectivity toward myosin II [Kovács et al. (2004) J. Biol. Chem. 279, 35557-35563], seem to overlap at least partly.

Topics: Adenosine Triphosphate; Binding Sites; Boronic Acids; Fluorescent Dyes; Heterocyclic Compounds, 4 or More Rings; Models, Molecular; Myosin Subfragments; Nucleotides; Phenylalanine; Protein Binding; Protein Conformation; Spectrometry, Fluorescence; Sulfonamides; Tryptophan; Tyrosine

We determined the tension over the entire surface of the sea urchin eggs during cytokinesis, on the basis of the intracellular pressure and cell shape. This allowed us to determine the temporal changes in both the distribution of local forces and the total force produced in the whole cell cortex. A spike-like peak at anaphase and a broader peak at the onset of furrowing were observed in the time-course of the total force. Treatment of the eggs with cytochalasin D, blebbistatin, ML-9, or ML-7 significantly lowered the total force when they inhibited cytokinesis, suggesting that the tension results mainly from the interaction between intact actin filaments and activated myosin II. Myosin II would function as a motor, not only in the furrow region, but over a wide area of the cell surface, because the sum of the tensions outside the furrow region was larger than that inside the furrow region throughout cytokinesis. The distribution of the local force revealed that a global increase in the cortical force started well before the onset of furrowing, and that the force inside the furrow region continued to increase despite the decrease in the force outside the furrow region after the onset of furrowing. The spatial and temporal patterns of the force over the entire surface support the hypothesis that there are two separate but coordinated actomyosin activation mechanisms, one of which induces global activation of the cortex and the other of which then maintains the contractility only inside the furrow region.

Topics: Animals; Cell Division; Cell Membrane; Cytochalasin D; Cytokinesis; Cytoskeleton; Heterocyclic Compounds, 4 or More Rings; Myosin Type II; Nucleic Acid Synthesis Inhibitors; Ovum; Sea Urchins; Spindle Apparatus; Surface Tension; Time Factors

Catch is characterized by maintenance of force with very low energy utilization in some invertebrate muscles. Catch is regulated by phosphorylation of the mini-titin, twitchin, and a catch component of force exists at all [Ca2+] except those resulting in maximum force. The mechanism responsible for catch force was characterized by determining how the effects of agents that inhibit the low to high force transition of the myosin cross-bridge (inorganic phosphate, butanedione monoxime, trifluoperazine, and blebbistatin) are modified by twitchin phosphorylation and [Ca2+]. In permeabilized anterior byssus retractor muscles from Mytilus edulis, catch force was identified as being sensitive to twitchin phosphorylation, whereas noncatch force was insensitive. In all cases, inhibition of the low to high force transition caused an increase in catch force. The same relationship exists between catch force and noncatch force whether force is varied by changes in [Ca2+] and/or agents that inhibit cross-bridge force production. This suggests that myosin in the high force state detaches catch force maintaining structures, whereas myosin in the low force state promotes their formation. It is unlikely that the catch structure is the myosin cross-bridge; rather, it appears that myosin interacts with the structure, most likely twitchin, and regulates its attachment and detachment.

Topics: Animals; Cyclic AMP; Diacetyl; Heterocyclic Compounds, 4 or More Rings; Muscle Contraction; Muscles; Myosins; Mytilus edulis; Organ Preservation Solutions; Phosphates; Tissue Culture Techniques; Trifluoperazine

Retrograde actin flow works in concert with cell adhesion to generate traction forces that are involved in axon guidance in neuronal growth cones. Myosins have been implicated in retrograde flow, but identification of the specific myosin subtype(s) involved has been controversial. Using fluorescent speckle microscopy (FSM) to assess actin dynamics, we report that inhibition of myosin II alone decreases retrograde flow by 51% and the remaining flow can be almost fully accounted for by the 'push' of plus-end actin assembly at the leading edge of the growth cone. Interestingly, actin bundles that are associated with filopodium roots elongated by approximately 83% after inhibition of myosin II. This unexpected result was due to decreased rates of actin-bundle severing near their proximal (minus or pointed) ends which are located in the transition zone of the growth cone. Our study reveals a mechanism for the regulation of actin-bundle length by myosin II that is dependent on actin-bundle severing, and demonstrate that retrograde flow is a steady state that depends on both myosin II contractility and actin-network treadmilling.

Topics: Actins; Animals; Aplysia; Axons; Cells, Cultured; Fluorescent Dyes; Growth Cones; Heterocyclic Compounds, 4 or More Rings; Immunohistochemistry; Microscopy, Fluorescence; Myosin Type II; Neurons; Pseudopodia

The adherens junction (AJ) densely associated with actin filaments is a major cell-cell adhesion structure. To understand the importance of actin filament association in AJ formation, we first analyzed punctate AJs in NRK fibroblasts where one actin cable binds to one AJ structure unit. The accumulation of AJ components such as the cadherin/catenin complex and vinculin, as well as the formation of AJ-associated actin cables depended on Rho activity. Inhibitors for the Rho target, ROCK, which regulates myosin II activity, and for myosin II ATPase prevented the accumulation of AJ components, indicating that myosin II activity is more directly involved than Rho activity. Depletion of myosin II by RNAi showed similar results. The inhibition of myosin II activity in polarized epithelial MTD-1A cells affected the accumulation of vinculin to circumferential AJ (zonula adherens). Furthermore, correct zonula occludens (tight junction) formation along the apicobasal axis that requires cadherin activity was also impaired. Although MDCK cells which are often used as typical epithelial cells do not have a typical zonula adherens, punctate AJs formed dependently on myosin II activity by inducing wound closure in a MDCK cell sheet. These findings suggest that tension generated by actomyosin is essential for correct AJ assembly.

Topics: Actomyosin; Adherens Junctions; Amides; Animals; Cell Adhesion Molecules; Cell Line; Chlorocebus aethiops; COS Cells; Cytoskeletal Proteins; Dogs; Enzyme Inhibitors; Heterocyclic Compounds, 4 or More Rings; Lysophospholipids; Membrane Proteins; Mice; Microscopy, Electron, Transmission; Myosin Type II; Nonmuscle Myosin Type IIB; Occludin; Platelet-Derived Growth Factor; Pyridines; Rats; rho GTP-Binding Proteins; RNA, Small Interfering; Tight Junctions; Vinculin

Activation of actomyosin II by phosphorylation of its regulatory light chain is one of the main factors involved in the regulation of cytoskeletal dynamics. Phosphorylation of myosin regulatory light chain may be mediated directly and indirectly by several kinases including myosin light chain kinase (MLCK) and kinases activated by small GTP-binding proteins. Most of the myosin kinases, including PAK, can also interact with other proteins through binding sites located outside of their catalytic domains. In an attempt to study the effects due only to phosphorylation of myosin light chain, we expressed the constitutively active catalytic domain of ameba PAK in HeLa cells. The catalytic domain phosphorylates myosin light chain in vitro with high specific activity but has none of the sequences that target mammalian PAK to other proteins and membranes. Expression of the catalytic domain caused disassembly of focal adhesions and stress fibers in the cell center and accumulation of focal adhesions and F-actin at the cell periphery. There was a twofold increase in the phosphorylation level of endogenous myosin light chain and changes in cell shape consistent with enhanced cell contractility. The phenotype was independent of MLCK, ROCK, MEK, Rac, and Rho activities but was abolished by blebbistatin, a specific inhibitor of myosin II activity. Our data are consistent with myosin being directly phosphorylated by the expressed catalytic domain of ameba PAK with the induced phenotype resulting from cell retraction driven by contraction of peripheral actomyosin. The phenotype induced by expression of the catalytic domain is reminiscent of that caused by expression of active mammalian PAK, suggesting that myosin phosphorylation may play an important role in PAK-induced cytoskeletal changes. The catalytic domain of ameba PAK may be a useful tool for studying the effects of myosin light chain phosphorylation in other cells.

Topics: Actins; Actomyosin; Amoeba; Animals; Catalytic Domain; Cell Nucleus; Cytokinesis; Focal Adhesions; HeLa Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Myosin Light Chains; Myosin Type II; Myosins; p21-Activated Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Stress Fibers; Transfection; Vinculin

During the initial phase of cardiac looping, known as c-looping, the heart bends and twists into a c-shaped tube with the convex outer curvature normally directed toward the right side of the embryo. Despite intensive study for more than 80 years, the biophysical mechanisms that drive and regulate looping remain poorly understood, although some investigators have speculated that differential cytoskeletal contraction supplies the driving force for c-looping. The purpose of this investigation was to test this hypothesis. To inhibit contraction, embryonic chick hearts at stages 10-12 (10-16 somites, 33-48 h) were exposed to the myosin inhibitors 2,3-butanedione monoxime (BDM), ML-7, Y-27632, and blebbistatin. Experiments were conducted in both whole embryo culture and, to focus on bending alone, isolated heart culture. Measurements of heart stiffness and phosphorylation of the myosin regulatory light chains showed that BDM, Y-27632, and blebbistatin significantly reduced myocardial contractility, while ML-7 had a lesser effect. None of these drugs significantly affected looping during the studied stages. These results suggest that active contraction is not required for normal c-looping of the embryonic chick heart between stages 10 and 12.

Topics: Amides; Animals; Azepines; Chick Embryo; Chickens; Cytoskeleton; Diacetyl; Enzyme Inhibitors; Heart; Heterocyclic Compounds, 4 or More Rings; Myocardial Contraction; Myosin Light Chains; Naphthalenes; Organ Culture Techniques; Phosphorylation; Pyridines; Somites; Torsion Abnormality

Stress fibers play a central role in adhesion, motility, and morphogenesis of eukaryotic cells, but the mechanism of how these and other contractile actomyosin structures are generated is not known. By analyzing stress fiber assembly pathways using live cell microscopy, we revealed that these structures are generated by two distinct mechanisms. Dorsal stress fibers, which are connected to the substrate via a focal adhesion at one end, are assembled through formin (mDia1/DRF1)-driven actin polymerization at focal adhesions. In contrast, transverse arcs, which are not directly anchored to substrate, are generated by endwise annealing of myosin bundles and Arp2/3-nucleated actin bundles at the lamella. Remarkably, dorsal stress fibers and transverse arcs can be converted to ventral stress fibers anchored to focal adhesions at both ends. Fluorescence recovery after photobleaching analysis revealed that actin filament cross-linking in stress fibers is highly dynamic, suggesting that the rapid association-dissociation kinetics of cross-linkers may be essential for the formation and contractility of stress fibers. Based on these data, we propose a general model for assembly and maintenance of contractile actin structures in cells.

Topics: Actin-Related Protein 2-3 Complex; Actinin; Actins; Adaptor Proteins, Signal Transducing; Cell Line, Tumor; Cell Movement; Cytoskeletal Proteins; Enzyme Inhibitors; Fluorescence Recovery After Photobleaching; Focal Adhesions; Formins; Glycoproteins; Heterocyclic Compounds, 4 or More Rings; Humans; Kinetics; Microscopy, Fluorescence; Models, Biological; Myosin Light Chains; Myosin Type II; Protein Subunits; RNA, Small Interfering; Stress Fibers; Transfection; Vinculin; Zyxin

Recent studies have demonstrated that nonmuscle (NM) myosin II forms filaments and can generate and maintain force in smooth muscle tissue [Lofgren et al. (2003) J Gen Physiol 121:301-310; Morano et al. (2000) Nat Cell Biol 2:371-375]. To further investigate the mechanical contribution of NM myosin to force maintenance during smooth muscle contraction, we utilized a selective inhibitor of the NM myosin ATPase, blebbistatin [Straight et al. (2003) Science 299:1743-1747]. Force and myosin light chain (MLC(20)) phosphorylation were measured during KCl stimulation of small strips of intact mouse bladder and aorta at 22 degrees C. The bladder strips contracted with a typical phasic force response, characterized by a large, rapid, transient increase in force followed by a decline to a lower, steady-state level. The addition of blebbistatin did not alter the peak force, but decreased force maintenance. KCl depolarization of aortic strips resulted in a tonic contraction; force increased to a sustained steady state. Similar to the bladder tissue, blebbistatin substantially decreased the steady-state force in the aorta. Blebbistatin did not influence the MLC(20) phosphorylation transient in either tissue type. Additionally, blebbistatin did not change the maximum shortening velocity (V (max)) during KCl depolarization of the aorta. Our results also suggest that NMIIA and NMIIB isoforms are differentially expressed. The expression of NMIIA is more prominent in the bladder, while NMIIB expression is predominant in the aorta. These results suggest that NM myosin contributes to the mechanism of force maintenance in smooth muscle, and could suggest that the expression of NMIIB is a factor for determining the tonic contractile phenotype.

Topics: Animals; Aorta, Thoracic; Dose-Response Relationship, Drug; Heterocyclic Compounds, 4 or More Rings; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Immunohistochemistry; Kinetics; Male; Mice; Mice, Inbred C57BL; Muscle Contraction; Muscle Tonus; Muscle, Smooth; Myosin Light Chains; Myosins; Phenotype; Phosphorylation; Potassium Chloride; Urinary Bladder

Heart growth in the embryo is achieved by division of differentiated cardiomyocytes. Around birth, cardiomyocytes stop dividing and heart growth occurs only by volume increase of the individual cells. Cardiomyocytes seem to lose their capacity for cytokinesis at this developmental stage. Septins are GTP-binding proteins that have been shown to be involved in cytokinesis from yeast to vertebrates. We wanted to determine whether septin expression patterns can be correlated to the cessation of cytokinesis during heart development. We found significant levels of expression only for SEPT2, SEPT6, SEPT7 and SEPT9 in heart, in a developmentally regulated fashion, with high levels in the embryonic heart, downregulation around birth and no detectable expression in the adult. In dividing embryonic cardiomyocytes, all septins localize to the cleavage furrow. We used drugs to probe for the functional interactions of SEPT2 in dividing embryonic cardiomyocytes. Differences in the effects on subcellular septin localization in cardiomyocytes were observed, depending whether a Rho kinase (ROCK) inhibitor was used or whether actin and myosin were targeted directly. Our data show a tight correlation of high levels of septin expression and the ability to undergo cytokinesis in cardiomyocytes. In addition, we were able to dissect the different contributions of ROCK signaling and the actomyosin cytoskeleton to septin localization to the contractile ring using cardiomyocytes as an experimental system.

Topics: Actinin; Amides; Animals; Cell Cycle Proteins; Cells, Cultured; Cytochalasin D; Cytokinesis; Cytoskeletal Proteins; Enzyme Inhibitors; GTP Phosphohydrolases; GTP-Binding Proteins; Heart; Heterocyclic Compounds, 4 or More Rings; Mice; Microtubules; Models, Biological; Myocardium; Myocytes, Cardiac; Nonmuscle Myosin Type IIB; Pyridines; Rats; Schizosaccharomyces pombe Proteins; Septins; Spindle Apparatus; Time Factors; Transcription Factors

Nonmuscle myosin IIA and IIB distribute preferentially toward opposite ends of migrating endothelial cells. To understand the mechanism and function of this behavior, myosin II was examined in cells treated with the motor inhibitor, blebbistatin. Blebbistatin at > or = 30 microM inhibited anterior redistribution of myosin IIA, with 100 microM blebbistatin causing posterior accumulation. Posterior accumulation of myosin IIB was unaffected. Time-lapse cinemicrography showed myosin IIA entering lamellipodia shortly after their formation, but failing to move into lamellipodia in blebbistatin. Thus, myosin II requires motor activity to move forward onto F-actin in protrusions. However, this movement is inhibited by myosin filament assembly, because whole myosin was delayed relative to a tailless fragment. Inhibiting myosin's forward movement reduced coupling between protrusive activity and translocation of the cell body: In untreated cells, body movement followed advancing lamellipodia, whereas blebbistatin-treated cells extended protrusions without displacement of the body or with a longer delay before movement. Anterior cytoplasm of blebbistatin-treated cells contained disorganized bundles of parallel microfilaments, but anterior F-actin bundles in untreated cells were mostly oriented perpendicular to movement. Myosin II may ordinarily move anteriorly on actin filaments and pull crossed filaments into antiparallel bundles, with the resulting realignment pulling the cell body forward.

Topics: Actin Cytoskeleton; Actins; Animals; Cattle; Cell Movement; Cell Polarity; Cells, Cultured; Cytoplasm; Dose-Response Relationship, Drug; Endothelial Cells; Heterocyclic Compounds, 4 or More Rings; Models, Biological; Myosin Type II; Protein Isoforms

Cytokinesis in early zebrafish embryos involves coordinated changes in the f-actin- and microtubule-based cytoskeleton, and the recruitment of adhesion junction components to the furrow. We show that exposure to inhibitors of non-muscle myosin II function does not affect furrow ingression during the early cleavage cycles but interferes with the recruitment of pericleavage f-actin and cortical beta-catenin aggregates to the furrow, as well as the remodeling of the furrow microtubule array. This remodeling is in turn required for the distal aggregation of the zebrafish germ plasm. Embryos with reduced myosin activity also exhibit at late stages of cytokinesis a stabilized contractile ring apparatus that appears as a ladder-like pattern of short f-actin cables, supporting a role for myosin function in the disassembly of the contractile ring after furrow formation. Our studies support a role for myosin function in furrow maturation that is independent of furrow ingression and which is essential for the recruitment of furrow components and the remodeling of the cytoskeleton during cytokinesis.

Topics: Actins; Animals; Azepines; beta Catenin; Cell Aggregation; Cytokinesis; Cytoskeleton; DEAD-box RNA Helicases; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Heterocyclic Compounds, 4 or More Rings; In Situ Hybridization; Myosin Type II; Myosin-Light-Chain Kinase; Naphthalenes; RNA, Messenger; Zebrafish; Zebrafish Proteins

Catch is a holding state of muscle where tension is maintained passively for long time periods in the absence of stimulation. The catch state becomes obvious after termination of activation; however, it is possible that catch linkages are already established during activation. To investigate this, skinned fibre bundles of the anterior byssus retractor muscle of Mytilus edulis were maximally activated with Ca(2+) and subsequently exposed to 10 mmol l(-1) orthovanadate (V(i)) or 5 mumol l(-1) blebbistatin to inhibit the force-generating myosin head cross-bridges. Repetitive stretches of about 0.1% fibre bundle length were applied to measure stiffness. Inhibitor application depressed force substantially but never resulted in a full relaxation. The remaining force was further decreased by moderate alkalization (change of pH from 6.7 to 7.4) or by cAMP. Furthermore, the stiffness/force ratio was higher during exposure to V(i) or blebbistatin than during partial Ca(2+) activation producing the same submaximal force. The increased stiffness/force ratio was abolished by moderate alkalization or cAMP. Finally, the stretch-induced delayed force increase (stretch activation) disappeared, and the force recovery following a quick release of the fibre length, was substantially reduced when the force was depressed by V(i) or blebbistatin. All these findings suggest that catch linkages are already established during maximal Ca(2+) activation. They seem to exhibit ratchet properties because they allow shortening and resist stretches. In isometric experiments a force decrease is needed to stress the catch linkages in the high resistance direction so that they contribute to force.

Topics: Animals; Calcium; Cyclic AMP; Heterocyclic Compounds, 4 or More Rings; Hydrogen-Ion Concentration; Muscle Fibers, Skeletal; Muscle Tonus; Myosins; Mytilus edulis; Vanadates

We prepared a cell-populated collagen-gel fiber including GbaSM-4 cells derived from the basilar artery of guinea pigs. This fiber tended to be a differentiated contractile phenotype in electron-microscope observations. Sphingosylphosphorylcholine (SPC) can induce contraction of the fiber (EC50 = 0.70 +/- 0.05 microM), and blebbistatin can inhibit the SPC-induced contraction (IC50 = 22.8 +/- 1.26 microM). Phosphorylation of the 20 kD myosin light chain (MLC20) significantly increased in GbaSM-4 cells provided with 1 microM SPC (P<0.05), which was maintained in the presence of 1 to 100 microM blebbistatin. These results suggest that vascular smooth muscle can relax even if MLC20 is phosphorylated.

Topics: Animals; Cells, Cultured; Collagen; Gels; Guinea Pigs; Heterocyclic Compounds, 4 or More Rings; Microscopy, Electron; Muscle Contraction; Muscle, Smooth, Vascular; Myosin Light Chains; Phosphorylcholine; Sphingosine

The migration of cells is a complex regulatory process which results in the generation of motor forces through the reorganization of the cytoskeleton. Here we present a comparative study of the expression and involvement of myosin in the regulation of the physiological migration of leukocytes and the pathological migration of tumor cells. We show that the involvement of myosin in the migration is distinct in these two cell types. In leukocytes, the activity of non-muscle myosin II is essential for both the spontaneous (matrix-induced) migration and the migration induced by ligands to G protein-coupled receptors, i.e. chemokines and neurotransmitters. In contrast, spontaneous tumor cell migration is largely independent of non-muscle myosin II activity, whereas the norepinephrine-induced migration is completely inhibited by either direct inhibition of non-muscle myosin II or of the kinases phosphorylating the myosin light chain, namely ROCK or the calcium/calmodulin-dependent myosin light-chain kinase.

Topics: Actins; Breast Neoplasms; Cell Movement; Collagen; Female; Heterocyclic Compounds, 4 or More Rings; Humans; Intracellular Signaling Peptides and Proteins; Leukocytes; Male; Myosin Type II; Myosin-Light-Chain Kinase; Norepinephrine; Phosphorylation; Prostatic Neoplasms; Protein Isoforms; Protein Serine-Threonine Kinases; rho-Associated Kinases; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

Blebbistatin is a small molecule inhibitor discovered in a screen for inhibitors of nonmuscle myosin IIA. Blebbistatin inhibits the actin-activated MgATPase activity and in vitro motility of class II myosins. In cells, it has been shown to inhibit contraction of the cytokinetic ring. Blebbistatin has some photochemical properties that may affect its behavior in cells. In particular, we have found that exposure to light at wavelengths below 488 nm rapidly inactivates the inhibitory action of blebbistatin using the in vitro motility of myosin as an assay. In addition, the inhibition of cytokinetic ring contraction can be reversed by exposure of the cells to blue light. This property may be useful in locally reversing the action of blebbistatin treatment in a cell. However, caution should be exercised as free radicals may be produced upon irradiation of blebbistatin that could result in cell damage.

Topics: Actins; Animals; Dose-Response Relationship, Radiation; Free Radicals; HeLa Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Light; Microscopy, Confocal; Microscopy, Fluorescence; Mitosis; Muscle Contraction; Muscle, Skeletal; Nonmuscle Myosin Type IIA; Photochemistry; Rabbits; Videotape Recording

Blebbistatin, a cell-permeable inhibitor of class-II myosins, was developed to provide a tool for studying the biologic roles of myosin II. Consistent with this use, we find that blebbistatin inhibits three myosin II-dependent processes in Dictyostelium (growth in suspension culture, capping of Con A receptors, and development to fruiting bodies) and does not inhibit growth on plates, which does not require myosin II. As expected, macropinocytosis (myosin I-dependent), contractile vacuole activity (myosin V-dependent), and phagocytosis (myosin VII-dependent), none of which requires myosin II, are not inhibited by blebbistatin in myosin II-null cells, but, unexpectedly, blebbistatin does inhibit macropinocytosis and phagocytosis by cells expressing myosin II. Expression of catalytically inactive myosin II in myosin II-null cells also inhibits macropinocytosis and phagocytosis. Both blebbistatin-inhibited myosin II and catalytically inactive myosin II form cytoplasmic aggregates, which may be why they inhibit myosin II-independent processes, but neither affects the distribution of actin filaments in vegetative cells or actin and myosin distribution in dividing or polarized cells. Blebbistatin also inhibits cell streaming and plaque expansion in myosin II-null cells. Our results are consistent with myosin II being the only Dictyostelium myosin that is inhibited by blebbistatin but also show that blebbistatin-inactivated myosin II inhibits some myosin II-independent processes and that blebbistatin inhibits other activities in the absence of myosin II.

Topics: Animals; Concanavalin A; Dictyostelium; Heterocyclic Compounds, 4 or More Rings; Kinetics; Myosin Type II; Phagocytosis; Pinocytosis; Receptors, Concanavalin A; Vacuoles

Cell migration is important in the development of atherosclerotic lesions. Macrophages and smooth muscle cells migrate into the subendothelial space of arteries, leading to plaque formation. Long-term inhibition of the activity of Rho-kinase induces a regression of atherosclerotic coronary lesions, probably by preventing migration of macrophages and smooth muscle cells. Previous reports concerning the effect of Rho-kinase inhibitors on cell migration are contradictory, however. We examined here the cell type specificity of Rho-kinase inhibitors and found that migration of endothelial cells, macrophages, and smooth muscle cells was inhibited by treatment with Rho-kinase inhibitors in a dose-dependent fashion in a three-dimensional migration assay, whereas that of fibroblasts and epithelial cells was not inhibited. Myosin II inhibitor prevented cell migration in a manner similar to Rho-kinase inhibitors. In contrast, in a two-dimensional migration assay, cell migration was not inhibited by Rho-kinase or myosin II inhibitors for any of the cell types examined. Taken together, these results indicate that Rho-kinase inhibitors suppress migration of specific cell types under specific conditions through the regulation of myosin II activity. Our findings suggest that Rho-kinase is the therapeutic target of atherosclerosis accompanied with invasion by leukocytes and smooth muscle cells.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amides; Animals; Cell Culture Techniques; Cell Movement; Cell Nucleus; Cells, Cultured; Endothelial Cells; Endothelium, Vascular; Epithelial Cells; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Humans; Intracellular Signaling Peptides and Proteins; Macrophages; Monomeric GTP-Binding Proteins; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Myosin Type II; Phosphorylation; Protein Serine-Threonine Kinases; Pyridines; rho-Associated Kinases

Mammalian cells have been reported to have a p53-dependent tetraploidy checkpoint that blocks cell cycle progression in G1 in response to failure of cell division. In most cases where the tetraploidy checkpoint has been observed cell division was perturbed by anti-cytoskeleton drug treatments. However, other evidence argues against the existence of a tetraploidy checkpoint. Cells that have failed to divide differ from normal cells in having two nuclei, two centrosomes, a decreased surface to volume ratio, and having undergone an abortive cytokinesis. We tested each of these to determine which, if any, cause a G1 cell cycle arrest.. Primary human diploid fibroblasts with intact cell cycle checkpoints were used in all experiments. Synchronized cells exhibited G1 arrest in response to division failure caused by treatment with either cytochalasin or the myosin II inhibitor blebbistatin. The role of tetraploidy, aberrant centrosome number, and increased cell size were tested by cell/cell and cell/cytoplast fusion experiments; none of these conditions resulted in G1 arrest. Instead we found that various drug treatments of the cells resulted in cellular damage, which was the likely cause of the arrest. When cytokinesis was blocked in the absence of damage-inducing drug treatments no G1 arrest was observed.. We show that neither tetraploidy, aberrant centrosome number, cell size, nor failure of cytokinesis lead to G1 arrest, suggesting that there is no tetraploidy checkpoint. Rather, certain standard synchronization treatments cause damage that is the likely cause of G1 arrest. Since tetraploid cells can cycle when created with minimal manipulation, previous reports of a tetraploidy checkpoint can probably be explained by side effects of the drug treatments used to observe them.

Topics: Cell Cycle; Cell Shape; Centrosome; Cytochalasins; Cytokinesis; Fibroblasts; G1 Phase; Heterocyclic Compounds, 4 or More Rings; Humans; Polyploidy

Actomyosin contractility is a mechanism by which cells exert locomotory force against their environment. Signalling downstream of the small GTPase Rho increases contractility through Rho-kinase (ROCK)-mediated regulation of myosin-II light chain (MLC2) phosphorylation. Cdc42 signalling has been shown to control cell polarity. Tumour cells can move through a three-dimensional matrix with either a rounded morphology characterized by Rho-ROCK dependence or with an elongated morphology characterized by Rho-ROCK independence. Here we show that contractility necessary for elongated morphology and invasion can be generated by Cdc42-MRCK signalling. MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) cooperates with ROCK in the maintenance of elongated morphology and invasion and either MRCK or ROCK is sufficient for MLC2 phosphorylation, through the inhibitory phosphorylation of myosin phosphatase. By contrast, in rounded ROCK-dependent movement, where MLC2 phosphorylation is higher, MRCK has a smaller role. Our data show that a Cdc42-MRCK signal mediates myosin-dependent cell motility and highlight convergence between Rho and Cdc42 signalling.

Topics: Actomyosin; Animals; Cattle; cdc42 GTP-Binding Protein; Cell Movement; Collagen; Genetic Vectors; Green Fluorescent Proteins; Heterocyclic Compounds, 4 or More Rings; Humans; Immunoblotting; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Microscopy, Phase-Contrast; Myosins; Myotonin-Protein Kinase; Neoplasm Invasiveness; Phosphorylation; Protein Serine-Threonine Kinases; rho GTP-Binding Proteins; rho-Associated Kinases; RNA, Small Interfering; Signal Transduction; Time Factors; Transfection

Myosin II plays important roles in many contractile-like cell functions, including cell migration, adhesion, and retraction. Myosin II is activated by regulatory light chain (RLC) phosphorylation whereas RLC dephosphorylation by myosin light chain phosphatase containing a myosin phosphatase targeting subunit (MYPT1) leads to myosin inactivation. HeLa cells contain MYPT1 in addition to a newly identified human variant 2 containing an internal deletion. RLC dephosphorylation, cell migration, and adhesion were inhibited when either or both MYPT1 isoforms were knocked down by RNA interference. RLC was highly phosphorylated (60%) when both isoforms were suppressed by siRNA treatment relative to control cells (10%) with serum-starvation and ROCK inhibition. Prominent stress fibers and focal adhesions were associated with the enhanced RLC phosphorylation. The reintroduction of MYPT1 or variant 2 in siRNA-treated cells decreased stress fibers and focal adhesions. MYPT1 knockdown also led to an increase of F-actin relative to G-actin in HeLa cells. The myosin inhibitor blebbistatin did not inhibit this effect, indicating MYPT1 likely affects actin assembly independent of RLC phosphorylation. Proper expression of MYPT1 or variant 2 is critical for RLC phosphorylation and actin assembly, thus maintaining normal cellular functions by simultaneously controlling cytoskeletal architecture and actomyosin activation.

Topics: Actins; Cell Movement; Culture Media, Serum-Free; Down-Regulation; Focal Adhesions; HeLa Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Intracellular Signaling Peptides and Proteins; Myosin Light Chains; Myosin Type II; Myosin-Light-Chain Phosphatase; Myosins; Phosphorylation; Protein Isoforms; Protein Serine-Threonine Kinases; rho-Associated Kinases; RNA Interference; Stress Fibers

Molecular motors play a central role in cytoskeletal-mediated cellular processes and thus present an excellent target for cellular control by pharmacological agents. Yet very few such compounds have been found. We report here the structure of blebbistatin, which inhibits specific myosin isoforms, bound to the motor domain of Dictyostelium discoideum myosin II. This reveals the structural basis for its specificity and provides insight into the development of new agents.

Topics: Animals; Binding Sites; Dictyostelium; Heterocyclic Compounds, 4 or More Rings; Models, Molecular; Myosin Type II; Protein Isoforms; Protein Structure, Tertiary; Substrate Specificity

Differentiation and polarization of epithelial cells depends on the formation of the apical junctional complex (AJC), which is composed of the tight junction (TJ) and the adherens junction (AJ). In this study, we investigated mechanisms of actin reorganization that drive the establishment of AJC. Using a calcium switch model, we observed that formation of the AJC in T84 intestinal epithelial cells began with the assembly of adherens-like junctions followed by the formation of TJs. Early adherens-like junctions and TJs readily incorporated exogenous G-actin and were disassembled by latrunculin B, thus indicating dependence on continuous actin polymerization. Both adherens-like junctions and TJs were enriched in actin-related protein 3 and neuronal Wiskott-Aldrich syndrome protein (N-WASP), and their assembly was prevented by the N-WASP inhibitor wiskostatin. In contrast, the formation of TJs, but not adherens-like junctions, was accompanied by recruitment of myosin II and was blocked by inhibition of myosin II with blebbistatin. In addition, blebbistatin inhibited the ability of epithelial cells to establish a columnar phenotype with proper apico-basal polarity. These findings suggest that actin polymerization directly mediates recruitment and maintenance of AJ/TJ proteins at intercellular contacts, whereas myosin II regulates cell polarization and correct positioning of the AJC within the plasma membrane.

Topics: Actins; Adherens Junctions; Bridged Bicyclo Compounds, Heterocyclic; Carbazoles; Cell Line; Cell Polarity; Epithelial Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Intestinal Mucosa; Microvilli; Models, Biological; Molecular Motor Proteins; Myosin Type II; Polymers; Propanolamines; Thiazoles; Thiazolidines; Tight Junctions

Three of seven recently identified genes mutated in nonsyndromic mental retardation are involved in Rho family signaling. Two of the gene products, alpha-p-21-activated kinase (PAK) interacting exchange factor (alphaPIX) and PAK3, form a complex with the synaptic adaptor protein G-protein-coupled receptor kinase-interacting protein 1 (GIT1). Using an RNA interference approach, we show that GIT1 is critical for spine and synapse formation. We also show that Rac is locally activated in dendritic spines using fluorescence resonance energy transfer. This local activation of Rac is regulated by PIX, a Rac guanine nucleotide exchange factor. PAK1 and PAK3 serve as downstream effectors of Rac in regulating spine and synapse formation. Active PAK promotes the formation of spines and dendritic protrusions, which correlates with an increase in the number of excitatory synapses. These effects are dependent on the kinase activity of PAK, and PAK functions through phosphorylating myosin II regulatory light chain (MLC). Activated MLC causes an increase in dendritic spine and synapse formation, whereas inhibiting myosin ATPase activity results in decreased spine and synapse formation. Finally, both activated PAK and activated MLC can rescue the defects of GIT1 knockdown, suggesting that PAK and MLC are downstream of GIT1 in regulating spine and synapse formation. Our results point to a signaling complex, consisting of GIT1, PIX, Rac, and PAK, that plays an essential role in the regulation of dendritic spine and synapse formation and provides a potential mechanism by which alphaPIX and PAK3 mutations affect cognitive functions in mental retardation.

Topics: Animals; Cell Cycle Proteins; Dendritic Spines; Diglycerides; Disks Large Homolog 4 Protein; Embryo, Mammalian; Fluorescence Resonance Energy Transfer; Gene Expression; Green Fluorescent Proteins; Guanine Nucleotide Exchange Factors; Heterocyclic Compounds, 4 or More Rings; Hippocampus; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Morphogenesis; Myosin Light Chains; Neurons; p21-Activated Kinases; Phosphoproteins; Protein Serine-Threonine Kinases; Rats; Rho Guanine Nucleotide Exchange Factors; RNA, Small Interfering; Synapses; Transfection

The assembly of an F-actin- and myosin-II-containing contractile ring (CR) is required for cytokinesis in eukaryotic cells. Interactions between myosin II and actin in the ring are believed to generate the force that constricts the cell into two daughters. The mechanism(s) that contribute to the spatially and temporally regulated assembly and disassembly of the CR at the cell equator are poorly understood.. We generated an LLCPK1 epithelial cell line that stably expresses GFP-actin. Live confocal imaging showed accumulation of GFP-actin in the equatorial cortex from late anaphase through cytokinesis. Fluorescence recovery after photobleaching (FRAP) experiments showed that actin in the CR is highly dynamic (t(1/2) = 26 s). In some cells, movement of GFP-actin toward the equatorial region was observed and contributed to FRAP. Blocking actin dynamic turnover with jasplakinolide demonstrates that dynamic actin is required for CR formation and cytokinesis. To test the role of myosin II in actin turnover and transport during CR formation, we inhibited myosin light-chain kinase with ML7 and myosin II ATPase activity with blebbistatin. Inhibition of myosin light-chain phosphorylation resulted in clearance of GFP-actin from the equatorial region, a reduction in myosin II in the furrow, and inhibition of cytokinesis. Treatment with blebbistatin did not block CR formation but reduced FRAP of GFP-actin and prevented completion of cytokinesis.. These results demonstrate that the majority of actin in the CR is highly dynamic and establish novel roles for myosin II in the retention and dynamic turnover of actin in the CR.

Topics: Actins; Animals; Azepines; Cytokinesis; Depsipeptides; Fluorescence Recovery After Photobleaching; Green Fluorescent Proteins; Heterocyclic Compounds, 4 or More Rings; LLC-PK1 Cells; Microscopy, Fluorescence; Myosin Type II; Naphthalenes; Phosphorylation; Swine

The involvement of myosin II in cytokinesis has been demonstrated with microinjection, genetic, and pharmacological approaches; however, the exact role of myosin II in cell division remains poorly understood. To address this question, we treated dividing normal rat kidney (NRK) cells with blebbistatin, a potent inhibitor of the nonmuscle myosin II ATPase. Blebbistatin caused a strong inhibition of cytokinesis but no detectable effect on the equatorial localization of actin or myosin. However, whereas these filaments dissociated from the equator in control cells during late cytokinesis, they persisted in blebbistatin-treated cells over an extended period of time. The accumulation of equatorial actin was caused by the inhibition of actin filament turnover, as suggested by a 2-fold increase in recovery half-time after fluorescence photobleaching. Local release of blebbistatin at the equator caused localized accumulation of equatorial actin and inhibition of cytokinesis, consistent with the function of myosin II along the furrow. However, treatment of the polar region also caused a high frequency of abnormal cytokinesis, suggesting that myosin II may play a second, global role. Our observations indicate that myosin II ATPase is not required for the assembly of equatorial cortex during cytokinesis but is essential for its subsequent turnover and remodeling.

Topics: Actins; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Cytokinesis; Fluorescence Recovery After Photobleaching; Heterocyclic Compounds, 4 or More Rings; Myosin Type II; Protein Transport; Protozoan Proteins; Quinolinium Compounds; Rats

We have determined the microrheological response of the actin meshwork for individual cells. We applied oscillating forces with an optical tweezer to a micrometric bead specifically bound to the actin meshwork of C2 myoblasts, and measured the amplitude and phase shift of the induced cell deformation. For a non-perturbed single cell, we have shown that the elastic and loss moduli G' and G'' behave as power laws f (alpha) and f (beta) of the frequency f (0.01

Topics: Animals; Cell Line; Cytoskeleton; Elasticity; Energy Transfer; Heterocyclic Compounds, 4 or More Rings; Mice; Microfluidics; Micromanipulation; Myoblasts; Myosin Type II; Physical Stimulation; Stress, Mechanical; Viscosity

Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles from an intracellular pool to the plasma membrane. The studies presented here show that inhibition of myosin II activity impairs GLUT4-mediated glucose uptake but not GLUT4 translocation to the plasma membrane. We also show that adipocytes express both myosin IIA and IIB isoforms, and that myosin IIA is recruited to the plasma membrane upon insulin stimulation. Taken together, the data presented here represent the first demonstration that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. Based on our findings, we hypothesize that myosin II is activated upon insulin stimulation and recruited to the cell cortex to facilitate GLUT4 fusion with the plasma membrane. The identification of myosin II as a key component of GLUT4-mediated glucose uptake represents an important advance in our understanding of the mechanisms regulating glucose homeostasis.

Topics: 3T3-L1 Cells; Adipocytes; Animals; Fluorescent Antibody Technique; Glucose; Glucose Transporter Type 4; Heterocyclic Compounds, 4 or More Rings; Homeostasis; Insulin; Mice; Monosaccharide Transport Proteins; Muscle Proteins; Myosin Type II

Myosin II-dependent contraction of the contractile ring drives equatorial furrowing during cytokinesis in animal cells. Nonetheless, myosin II-null cells of the cellular slime mold Dictyostelium divide efficiently when adhering to substrates by making use of polar traction forces. Here, we show that in the presence of 30 microM blebbistatin, a potent myosin II inhibitor, normal rat kidney (NRK) cells adhering to fibronectin-coated surfaces formed equatorial furrows and divided in a manner strikingly similar to myosin II-null Dictyostelium cells. Such blebbistatin-resistant cytokinesis was absent in partially detached NRK cells and was disrupted in adherent cells if the advance of their polar lamellipodia was disturbed by neighboring cells. Y-27632 (40 microM), which inhibits Rho-kinase, was similar to 30 microM blebbistatin in that it inhibited cytokinesis of partially detached NRK cells but only prolonged furrow ingression in attached cells. In the presence of 100 microM blebbistatin, most NRK cells that initiated anaphase formed tight furrows, although scission never occurred. Adherent HT1080 fibrosarcoma cells also formed equatorial furrows efficiently in the presence of 100 microM blebbistatin. These results provide direct evidence for adhesion-dependent, contractile ring-independent equatorial furrowing in mammalian cells and demonstrate the importance of substrate adhesion for cytokinesis.

Topics: Amides; Animals; Cell Adhesion; Cell Line; Cytokinesis; Dictyostelium; Fibronectins; Heterocyclic Compounds, 4 or More Rings; Humans; Pyridines; Rats

NCAM 180 isoform null neuromuscular junctions are unable to effectively mobilize and exocytose synaptic vesicles and thus exhibit periods of total transmission failure during high-frequency repetitive stimulation. We have identified a highly conserved C-terminal (KENESKA) domain on NCAM that is required to maintain effective transmission and demonstrate that it acts via a pathway involving MLCK and probably myosin light chain (MLC) and myosin II. By perfecting a method of introducing peptides into adult NMJs, we tested the hypothesized role of proteins in this pathway by competitive disruption of protein-protein interactions. The effects of KENESKA and other peptides on MLCK and MLC activation and on failures in both wild-type and NCAM 180 null junctions supported this pathway, and serine phosphorylation of KENESKA was critical. We propose that this pathway is required to replenish synaptic vesicles utilized during high levels of exocytosis by facilitating myosin-driven delivery of synaptic vesicles to active zones or their subsequent exocytosis.

Topics: 4-Aminopyridine; Action Potentials; Amifampridine; Animals; Animals, Newborn; Blotting, Western; Bungarotoxins; Computational Biology; Cysteamine; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Electric Stimulation; Heterocyclic Compounds, 4 or More Rings; Humans; Immunohistochemistry; In Vitro Techniques; Mice; Mice, Knockout; Models, Neurological; Myosins; Neural Cell Adhesion Molecules; Neuromuscular Junction; Oligopeptides; PC12 Cells; Peptide Fragments; Peptides; Potassium Channel Blockers; Protein Structure, Tertiary; Rats; Signal Transduction; Synaptic Transmission; Xenopus

Viruses have often been observed in association with the dense microvilli of polarized epithelia as well as the filopodia of nonpolarized cells, yet whether interactions with these structures contribute to infection has remained unknown. Here we show that virus binding to filopodia induces a rapid and highly ordered lateral movement, "surfing" toward the cell body before cell entry. Virus cell surfing along filopodia is mediated by the underlying actin cytoskeleton and depends on functional myosin II. Any disruption of virus cell surfing significantly reduces viral infection. Our results reveal another example of viruses hijacking host machineries for efficient infection by using the inherent ability of filopodia to transport ligands to the cell body.

Topics: Actins; Animals; Avian Leukosis Virus; Cell Line; Cell Membrane; Cytochalasin D; Heterocyclic Compounds, 4 or More Rings; Humans; Leukemia Virus, Murine; Mice; Microscopy, Electron; Myosins; Pseudopodia

Classical cadherins accumulate at cell-cell contacts as a characteristic response to productive adhesive ligation. Such local accumulation of cadherins is a developmentally regulated process that supports cell adhesiveness and cell-cell cohesion. Yet the molecular effectors responsible for cadherin accumulation remain incompletely understood. We now report that Myosin 2 is critical for cells to concentrate E-cadherin at cell-cell contacts. Myosin 2 is found at cadherin-based cell-cell contacts and its recruitment requires E-cadherin activity. Indeed, both Myosin 2 recruitment and its activation were stimulated by E-cadherin homophilic ligation alone. Inhibition of Myosin 2 activity by blebbistatin or ML-7 rapidly impaired the ability of cells to concentrate E-cadherin at adhesive contacts, accompanied by decreased cadherin-based cell adhesiveness. The total surface expression of cadherins was unaffected, suggesting that Myosin 2 principally regulates the regional distribution of cadherins at the cell surface. The recruitment of Myosin 2 to cadherin contacts, and its activation, required Rho kinase; furthermore, inhibition of Rho kinase signaling effectively phenocopied the effects of Myosin 2 inhibition. We propose that Myosin 2 is a key effector of Rho-Rho kinase signaling that regulates cell-cell adhesion by determining the ability of cells to concentrate cadherins at contacts in response to homophilic ligation.

Topics: Animals; Azepines; Cadherins; Cell Line; Cell Membrane; Cricetinae; Cricetulus; Heterocyclic Compounds, 4 or More Rings; Humans; Intercellular Junctions; Myosin Type II; Naphthalenes; Nonmuscle Myosin Type IIA; rho GTP-Binding Proteins; Signal Transduction

Actin inhibitors block or slow anaphase chromosome movements in crane-fly spermatocytes, but stopping of movement is only temporary; we assumed that cells adapt to loss of actin by switching to mechanism(s) involving only microtubules. To test this, we produced actin-filament-free spindles: we added latrunculin B during prometaphase, 9-80 min before anaphase, after which chromosomes generally moved normally during anaphase. We confirmed the absence of actin filaments by staining with fluorescent phalloidin and by showing that cytochalasin D had no effect on chromosome movement. Thus, actin filaments are involved in normal anaphase movements, but in vivo, spindles nonetheless can function normally without them. We tested whether chromosome movements in actin-filament-free spindles arise via microtubules by challenging such spindles with anti-myosin drugs. Y-27632 and BDM (2,3-butanedione monoxime), inhibitors that affect myosin at different regulatory levels, blocked chromosome movement in normal spindles and in actin-filament-free spindles. We tested whether BDM has side effects on microtubule motors. BDM had no effect on ciliary and sperm motility or on ATPase activity of isolated ciliary axonemes, and thus it does not directly block dynein. Nor does it block kinesin, assayed by a microtubule sliding assay. BDM could conceivably indirectly affect these microtubule motors, though it is unlikely that it would have the same side effect on the motors as Y-27632. Since BDM and Y-27632 both affect chromosome movement in the same way, it would seem that both affect spindle myosin; this suggests that spindle myosin interacts with kinetochore microtubules, either directly or via an intermediate component.

Topics: Actin Cytoskeleton; Amides; Anaphase; Animals; Azepines; Chromosomes; Diacetyl; Diptera; Drosophila melanogaster; Heterocyclic Compounds, 4 or More Rings; Locusta migratoria; Male; Naphthalenes; Pyridines; Sea Urchins; Spermatocytes; Spindle Apparatus

To investigate the specific role of myosin II, a critical biochemical determinant of cellular contraction, in modulation of aqueous humor outflow facility through the trabecular meshwork (TM) pathway.. Expression of the nonmuscle myosin II heavy chains (IIA, IIB, and IIC) in human TM and ciliary body (CB) cells was determined by RT-PCR analyses. The effects of inhibition of myosin II on cell morphology, actomyosin organization, and cell adhesions were evaluated in porcine TM and CB cells treated with blebbistatin, a cell-permeable, specific inhibitor of myosin II adenosine triphosphatase (ATPase) activity. Changes in aqueous humor outflow facility were determined in enucleated porcine eyes by using a constant-pressure Grant perfusion model system. Ultrastructural integrity of the outflow pathway in drug-perfused eyes was analyzed by transmission electron microscopy.. Expression of nonmuscle myosin IIA and IIB was confirmed in both human TM and CB cells. Confluent cultures of primary porcine TM and CB cells treated with blebbistatin in the presence of serum revealed dose (10-200 microM)-dependent changes in cell morphology, decreases in actin stress fiber content and in focal adhesions and adherens junctions. These changes were found to be reversible within 24 hours of drug withdrawal from the cell culture media. Blebbistatin did not affect the status of myosin light chain phosphorylation in TM cells. Perfusion of enucleated porcine eyes for 5 hours with 100 and 200 microM blebbistatin produced a significant increase (P < 0.01, n = 7) in aqueous outflow facility (53% and 64%, respectively) from the baseline facility, compared with a 21% facility increase in sham control specimens. The integrity of the inner wall of aqueous plexi in drug-perfused porcine eyes was found to be intact, and TM cell morphology appeared to be similar to that noted in sham-treated eyes.. These data demonstrate that selective inhibition of myosin II in the aqueous humor outflow pathway leads to increased aqueous outflow facility, suggesting a critical role for myosin II in the regulation of aqueous humor outflow facility. This study also suggests myosin II as a potential therapeutic target for lowering intraocular pressure in patients with glaucoma.

Topics: Adenosine Triphosphatases; Animals; Aqueous Humor; Cell Adhesion; Cell Culture Techniques; Cell Survival; Cells, Cultured; Ciliary Body; Dose-Response Relationship, Drug; Eye Enucleation; Heterocyclic Compounds, 4 or More Rings; Nonmuscle Myosin Type IIA; Nonmuscle Myosin Type IIB; Perfusion; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; Swine; Trabecular Meshwork

Blebbistatin is a novel 1-phenyl-2-pyrrolidinone derivative capable of inhibiting non-muscle myosin II activity with a high degree of specificity. We examined the effects of blebbistatin on pancreatic adenocarcinoma cellular migration, invasion, adhesion, and spreading. Blebbistatin dose-dependently inhibited cellular migration and invasiveness, quantified by modified Boyden chamber assay. Matrix metalloproteinase 2 and 9 activities were unaffected by blebbistatin and cellular proliferation was inhibited only by concentrations of blebbistatin exceeding those required to inhibit myosin II activity and to interfere with migration and invasion. While blebbistatin treatment did not affect cell adhesion to the extracellular matrix component fibronectin, it markedly impaired cell spreading on this substrate. Cell surface expression of the archetypal fibronectin receptor (alpha(5)beta(1) integrin) was unaffected by blebbistatin. Our observations illustrate the critical role of non-muscle myosin II in pancreatic adenocarcinoma cellular invasiveness and extracellular matrix interaction and suggest that therapeutic strategies targeting myosin II warrant further investigation.

Topics: Actins; Adenocarcinoma; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Survival; Heterocyclic Compounds, 4 or More Rings; Humans; Myosin Type II; Neoplasm Invasiveness; Pancreatic Neoplasms

Although cortical nonmuscle myosin II has long been implicated in cytokinetic aspects of cell division, there has never been strong evidence that it plays a role in the organization of the mitotic spindle. ([this issue of Cell]) use a number of methods to show that cortical myosin II is in fact important for spindle assembly in higher eukaryotic cells, specifically for the complete separation of centrosomes after nuclear envelope breakdown.

Topics: Actins; Amides; Centrosome; Enzyme Inhibitors; Eukaryotic Cells; Heterocyclic Compounds, 4 or More Rings; Models, Biological; Myosin Type II; Nuclear Envelope; Pyridines; RNA Interference; Spindle Apparatus; Time Factors

The role of myosin II in mitosis is generally thought to be restricted to cytokinesis. We present surprising new evidence that cortical myosin II is also required for spindle assembly in cells. Drug- or RNAi-mediated disruption of myosin II in cells interferes with normal spindle assembly and positioning. Time-lapse movies reveal that these treatments block the separation and positioning of duplicated centrosomes after nuclear envelope breakdown (NEBD), thereby preventing the migration of the microtubule asters to opposite sides of chromosomes. Immobilization of cortical movement with tetravalent lectins produces similar spindle defects to myosin II disruption and suggests that myosin II activity is required within the cortex. Latex beads bound to the cell surface move in a myosin II-dependent manner in the direction of the separating asters. We propose that after NEBD, completion of centrosome separation and positioning around chromosomes depends on astral microtubule connections to a moving cell cortex.

Topics: Actins; Amides; Animals; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Cell Polarity; Centrosome; Cross-Linking Reagents; Drosophila; Enzyme Inhibitors; Heterocyclic Compounds, 4 or More Rings; Hybridomas; Lectins; Marine Toxins; Marsupialia; Mitosis; Models, Biological; Movement; Myosin Type II; Nuclear Envelope; Pyridines; RNA Interference; Spindle Apparatus; Thiazoles; Thiazolidines; Time Factors

Failure of cells to cleave at the end of mitosis is dangerous to the organism because it immediately produces tetraploidy and centrosome amplification, which is thought to produce genetic imbalances. Using normal human and rat cells, we reexamined the basis for the attractive and increasingly accepted proposal that normal mammalian cells have a "tetraploidy checkpoint" that arrests binucleate cells in G1, thereby preventing their propagation. Using 10 microM cytochalasin to block cleavage, we confirm that most binucleate cells arrest in G1. However, when we use lower concentrations of cytochalasin, we find that binucleate cells undergo DNA synthesis and later proceed through mitosis in >80% of the cases for the hTERT-RPE1 human cell line, primary human fibroblasts, and the REF52 cell line. These observations provide a functional demonstration that the tetraploidy checkpoint does not exist in normal mammalian somatic cells.

Topics: Animals; Cell Line, Transformed; Cell Nucleus; Cytochalasin D; DNA; Fibroblasts; G1 Phase; Genes, cdc; Heterocyclic Compounds, 4 or More Rings; Humans; Mitosis; Polyploidy; Rats

Blebbistatin is a recently discovered small molecule inhibitor showing high affinity and selectivity toward myosin II. Here we report a detailed investigation of its mechanism of inhibition. Blebbistatin does not compete with nucleotide binding to the skeletal muscle myosin subfragment-1. The inhibitor preferentially binds to the ATPase intermediate with ADP and phosphate bound at the active site, and it slows down phosphate release. Blebbistatin interferes neither with binding of myosin to actin nor with ATP-induced actomyosin dissociation. Instead, it blocks the myosin heads in a products complex with low actin affinity. Blind docking molecular simulations indicate that the productive blebbistatin-binding site of the myosin head is within the aqueous cavity between the nucleotide pocket and the cleft of the actin-binding interface. The property that blebbistatin blocks myosin II in an actin-detached state makes the compound useful both in muscle physiology and in exploring the cellular function of cytoplasmic myosin II isoforms, whereas the stabilization of a specific myosin intermediate confers a great potential in structural studies.

Topics: Adenosine Diphosphate; Adenosine Triphosphatases; Animals; Binding Sites; Heterocyclic Compounds, 4 or More Rings; Models, Molecular; Myosin Type II; Protein Binding; Rabbits

Blebbistatin was recently identified as a selective, cell-permeant inhibitor of myosin II. Because blebbistatin is likely to be used extensively with fluorescence imaging in studies of cytoskeletal dynamics, its compatibility with common excitation wavelengths was examined. Illumination of blebbistatin-treated bovine aortic endothelial cells at 365 and 450-490 nm, but not 510-560 or 590-650 nm, caused dose-dependent cell death. Illumination of blebbistatin alone at 365 and 450-490 nm changed its absorption and emission spectra, but the resultant compounds were not toxic. In addition, photoreacted blebbistatin no longer disrupted myosin distribution in cells, indicating loss of pharmacological activity. Fluorescence microscopy showed that upon illumination, blebbistatin became bound to cells and to protein-coated glass, suggesting that toxicity may arise from light-induced reaction of blebbistatin with cell proteins. Blebbistatin should be used only with careful consideration of these photochemical effects.

Topics: Animals; Aorta; Cattle; Cells, Cultured; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Drug Resistance; Endothelium, Vascular; Heterocyclic Compounds, 4 or More Rings; Light; Radiation Tolerance; Tissue Distribution; Ultraviolet Rays

Understanding the mechanistic basis of the response of neurons to injury is directly relevant to the development of effective therapeutic approaches aimed at the amelioration of nervous system damage. Axons retract in response to severing. We investigated the mechanism of axon retraction in response to severing in vitro, testing the hypothesis that actomyosin contractility drives severing-induced axon retraction. Axon retraction commenced within 5 min following severing and correlated with actin filament accumulation at the site of severing. Depolymerization of actin filaments prevented retraction, demonstrating that actin filaments are required for severing-induced axon retraction. Direct inhibition of myosin II, using blebbistatin, minimized axon retraction in response to severing. Blocking RhoA-kinase (ROCK), a modulator of myosin II activity, inhibited axon retraction. Similarly, inhibiting myosin light chain kinase (MLCK) with a cell-permeable pseudo-substrate peptide also inhibited axon retraction. These data demonstrate that myosin II activity is required for severing-induced axon retraction in vitro, and suggest myosin II as a target for therapeutic interventions aimed at minimizing retraction following severing in vivo.

Topics: Actins; Amides; Animals; Axons; Axotomy; Bridged Bicyclo Compounds, Heterocyclic; Chick Embryo; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Ganglia, Spinal; Growth Cones; Heterocyclic Compounds, 4 or More Rings; Immunohistochemistry; Microscopy, Video; Myosin Type II; Neurons; Organ Culture Techniques; Peptides; Pyridines; Retina; Retrograde Degeneration; Thiazoles; Thiazolidines; Time Factors; Tubulin

Rho family GTPases play pivotal roles in cytokinesis. By using probes based on the principle of fluorescence resonance energy transfer (FRET), we have shown that in HeLa cells RhoA activity increases with the progression of cytokinesis. Here we show that in Rat1A cells RhoA activity remained suppressed during most of the cytokinesis. Consistent with this observation, the expression of C3 toxin inhibited cytokinesis in HeLa cells but not in Rat1A cells. Furthermore, the expression of a dominant negative mutant of Ect2, a Rho GEF, or Y-27632, an inhibitor of the Rho-dependent kinase ROCK, inhibited cytokinesis in HeLa cells but not in Rat1A cells. In contrast to the activity of RhoA, the activity of Rac1 was suppressed during cytokinesis and started increasing at the plasma membrane of polar sides before the abscission of the daughter cells in both HeLa and Rat1A cells. This type of Rac1 suppression was shown to be essential for cytokinesis because a constitutively active mutant of Rac1 induced a multinucleated phenotype in both HeLa and Rat1A cells. Moreover, the involvement of MgcRacGAP/CYK-4 in this suppression of Rac1 during cytokinesis was shown by the use of a dominant negative mutant. Because ML-7, an inhibitor of myosin light chain kinase, delayed the cytokinesis of Rat1A cells and because Pak, a Rac1 effector, is known to suppress myosin light chain kinase, the suppression of the Rac1-Pak pathway by MgcRacGAP may play a pivotal role in the cytokinesis of Rat1A cells.

Topics: Adenoviridae; ADP Ribose Transferases; Amides; Animals; Azepines; Botulinum Toxins; Cell Line; Cell Membrane; Cytokinesis; DNA, Complementary; Fluorescence Resonance Energy Transfer; G1 Phase; G2 Phase; Gene Expression Regulation; Genes, Dominant; Green Fluorescent Proteins; HeLa Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Mice; Mutation; Naphthalenes; NIH 3T3 Cells; Plasmids; Proto-Oncogene Proteins; Pyridines; Rats; rhoA GTP-Binding Protein; Time Factors

Cell migration initiates by extension of the actin cytoskeleton at the leading edge. Computational analysis of fluorescent speckle microscopy movies of migrating epithelial cells revealed this process is mediated by two spatially colocalized but kinematically, kinetically, molecularly, and functionally distinct actin networks. A lamellipodium network assembled at the leading edge but completely disassembled within 1 to 3 micrometers. It was weakly coupled to the rest of the cytoskeleton and promoted the random protrusion and retraction of the leading edge. Productive cell advance was a function of the second colocalized network, the lamella, where actomyosin contraction was integrated with substrate adhesion.

Topics: Actin Cytoskeleton; Actins; Animals; Cell Line; Cell Movement; Cells, Cultured; Cytochalasin D; Depsipeptides; Epithelial Cells; Heterocyclic Compounds, 4 or More Rings; Kinetics; Macropodidae; Microscopy, Fluorescence; Motion Pictures; Peptides, Cyclic; Pseudopodia; Salamandridae

Tissue cells generally pull on their matrix attachments and balance a quasi-static contractility against adequate adhesion, but any correlation with and/or influence on phenotype are not yet understood. Here, we begin to demonstrate how differentiation state couples to actomyosin-based contractility through adhesion and substrate compliance. Myotubes are differentiated from myoblasts on collagen-patterned coverslips that allow linear fusion but prevent classic myotube branching. Post-fusion, myotubes adhere to the micro-strips but lock into a stress fiber-rich state and do not differentiate significantly further. In contrast, myotubes grown on top of such cells do progress through differentiation, exhibiting actomyosin striations within one week. A compliant adhesion to these lower cells is suggested to couple to contractility and accommodate the reorganization needed for upper cell striation. Contractility is assessed in these adherent cells by mechanically detaching one end of the myotubes. All myotubes, whether striated or not, shorten with an exponential decay. The cell-on-cell myotubes relax more, which implies a greater contractile stress. The non-muscle myosin II inhibitor blebbistatin inhibits relaxation for either case. Myotubes in culture are thus clearly prestressed by myosin II, and this contractility couples to substrate compliance and ultimately influences actomyosin striation.

Topics: Actins; Actomyosin; Animals; Cell Adhesion; Cell Differentiation; Cell Line; Collagen; Heterocyclic Compounds, 4 or More Rings; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Models, Biological; Muscle Cells; Muscle Contraction; Muscle Fibers, Skeletal; Myosin Type II; Myosins; Time Factors

We examined the effect of blebbistatin on the kinetic properties of nonmuscle myosin IIB subfragment 1 (NMIIB S1). Blebbistatin is a small molecule that affects cell blebbing during the process of cell division, which has been shown to decrease the myosin ATPase activity of a number of myosins [Straight et al. (2003) Science 299, 1743-1747]. The steady-state actin-activated ATPase activity of NMIIB S1 was decreased approximately 90% at 40 microM actin in the presence of blebbistatin. Stopped-flow techniques were employed to elucidate the effect of blebbistatin on the various steps of the NMIIB S1 cross-bridge cycle. Blebbistatin did not affect ATP binding and hydrolysis. Binding to actin in the presence of ADP (0.57 +/-0.08 microM(-1) s(-1)) was reduced slightly in the presence of blebbistatin (0.38 +/- 0.03 microM(-1) s(-1)), while mantADP dissociation from acto-NMIIB S1 was reduced (approximately 30%). P(i) release was blocked in the presence of blebbistatin. Accordingly, the apparent affinity of NMIIB S1 for actin in the presence of ATP was greatly reduced. Based on the above data, we surmise that blebbistatin inhibits the ATPase activity of NMIIB S1 primarily by blocking entry into the strong binding state; secondarily, it reduces the rate of ADP release. These effects are likely mediated by binding of blebbistatin within the myosin cleft that progressively closes in forming the acto-myosin rigor state.

Topics: Actins; Adenosine Diphosphate; Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Chickens; Heterocyclic Compounds, 4 or More Rings; Hydrolysis; Kinetics; Myosin Subfragments; Nonmuscle Myosin Type IIB; ortho-Aminobenzoates; Peptide Fragments; Phosphates; Photochemistry; Protein Binding; Pyrenes

Blebbistatin is a small molecule inhibitor discovered in a screen for inhibitors of nonmuscle myosin IIA. We have examined the specificity and potency of the drug by assaying its effects on the actin-activated MgATPase assay of diverse members of the myosin superfamily. Blebbistatin potently inhibits several striated muscle myosins as well as vertebrate nonmuscle myosin IIA and IIB with IC50 values ranging from 0.5 to 5 microM. Interestingly, smooth muscle which is highly homologous to vertebrate nonmuscle myosin is only poorly inhibited (IC50=80 microM). The drug potently inhibits Dictyostelium myosin II, but poorly inhibits Acanthamoeba myosin II. Blebbistatin did not inhibit representative myosin superfamily members from classes I, V, and X.

Topics: Actins; Adenosine Triphosphate; Animals; Fluorescent Dyes; Heterocyclic Compounds, 4 or More Rings; Humans; Myosin Type II; Myosins

The molecular mechanisms by which neurotrophins regulate growth cone motility are not well understood. This study investigated the signaling involved in transducing BDNF-induced increases of filopodial dynamics. Our results indicate that BDNF regulates filopodial length and number through a Rho kinase-dependent mechanism. Additionally, actin depolymerizing factor (ADF)/cofilin activity is necessary and sufficient to transduce the effects of BDNF. Our data indicate that activation of ADF/cofilin mimics the effects of BDNF on filopodial dynamics, whereas ADF/cofilin inactivity blocks the effects of BDNF. Furthermore, BDNF promotes the activation of ADF/cofilin by reducing the phosphorylation of ADF/cofilin. Although inhibition of myosin II also enhances filopodial length, our results indicate that BDNF signaling is independent of myosin II activity and that the two pathways result in additive effects on filopodial length. Thus, filopodial extension is regulated by at least two independent mechanisms. The BDNF-dependent pathway works via regulation of ADF/cofilin, independently of myosin II activity.

Topics: 14-3-3 Proteins; Actin Depolymerizing Factors; Animals; Brain-Derived Neurotrophic Factor; Chick Embryo; Destrin; Growth Cones; Heterocyclic Compounds, 4 or More Rings; Intracellular Signaling Peptides and Proteins; Microfilament Proteins; Myosin Type II; Phosphorylation; Protein Serine-Threonine Kinases; Pseudopodia; Retina; rho-Associated Kinases; Signal Transduction; Tissue Culture Techniques

Completion of cell division during cytokinesis requires temporally and spatially regulated communication from the microtubule cytoskeleton to the actin cytoskeleton and the cell membrane. We identified a specific inhibitor of nonmuscle myosin II, blebbistatin, that inhibited contraction of the cleavage furrow without disrupting mitosis or contractile ring assembly. Using blebbistatin and other drugs, we showed that exit from the cytokinetic phase of the cell cycle depends on ubiquitin-mediated proteolysis. Continuous signals from microtubules are required to maintain the position of the cleavage furrow, and these signals control the localization of myosin II independently of other furrow components.

Topics: Anaphase; Aurora Kinases; Cell Division; Cell Movement; Contractile Proteins; Cyclin-Dependent Kinases; DNA Replication; Enzyme Inhibitors; HeLa Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Kinesins; Leupeptins; Microtubules; Mitosis; Myosin Type II; Nocodazole; Protein Serine-Threonine Kinases; Staurosporine; Ubiquitin

Fibroblast-collagen matrix contraction has been used as a model system to study how cells organize connective tissue. Previous work showed that lysophosphatidic acid (LPA)-stimulated floating collagen matrix contraction is independent of Rho kinase, whereas platelet-derived growth factor (PDGF)-stimulated contraction is Rho kinase-dependent. The current studies were carried out to learn more about the molecular motors responsible for LPA- and PDGF-stimulated contraction. We found that neither PDGF nor LPA-dependent contractile mechanisms require myosin II regulatory light chain kinase or increased phosphorylation of myosin II regulatory light chain (measured as diphosphorylation). Low concentrations of the specific myosin II inhibitor blebbistatin blocked PDGF-stimulated matrix contraction and LPA-stimulated retraction of fibroblast dendritic extensions but not LPA-stimulated matrix contraction. These data suggest that PDGF- and LPA-stimulated floating matrix contraction utilize myosin II-dependent and -independent mechanisms, respectively. LPA-dependent, Rho kinase-independent force generation also was detected during fibroblast spreading on collagen-coated coverslips.

Topics: Cells, Cultured; Collagen; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Humans; Immunoblotting; Intracellular Signaling Peptides and Proteins; Lysophospholipids; Microscopy, Fluorescence; Myosin Light Chains; Myosin Type II; Phosphorylation; Platelet-Derived Growth Factor; Protein Serine-Threonine Kinases; rho-Associated Kinases; Time Factors; Transfection; Wound Healing