latrunculin-a and jasplakinolide

The F-actin cytoskeleton and its connection to the plasma membrane provide structure and shape of epithelial cells. In this study we focus on the impact of the F-actin cytoskeleton on the morphology and mechanical behaviour of confluent epithelial cells. F-actin depolymerisation was fostered by Latrunculin A, while depolymerisation was allayed by Jasplakinolide. The impact of drug treatment on cellular mechanics was measured using atomic force microscopy based active microrheology and force-indentation curves, while morphology was monitored by AFM imaging, electric cell-substrate impedance sensing (ECIS) experiments and fluorescence microscopy. A softening and fluidisation of the cells upon dissolution of F-actin was observed, accompanied by reduction of cell-substrate and cell-cell contacts and an altered topography. The strengthening of actin filaments upon Jasplakinolide treatment was mirrored in several mechanical properties. The largest impact was on the cellular viscosity. The cells were, however, capable of restoring their initial phenotypes, e.g., amount of actin, intercellular and cell-substrate interactions.

Topics: Actins; Animals; Biomechanical Phenomena; Bridged Bicyclo Compounds, Heterocyclic; Cytoskeleton; Depsipeptides; Dogs; Epithelial Cells; Kinetics; Madin Darby Canine Kidney Cells; Mechanical Phenomena; Phenotype; Thiazolidines

To investigate plasmodesmata (PD) function, a useful technique is to monitor the effect on cell-to-cell transport of applying an inhibitor of a physiological process, protein, or other cell component of interest. Changes in PD transport can then be monitored in one of several ways, most commonly by measuring the cell-to-cell movement of fluorescent tracer dyes or of free fluorescent proteins. Effects on PD structure can be detected in thin sections of embedded tissue observed using an electron microscope, most commonly a Transmission Electron Microscope (TEM). This chapter outlines commonly used inhibitors, methods for treating different tissues, how to detect altered cell-to-cell transport and PD structure, and important caveats.

Topics: Actins; Arabidopsis; Biological Transport; Bridged Bicyclo Compounds, Heterocyclic; Cytochalasin B; Cytotoxins; Depsipeptides; Fixatives; Fluorescent Dyes; Gene Expression; Green Fluorescent Proteins; Image Processing, Computer-Assisted; Microinjections; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Microtomy; Phalloidine; Plant Roots; Plasmodesmata; Profilins; Recombinant Fusion Proteins; Thiazolidines; Tissue Fixation; Tradescantia

Cell-shape changes associated with processes like cytokinesis and motility proceed on several-second timescales but are derived from molecular events, including protein-protein interactions, filament assembly, and force generation by molecular motors, all of which occur much faster [1-4]. Therefore, defining the dynamics of such molecular machinery is critical for understanding cell-shape regulation. In addition to signaling pathways, mechanical stresses also direct cytoskeletal protein accumulation [5-7]. A myosin-II-based mechanosensory system controls cellular contractility and shape during cytokinesis and under applied stress [6, 8]. In Dictyostelium, this system tunes myosin II accumulation by feedback through the actin network, particularly through the crosslinker cortexillin I. Cortexillin-binding IQGAPs are major regulators of this system. Here, we defined the short timescale dynamics of key cytoskeletal proteins during cytokinesis and under mechanical stress, using fluorescence recovery after photobleaching and fluorescence correlation spectroscopy, to examine the dynamic interplay between these proteins. Equatorially enriched proteins including cortexillin I, IQGAP2, and myosin II recovered much more slowly than actin and polar crosslinkers. The mobility of equatorial proteins was greatly reduced at the furrow compared to the interphase cortex, suggesting their stabilization during cytokinesis. This mobility shift did not arise from a single biochemical event, but rather from a global inhibition of protein dynamics by mechanical-stress-associated changes in the cytoskeletal structure. Mechanical tuning of contractile protein dynamics provides robustness to the cytoskeletal framework responsible for regulating cell shape and contributes to cytokinesis fidelity.

Topics: Analysis of Variance; Biomechanical Phenomena; Bridged Bicyclo Compounds, Heterocyclic; Cell Cycle Proteins; Cell Shape; Cytokinesis; Cytoskeleton; Depsipeptides; Dictyostelium; Fluorescence Recovery After Photobleaching; Microfilament Proteins; Myosin Type II; Protozoan Proteins; ras GTPase-Activating Proteins; Signal Transduction; Spectrometry, Fluorescence; Stress, Mechanical; Thiazolidines

Functional selectivity, a process by which G-protein coupled receptors (GPCRs) can activate one signaling route while avoiding another, is regulated by ligand-mediated stabilization of specific receptor states that modulate different downstream signaling events. We propose a novel mechanism for functional selectivity, induced by the endogenous P2Y2R agonist ATP and regulated at the signaling interface by the cytoskeleton. Upon ATP stimulation of human neutrophils, a transient rise in the cytosolic concentration of free Ca(2+) was not followed by activation of the superoxide anion-generating NADPH-oxidase. This was in contrast to signals generated through the formyl peptide receptor 1 (FPR1), as its activation was accompanied by both a mobilization of Ca(2+) and activation of the NADPH-oxidase. The phospholipase C/Ca(2+) signaling route is not modulated by the cytoskeleton-disrupting drug latrunculin A, but this drug was able to launch a new signaling route downstream of P2Y2R that led to NADPH-oxidase activation. The signaling downstream of P2Y2R was rapidly terminated and the receptors were desensitized; however, in contrast to desensitized FPR1, no P2Y2 receptor reactivation could be induced by latrunculin A. Thus, P2Y2R desensitization does not appear to involve the cytoskeleton, contrary to FPR1 desensitization. In summary, we hereby describe how ATP regulates functional selectivity via the cytoskeleton, leading to intracellular Ca(2+) increase, alone or with simultaneous NADPH-oxidase activation in neutrophils.

Topics: Adenosine Triphosphate; Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cells, Cultured; Cytoskeleton; Depsipeptides; Enzyme Activation; Humans; Membrane Potentials; NADPH Oxidases; Neutrophil Activation; Neutrophils; Pertussis Toxin; Receptors, Formyl Peptide; Receptors, Purinergic P2Y2; Signal Transduction; Thiazolidines; Type C Phospholipases

We previously demonstrated that many aspects of the intracellular Ca(2+) increase in fertilized eggs of starfish are significantly influenced by the state of the actin cytoskeleton. In addition, the actin cytoskeleton appeared to play comprehensive roles in modulating cortical granules exocytosis and sperm entry during the early phase of fertilization. In the present communication, we have extended our work to sea urchin which is believed to have bifurcated from the common ancestor in the phylogenetic tree some 500 million years ago. To corroborate our earlier findings in starfish, we have tested how the early events of fertilization in sea urchin eggs are influenced by four different actin-binding drugs that promote either depolymerization or stabilization of actin filaments. We found that all the actin drugs commonly blocked sperm entry in high doses and significantly reduced the speed of the Ca(2+) wave. At low doses, however, cytochalasin B and phalloidin increased the rate of polyspermy. Overall, certain aspects of Ca(2+) signaling in these eggs were in line with the morphological changes induced by the actin drugs. That is, the time interval between the cortical flash and the first Ca(2+) spot at the sperm interaction site (the latent period) was significantly prolonged in the eggs pretreated with cytochalasin B or latrunculin A, whereas the Ca(2+) decay kinetics after the peak was specifically attenuated in the eggs pretreated with jasplakinolide or phalloidin. In addition, the sperm interacting with the eggs pretreated with actin drugs often generated multiple Ca(2+) waves, but tended to fail to enter the egg. Thus, our results indicated that generation of massive Ca(2+) waves is neither indicative of sperm entry nor sufficient for cortical granules exocytosis in the inseminated sea urchin eggs, whereas the structure and functionality of the actin cytoskeleton are the major determining factors in the two processes.

Topics: Actin Cytoskeleton; Animals; Bridged Bicyclo Compounds, Heterocyclic; Calcium Signaling; Cytochalasin B; Depsipeptides; Exocytosis; Female; Fertilization; Male; Microfilament Proteins; Oocytes; Paracentrotus; Phalloidine; Thiazolidines; Zygote

Neutrophils are cells of the innate immune system that hunt and kill pathogens using directed migration. This process, known as chemotaxis, requires the regulation of actin polymerization downstream of chemoattractant receptors. Reciprocal interactions between actin and intracellular signals are thought to underlie many of the sophisticated signal processing capabilities of the chemotactic cascade including adaptation, amplification and long-range inhibition. However, with existing tools, it has been difficult to discern actin's role in these processes. Most studies investigating the role of the actin cytoskeleton have primarily relied on actin-depolymerizing agents, which not only block new actin polymerization but also destroy the existing cytoskeleton. We recently developed a combination of pharmacological inhibitors that stabilizes the existing actin cytoskeleton by inhibiting actin polymerization, depolymerization and myosin-based rearrangements; we refer to these processes collectively as actin dynamics. Here, we investigated how actin dynamics influence multiple signalling responses (PI3K lipid products, calcium and Pak phosphorylation) following acute agonist addition or during desensitization. We find that stabilized actin polymer extends the period of receptor desensitization following agonist binding and that actin dynamics rapidly reset receptors from this desensitized state. Spatial differences in actin dynamics may underlie front/back differences in agonist sensitivity in neutrophils.

Topics: Actins; Amides; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Chemotaxis; Cytoskeleton; Depsipeptides; Humans; Myosins; Neutrophils; Phosphorylation; Polymerization; Pyridines; Signal Transduction; Thiazolidines

The deformability of circulating leukocytes plays an important role in the physiopathology of several diseases like sepsis or acute respiratory distress syndrome (ARDS). We present here a microfluidic method for the passive testing, sorting and separating of non-adherent cell populations by deformability. It consists of microfluidic sieves in series with pore sizes decreasing from the upstream to the downstream. The method capabilities are demonstrated with monocytic cell lines (THP-1) treated by Jasplakinolide (a stabilizer of polymerized actin), LatrunculinA (an inhibitor of actin polymerization), and with the plasma of patients suffering from ARDS. Simple sample injection with standard syringes and pumps makes the method readily adapted for experimentation in hospitals.

Topics: Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Cell Separation; Cells, Cultured; Depsipeptides; Filtration; Hemorheology; Humans; Leukocytes; Microfluidic Analytical Techniques; Porosity; Respiratory Distress Syndrome; Thiazolidines

Insulin secretion (IS) triggered by β-cell [Ca(2+)](c) is amplified by metabolic and receptor-generated signals. Diacylglycerol largely mediates acetylcholine (ACh) effects through protein-kinase C and other effectors, which can be directly activated by phorbol-ester (PMA). Using mouse islets, we investigated the possible role of microfilaments in ACh/PMA-mediated amplification of IS. PMA had no steady-state impact on actin microfilaments. Although ACh slightly augmented and PMA diminished glucose- and tolbutamide-induced increases in β-cell [Ca(2+)](c), both amplified IS in control islets and after microfilament disruption (latrunculin) or stabilization (jasplakinolide). Both phases of IS were larger in response to glucose than tolbutamide, although [Ca(2+)](c) was lower. This difference in secretion, which reflects metabolic amplification, persisted in presence of ACh/PMA and was independent of microfilaments. Amplification of IS by ACh/PMA is thus distinct from metabolic amplification, but both pathways promote acquisition of release competence by insulin granules, which can access exocytotic sites without intervention of microfilaments.

Topics: Acetylcholine; Actin Cytoskeleton; Animals; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Depsipeptides; Female; Glucose; Insulin; Insulin Secretion; Insulin-Secreting Cells; Mice; Mice, Inbred C57BL; Phorbol Esters; Polymerization; Thiazolidines; Tolbutamide

We investigated actin's function in vesicle recycling and exocytosis at lamprey synapses and show that FM1-43 puncta and phalloidin-labeled filamentous actin (F-actin) structures are colocalized, yet recycling vesicles are not contained within F-actin clusters. Additionally, phalloidin also labels a plasma membrane-associated cortical actin. Injection of fluorescent G-actin revealed activity-independent dynamic actin incorporation into presynaptic synaptic vesicle clusters but not into cortical actin. Latrunculin-A, which sequesters G-actin, dispersed vesicle-associated actin structures and prevented subsequent labeled G-actin and phalloidin accumulation at presynaptic puncta, yet cortical phalloidin labeling persisted. Dispersal of presynaptic F-actin structures by latrunculin-A did not disrupt vesicle clustering or recycling or alter the amplitude or kinetics of excitatory postsynaptic currents (EPSCs). However, it slightly enhanced release during repetitive stimulation. While dispersal of presynaptic actin puncta with latrunculin-A failed to disperse synaptic vesicles or inhibit synaptic transmission, presynaptic phalloidin injection blocked exocytosis and reduced endocytosis measured by action potential-evoked FM1-43 staining. Furthermore, phalloidin stabilization of only cortical actin following pretreatment with latrunculin-A was sufficient to inhibit synaptic transmission. Conversely, treatment of axons with jasplakinolide, which induces F-actin accumulation but disrupts F-actin structures in vivo, resulted in increased synaptic transmission accompanied by a loss of phalloidin labeling of cortical actin but no loss of actin labeling within vesicle clusters. Marked synaptic deficits seen with phalloidin stabilization of cortical F-actin, in contrast to the minimal effects of disruption of a synaptic vesicle-associated F-actin, led us to conclude that two structurally and functionally distinct pools of actin exist at presynaptic sites.

Topics: Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Depsipeptides; Endocytosis; Exocytosis; Lampreys; Phalloidine; Presynaptic Terminals; Pyridinium Compounds; Quaternary Ammonium Compounds; Synaptic Transmission; Synaptic Vesicles; Thiazolidines

Fertilization changes the structure and function of the cell surface. In sea urchins, these changes include polymerization of cortical actin and a coincident, switch-like increase in the activity of the multidrug efflux transporter ABCB1a. However, it is not clear how cortical reorganization leads to changes in membrane transport physiology. In this study, we used three-dimensional superresolution fluorescence microscopy to resolve the fine-scale movements of the transporter along polymerizing actin filaments, and we show that efflux activity is established after ABCB1a translocates to the tips of the microvilli. Inhibition of actin polymerization or bundle formation prevents tip localization, resulting in the patching of ABCB1a at the cell surface and decreased efflux activity. In contrast, enhanced actin polymerization promotes tip localization. Finally, interference with Rab11, a regulator of apical recycling, inhibits activation of efflux activity in embryos. Together our results show that actin-mediated, short-range traffic and positioning of transporters at the cell surface regulates multidrug efflux activity and highlight the multifaceted roles of microvilli in the spatial distribution of membrane proteins.

Topics: Actins; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Cell Membrane; Cytochalasin D; Depsipeptides; Embryo, Nonmammalian; Female; Fertilization; Male; Microscopy, Confocal; Microscopy, Fluorescence; Microvilli; Ovum; Phylogeny; Polymerization; Protein Transport; rab GTP-Binding Proteins; Sea Urchins; Thiazolidines

In migrating fibroblasts, RhoA and its effector mDia1 regulate the selective stabilization of microtubules (MTs) polarized in the direction of migration. The conserved formin homology 2 domain of mDia1 is involved both in actin polymerization and MT stabilization, and the relationship between these two activities is unknown. We found that latrunculin A (LatA) and jasplakinolide, actin drugs that release mDia1 from actin filament barbed ends, stimulated stable MT formation in serum-starved fibroblasts and caused a redistribution of mDia1 onto MTs. Knockdown of mDia1 by small interfering RNA (siRNA) prevented stable MT induction by LatA, whereas blocking upstream Rho or integrin signaling had no effect. In search of physiological regulators of mDia1, we found that actin-capping protein induced stable MTs in an mDia1-dependent manner and inhibited the translocation of mDia on the ends of growing actin filaments. Knockdown of capping protein by siRNA reduced stable MT levels in proliferating cells and in starved cells stimulated with lysophosphatidic acid. These results show that actin-capping protein is a novel regulator of MT stability that functions by antagonizing mDia1 activity toward actin filaments and suggest a novel form of actin-MT cross-talk in which a single factor acts sequentially on actin and MTs.

Topics: Actin Capping Proteins; Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Carrier Proteins; Depsipeptides; Formins; Genes, Dominant; Glutamic Acid; Integrins; Mice; Microtubule-Associated Proteins; Microtubules; NIH 3T3 Cells; rho GTP-Binding Proteins; Signal Transduction; Thiazolidines; Tyrosine

Clasmatodendrosis is an irreversible astroglial degenerative change, which includes extensive swelling and vacuolization of cell bodies and disintegrated and beaded processes. Since alteration in F-actin level influences on the formation of vacuoles/vesicles during exocytosis/endocytosis in astrocytes, we investigated whether F-actin polymerization involves clasmatodendrosis in the rat hippocampus following status epilepticus (SE). In the present study, vacuoles in clasmatodendrotic astrocytes showed LAMP-1 and LC3-II (a marker for autophagy) immunoreactivity. These findings reveal that clasmatodendrosis may be lysosome-derived autophagic astroglial death. Jasplakinolide (an F-actin stabilizer) infusion significantly decreased the size and the number of medium/large-sized vacuoles in each clasmatodendritic astrocyte accompanied by enhancement of phalloidin signals, as compared to vehicle-infusion. In contrast, latrunculin A (an F-actin-depolymerizing agent) infusion increased the size and the number of medium/large-sized vacuoles, which were dissociated adjacent to cell membrane. Therefore, our findings suggest that F-actin stabilization may inhibit lysosome-derived autophagic astroglial death during clasmatodendrosis.

Topics: Actins; Animals; Antineoplastic Agents; Astrocytes; Autophagy; Biomarkers; Bridged Bicyclo Compounds, Heterocyclic; Cell Death; Depsipeptides; Lysosomes; Male; Rats; Rats, Sprague-Dawley; Seizures; Thiazolidines

Numerous studies indicate a role for the actin cytoskeleton in secretion. Here, we have used evanescent wave and widefield fluorescence microscopy to study the involvement of the actin cytoskeleton in secretion from PC12 cells. Secretion was assayed as loss of ANF-EmGFP in widefield mode. Under control conditions, depolarization induced secretion showed two phases: an initial rapid rate of loss of vesicular cargo (tau = 1.4 s), followed by a slower, sustained drop in fluorescence (tau = 34.1 s). Pretreatment with Latrunculin A changed the kinetics to a single exponential, slightly faster than the fast component of control cells (1.2 s). Evanescent wave microscopy allowed us to examine this at the level of individual events, and revealed equivalent changes in the rates of vesicular arrival at the plasma membrane immediately following and during the sustained phase of release. Co-transfection of mCherry labeled β-actin and ANF-EmGFP demonstrated that sites of exocytosis had an inverse relationship with sites of actin enrichment. Disruption of visualized actin at the membrane resulted in the loss of specificity of exocytic site.

Topics: Actin Cytoskeleton; Actins; Animals; Atrial Natriuretic Factor; Bridged Bicyclo Compounds, Heterocyclic; Depsipeptides; Exocytosis; Green Fluorescent Proteins; Membrane Fusion; Models, Biological; PC12 Cells; Protein Transport; Rats; Secretory Vesicles; Staining and Labeling; Thiazolidines; Utrophin

Paramyxovirus fusion (F) proteins promote both virus-cell fusion, required for viral entry, and cell-cell fusion, resulting in syncytia formation. We used the F-actin stabilizing drug, jasplakinolide, and the G-actin sequestrant, latrunculin A, to examine the role of actin dynamics in cell-cell fusion mediated by the parainfluenza virus 5 (PIV5) F protein. Jasplakinolide treatment caused a dose-dependent increase in cell-cell fusion as measured by both syncytia and reporter gene assays, and latrunculin A treatment also resulted in fusion stimulation. Treatment with jasplakinolide or latrunculin A partially rescued a fusion pore opening defect caused by deletion of the PIV5 F protein cytoplasmic tail, but these drugs had no effect on fusion inhibited at earlier stages by either temperature arrest or by a PIV5 heptad repeat peptide. These data suggest that the cortical actin cytoskeleton is an important regulator of fusion pore enlargement, an energetically costly stage of viral fusion protein-mediated membrane merger.

Topics: Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Cell Fusion; Cell Line; Chlorocebus aethiops; Cricetinae; Cytoskeleton; Depsipeptides; Humans; Models, Biological; Paramyxoviridae; Thiazolidines; Viral Fusion Proteins

Although patients with acute respiratory distress syndrome require mechanical ventilation, these ventilators often exacerbate the existing lung injury. For example, the cyclic closure and reopening of fluid-filled airways during ventilation can cause epithelial cell (EpC) necrosis and barrier disruption. Although much work has focused on minimizing the injurious mechanical forces generated during ventilation, an alternative approach is to make the EpC less susceptible to injury by altering the cell's intrinsic biomechanical/biostructural properties. In this study, we hypothesized that alterations in cytoskeletal structure and mechanics can be used to reduce the cell's susceptibility to injury during airway reopening. EpC were treated with jasplakinolide to stabilize actin filaments or latrunculin A to depolymerize actin and then exposed to cyclic airway reopening conditions at room temperature using a previously developed in vitro cell culture model. Actin stabilization did not affect cell viability but significantly improved cell adhesion primarily due to the development of more numerous focal adhesions. Surprisingly, actin depolymerization significantly improved both cell viability and cell adhesion but weakened focal adhesions. Optical tweezer based measurements of the EpC's micromechanical properties indicate that although latrunculin-treated cells are softer, they also have increased viscous damping properties. To further investigate the effect of "fluidization" on cell injury, experiments were also conducted at 37 degrees C. Although cells held at 37 degrees C exhibited no changes in cytoskeletal structure, they did exhibit increased viscous damping properties and improved cell viability. We conclude that fluidization of the actin cytoskeleton makes the EpC less susceptible to the injurious mechanical forces generated during cyclic airway reopening.

Topics: Actins; Biomechanical Phenomena; Bridged Bicyclo Compounds, Heterocyclic; Cell Adhesion; Cell Death; Cell Line; Cytoskeleton; Depsipeptides; Elastic Modulus; Epithelial Cells; Humans; Microscopy, Fluorescence; Optical Tweezers; Stress, Mechanical; Temperature; Thiazolidines; Vinculin

We studied the dynamics of ballistically injected latex particles (BIP) inside endothelial cells, using video particle tracking to measure the mean squared displacement (MSD) as a function of lag time. The MSD shows a plateau at short times and a linear behavior at longer times, indicating that the BIP are trapped into a viscoelastic network. To reveal more about the molecular constituents and the dynamics of this actin network, we added a variety of drugs. Latrunculin and Jasplakinolide aimed at intervening with the actin network caused a strong increase in MSD, whereas Taxol aimed at microtubules gave no measurable change in MSD. Additional corroborating information about these drug effects were obtained from MSD amplitude and exponent distributions and from fluorescent staining images of the actin and microtubule networks. Our evidence strongly suggests that BIP are primarily embedded in the actin network. Additional drug interventions aimed at disabling non-thermal forces could not conclusively resolve the nature of the forces driving BIP dynamics.

Topics: Actins; Adenosine Triphosphate; Biolistics; Bridged Bicyclo Compounds, Heterocyclic; Cells, Cultured; Depsipeptides; Endothelium, Vascular; Fluorescent Dyes; Humans; Latex; Microscopy, Fluorescence; Nocodazole; Paclitaxel; Thiazolidines

Microfilaments play a determinant role in different cell processes such as: motility, cell division, phagocytosis and intracellular transport; however, these structures are poorly understood in the parasite Giardia lamblia.. By confocal microscopy using TRITC-phalloidin, we found structured actin distributed in the entire trophozoite, the label stand out at the ventral disc, median body, flagella and around the nuclei. During Giardia encystation, a sequence of morphological changes concurrent to modifications on the distribution of structured actin and in the expression of actin mRNA were observed. To elucidate whether actin participates actively on growth and encystation, cells were treated with Cytochalasin D, Latrunculin A and Jasplakinolide and analyzed by confocal and scanning electron microscopy. All drugs caused a growth reduction (27 to 45%) and changes on the distribution of actin. Besides, 60 to 80% of trophozoites treated with the drugs, exhibited damage at the caudal region, alterations in the flagella and wrinkles-like on the plasma membrane. The drugs also altered the cyst-yield and the morphology, scanning electron microscopy revealed diminished cytokinesis, cysts with damages in the wall and alterations in the size and on the intermembranal space. Furthermore, the drugs caused a significant reduction of the intensity of fluorescence-labeled CWP1 on ESV and on cyst wall, this was coincident with a reduction of CWP1 gene expression (34%).. All our results, indicated an important role of actin in the morphology, growth and encystation and indirectly suggested an actin role in gene expression.

Topics: Actin Cytoskeleton; Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Cytochalasin D; Depsipeptides; Flagella; Giardia lamblia; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Microscopy, Electron, Scanning; Models, Biological; Phalloidine; Rats; Rats, Wistar; Rhodamines; Thiazolidines

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

The early events in Junín virus (JUNV) infection are not thoroughly understood. We have previously shown that JUNV enter cells by clathrin-mediated endocytosis. In this report we examine the role of microfilaments and microtubules during early virus infection. Inhibitory effects of drugs affecting main cytoskeletal components on JUNV entry into Vero cells were analyzed. Drugs that disrupted microfilaments or stabilized microtubules inhibited early steps of virus entry. In contrast, drugs that stabilized microfilaments or depolymerized microtubules were not able to block virus entry very efficiently. Furthermore, real time PCR was performed to detect viral entry and we found more than 10-fold less RNA when microfilaments were depolymerized while a 100-fold diminution was seen when microtubules were stabilized. Taken together our results demonstrate that JUNV relies on an intact actin network during early infection in Vero cells while a dynamic microtubule network is also needed. This represents an important contribution to the characterization of arenavirus multiplication cycle.

Topics: Animals; Arenaviridae Infections; Bridged Bicyclo Compounds, Heterocyclic; Chlorocebus aethiops; Cytoskeleton; Depsipeptides; Humans; Junin virus; Thiazolidines; Vero Cells; Virus Internalization

Cell-cell fusion in animal development and in pathophysiology involves expansion of nascent fusion pores formed by protein fusogens to yield an open lumen of cell-size diameter. Here we explored the enlargement of micron-scale pores in syncytium formation, which was initiated by a well-characterized fusogen baculovirus gp64. Radial expansion of a single or, more often, of multiple fusion pores proceeds without loss of membrane material in the tight contact zone. Pore growth requires cell metabolism and is accompanied by a local disassembly of the actin cortex under the pores. Effects of actin-modifying agents indicate that the actin cortex slows down pore expansion. We propose that the growth of the strongly bent fusion-pore rim is restricted by a dynamic resistance of the actin network and driven by membrane-bending proteins that are involved in the generation of highly curved intracellular membrane compartments.

Topics: Actins; Animals; Baculoviridae; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Cell Membrane; Cellular Structures; Cytoskeleton; Depsipeptides; Giant Cells; Insecta; Membrane Fusion; Microscopy, Confocal; Models, Biological; Thiazolidines

Activation of cytoskeleton regulator Rho-kinase during ischemia-reperfusion (I/R) plays a major role in I/R injury and apoptosis. Since Rho-kinase is a negative regulator of the pro-survival phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, we hypothesized that inhibition of Rho-kinase can prevent I/R-induced endothelial cell apoptosis by maintaining PI3-kinase/Akt activity and that protective effects of Rho-kinase inhibition are facilitated by prevention of F-actin rearrangement. Human umbilical vein endothelial cells were subjected to 1 h of simulated ischemia and 1 or 24 h of simulated reperfusion after treatment with Rho-kinase inhibitor Y-27632, PI3-kinase inhibitor wortmannin, F-actin depolymerizers cytochalasinD and latrunculinA and F-actin stabilizer jasplakinolide. Intracellular ATP levels decreased following I/R. Y-27632 treatment reduced I/R-induced apoptosis by 31% (P < 0.01) and maintained Akt activity. Both effects were blocked by co-treatment with wortmannin. Y-27632 treatment prevented the formation of F-actin bundles during I/R. Similar results were observed with cytochalasinD treatment. In contrast, latrunculinA and jasplakinolide treatment did not prevent the formation of F-actin bundles during I/R and had no effect on I/R-induced apoptosis. Apoptosis and Akt activity were inversely correlated (R (2) = 0.68, P < 0.05). In conclusion, prevention of F-actin rearrangement by Rho-kinase inhibition or by cytochalasinD treatment attenuated I/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity.

Topics: Actins; Amides; Androstadienes; Apoptosis; Bridged Bicyclo Compounds, Heterocyclic; Cells, Cultured; Cytochalasin D; Depsipeptides; Endothelium, Vascular; Humans; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyridines; Reperfusion Injury; rho-Associated Kinases; Thiazolidines; Wortmannin

In Entamoeba histolytica little is known about the microfilament rearrangements formed by actin and ABPs. Fibronectin regulates many aspects of cell behavior involving the actin cytoskeleton and members of the Rho family of small GTPases. Using trophozoites interacted with fibronectin and glass, we present evidence related with the formation and regulation of different microfilament rearrangements and their cellular distribution, the effect of actin affecting drugs on these arrangements, and on trophozoites adhesion; we also demonstrate that actin isoforms are induced after adhesion, and also the selective participation of specific actin binding proteins such as ABP-120 and phospho-paxillin, regarding their location in the different actin structures. In addition, we show results that confirm the participation of EhRho, ROCK-2, and GAP activities. We propose that fibronectin induced signaling in E. histolytica trophozoites have important consequences in the actin cytoskeleton that may affect its behavior during the invasive process in the host.

Topics: Actin Cytoskeleton; Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Cytochalasin D; Cytoskeleton; Depsipeptides; Entamoeba histolytica; Fibronectins; GTPase-Activating Proteins; Humans; Nucleic Acid Synthesis Inhibitors; Protein Isoforms; rho GTP-Binding Proteins; rho-Associated Kinases; Signal Transduction; Thiazolidines; Trophozoites

Airway hyperresponsiveness (AHR) is associated with airway wall structural remodeling and alterations in airway smooth muscle (ASM) function. Previously, in bronchioles from Brown Norway rats challenged by repeated ovalbumin (OVA) inhalation, we have reported increased force generation and depletion of smooth muscle contractile proteins. Here, we investigated if cytoskeletal changes in smooth muscle could account for this paradox. Sensitized rats (n = 5/group) were repeatedly challenged with OVA or saline, and the lungs were removed 24 h after the last challenge. Levels of globular (G) and filamentous (F) actin in bronchioles were determined by DNase I inhibition and contraction assessed in intact small bronchioles using a myograph. DNase I inhibition assays showed that G-actin monomers were more abundant ( approximately 1F:2G) in extracts from resting small bronchioles from OVA- or saline-challenged animals. However, while contractile protein levels in bronchioles were reduced by OVA (P < 0.05), the proportion of F:G actin was 1.8-fold greater compared with saline challenge (P < 0.05). Consistent with induction of F-actin after OVA challenge, increases in maximum tension development to carbachol or KCl in small bronchioles from OVA-challenged animals were abrogated (P < 0.01) by actin cytoskeleton disruption with 0.5 microM latrunculin A. Cytoskeletal stabilization of F-actin with 0.1 microM jasplakinolide potentiated maximum contractions to carbachol or KCl (P < 0.05) in bronchioles from OVA- but not saline-treated rats. We conclude that alterations in the composition and/or arrangement of the contractile apparatus after OVA exposure confer enhanced contractile responses, possibly as a result of increased F-actin content. Such a mechanism may have relevance for AHR found in allergic asthma.

Topics: Actins; Allergens; Animals; Antineoplastic Agents; Asthma; Bridged Bicyclo Compounds, Heterocyclic; Bronchi; Carbachol; Cells, Cultured; Cytoskeleton; Depsipeptides; Inhalation Exposure; Male; Muscle Contraction; Muscle, Smooth; Myocytes, Smooth Muscle; Ovalbumin; Rats; Thiazolidines

We have previously demonstrated a role for the reorganization of the actin cytoskeleton in store-operated calcium entry (SOCE) in human platelets and interpreted this as evidence for a de novo conformational coupling step in SOCE activation involving the type II IP(3) receptor and the platelet hTRPC1-containing store-operated channel (SOC). Here, we present evidence challenging this model. The actin polymerization inhibitors cytochalasin D or latrunculin A significantly reduced Ca2+ but not Mn2+ or Na+ entry into thapsigargin (TG)-treated platelets. Jasplakinolide, which induces actin polymerization, also inhibited Ca2+ but not Mn2+ or Na+ entry. However, an anti-hTRPC1 antibody inhibited TG-evoked entry of all three cations, indicating that they all permeate an hTRPC1-containing store-operated channel (SOC). These results indicate that the reorganization of the actin cytoskeleton is not involved in SOC activation. The inhibitors of the Na+/Ca2+ exchanger (NCX), KB-R7943 or SN-6, caused a dose-dependent inhibition of Ca2+ but not Mn2+ or Na+ entry into TG-treated platelets. The effects of the NCX inhibitors were not additive with those of actin polymerization inhibitors, suggesting a common point of action. These results indicate a role for two Ca2+ permeable pathways activated following Ca2+ store depletion in human platelets: A Ca2+-permeable, hTRPC1-containing SOC and reverse Na+/Ca2+ exchange, which is activated following Na+ entry through the SOC and requires a functional actin cytoskeleton.

Topics: Actins; Benzyl Compounds; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cytochalasin D; Cytoskeleton; Depsipeptides; Humans; Manganese; Potassium; Sodium; Sodium-Calcium Exchanger; Thapsigargin; Thiazolidines; Thiourea

Changes in gene expression during reversible bud-hypha transitions of the opportunistic fungal pathogen Candida albicans permit adaptation to environmental conditions that are critical for proliferation in host tissues. Our previous work has shown that the hypha-specific adhesin gene HWP1 is up-regulated by the cyclic AMP (cAMP) signaling pathway. However, little is known about the potential influences of determinants of cell morphology on HWP1 gene expression. We found that blocking hypha formation with cytochalasin A, which destabilizes actin filaments, and with latrunculin A, which sequesters actin monomers, led to a loss of HWP1 gene expression. In contrast, high levels of HWP1 gene expression were observed when the F-actin stabilizer jasplakinolide was used to block hypha formation, suggesting that HWP1 expression could be regulated by actin structures. Mutants defective in formin-mediated nucleation of F-actin were reduced in HWP1 gene expression, providing genetic support for the importance of actin structures. Kinetic experiments with wild-type and actin-deficient cells revealed two distinct phases of HWP1 gene expression, with a slow, actin-independent phase preceding a fast, actin-dependent phase. Low levels of HWP1 gene expression that appeared to be independent of stabilized actin and cAMP signaling were detected using indirect immunofluorescence. A connection between actin structures and the cAMP signaling pathway was shown using hyper- and hypomorphic cAMP mutants, providing a possible mechanism for up-regulation of HWP1 gene expression by stabilized actin. The results reveal a new role for F-actin as a regulatory agent of hypha-specific gene expression at the bud-hypha transition.

Topics: Actins; Bridged Bicyclo Compounds, Heterocyclic; Candida albicans; Cell Polarity; Cyclic AMP; Cytochalasins; Cytoskeleton; Depsipeptides; Fluorescent Antibody Technique; Fungal Proteins; Gene Expression Regulation, Fungal; Hyphae; Kinetics; Membrane Glycoproteins; Models, Biological; Mutation; Protein Transport; Thiazolidines

Metastases kill 90% of cancer patients. It is thus a major challenge in cancer therapy to inhibit the spreading of tumor cells from primary tumor sites to those particular organs where metastases are likely to occur. Whereas the actin cytoskeleton is a key component involved in cell migration, agents targeting actin dynamics have been relatively poorly investigated. Consequently, valuable in vitro pharmacological tools are needed to selectively identify this type of agent. In response to the absence of any standardized process, the present work aims to develop a multi-assay strategy for screening actin-affecting drugs with anti-migratory potentials. To validate our approach, we used two cancer cell lines (MCF7 and A549) and three actin-affecting drugs (cytochalasin D, latrunculin A, and jasplakinolide). We quantified the effects of these drugs on the kinetics of actin polymerization in tubes (by means of spectrofluorimetry) and on the dynamics of actin cytoskeletons within whole cells (by means of fluorescence microscopy). Using quantitative videomicroscopy, we investigated the actual effects of the drugs on cell motility. Finally, the combined drug effects on cell motility and cell growth were evaluated by means of a scratch-wound assay. While our results showed concordant drug-induced effects on actin polymerization occurring in vitro in test tubes and within whole cells, the whole cell assay appeared more sensitive than the tube assay. The inhibition of actin polymerization induced by cytochalasin D was paralleled by a decrease in cell motility for both cell types. In the case of jasplakinolide, which induces actin polymerization, while it significantly enhanced the locomotion of the A549 cells, it significantly inhibited that of the MCF-7 ones. All these effects were confirmed by means of the scratch-wound assay except of the jasplakinolide-induced effects on MCF-7 cell motility. These later seemed compensated by an additional effect occurring during wound recolonization (possibly acting on the cell growth features). In conclusion, the use of multi-assays with different levels of sophistication and biological relevance is recommended in the screening of new actin-affecting drugs with potentially anti-migratory effects.

Topics: Actins; Adenocarcinoma; Antineoplastic Agents; Breast Neoplasms; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cytochalasin D; Cytoskeleton; Depsipeptides; Humans; Inhibitory Concentration 50; Lung Neoplasms; Neoplasm Invasiveness; Statistics, Nonparametric; Thiazoles; Thiazolidines

Ligand-gated ion channels (ionotropic receptors) link to the cortical cytoskeleton via specialized scaffold proteins and thereby to appropriate signal transduction pathways in the cell. We studied the role of filamentous actin in the regulation of Ca influx through glutamate receptor-activated channels in third-order neurons of salamander retina. Staining by Alexa-Fluor 488-phalloidin, to visualize polymerized actin, we show localization of filamentous actin in neurites, and the membrane surrounding the cell soma. With Ca(2+) imaging we found that in dissociated neurons, depolymerization of filamentous actin by latrunculin A, or cytochalasin D significantly reduced glutamate-induced intracellular Ca(2+) accumulation to 53+/-7% of control value. Jasplakinolide, a stabilizer of filamentous actin, by itself slightly increased the glutamate-induced Ca(2+) signal and completely attenuated the inhibitory effect when applied in combination with actin depolymerizing agents. These results indicate that in salamander retinal neurons the actin cytoskeleton regulates Ca(2+) influx through ionotropic glutamate receptor-activated channels, suggesting regulatory roles for filamentous actin in a number of Ca(2+)-dependent physiological and pathological processes.

Topics: Actins; Ambystoma; Animals; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cells, Cultured; Cytochalasin D; Cytoskeleton; Data Interpretation, Statistical; Depsipeptides; Glutamic Acid; In Vitro Techniques; Microscopy, Confocal; Receptors, Glutamate; Retina; Retinal Ganglion Cells; Thiazoles; Thiazolidines

The role the cytoskeleton plays in generating and/or maintaining gephyrin-dependent receptor clusters at inhibitory synapses is poorly understood. Here, the effects of actin cytoskeleton disruption were investigated in eGFP-gephyrin-transfected cells and hippocampal neurons. While gephyrin was not associated with microfilaments in transfected cells, it colocalized with G-actin and cytochalasin-D-induced F-actin patches. The linker region between the MoeA and MogA homology domains of gephyrin was required for colocalization with F-actin patches and for the binding of gephyrin to ena/VASP, an actin anti-capping factor that, in vitro, caused gephyrin binding to polymerized actin. In hippocampal neurons, treatment with cytochalasin D resulted in the redistribution of the neuronal ena/VASP homologue Mena into actin patches and, at early stages of development, a reduction in the number of gephyrin clusters. Our data suggest that Mena binding to F-actin allows for gephyrin recruitment to the leading edge of uncapped actin filaments.

Topics: Actin Cytoskeleton; Actins; Animals; Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Carrier Proteins; Cell Adhesion Molecules; Cells, Cultured; Cytochalasin D; Cytoskeleton; Depsipeptides; Hippocampus; Humans; Membrane Proteins; Microfilament Proteins; Neurons; Nucleic Acid Synthesis Inhibitors; Phosphoproteins; Protein Structure, Tertiary; Rats; Recombinant Fusion Proteins; Synapses; Thiazoles; Thiazolidines

The activity and the membrane expression of EAAT3 glutamate transporter are stimulated upon PKC activation by phorbol esters in C6 rat glioma cells. To investigate the role of cytoskeleton in these effects, we have employed actin-perturbing toxins and found that the perturbation of actin cytoskeleton inhibits basal but not phorbol-stimulated EAAT3 activity and membrane trafficking. In the absence of phorbols, latrunculin A, a toxin that disassembles actin cytoskeleton, produced a rapid inhibition of EAAT3 activity, due to a decrease in transport V(max). The inhibitory effect was fully reversible and was not detected for other sodium dependent transport systems for amino acids. However, latrunculin did not prevent the increase in transport caused by phorbol esters and, moreover, cells pre-treated with phorbols were resistant to the inhibitory effect of the toxin on EAAT3 activity. Biotinylation experiments indicated that the inhibitory effect of latrunculin was attributable to a decreased expression of the carrier on the membrane, while the toxin did not suppress the PKC-dependent increase in EAAT3 membrane abundance. Latrunculin A effects on EAAT3 were shared by cytochalasin D, a toxin that disorganizes actin filaments with a distinct mechanism of action. On the contrary, a small, but significant, increase of EAAT3 activity was observed upon incubation with jasplakinolide, a drug that stabilizes actin microfilaments. Also jasplakinolide, however, did not hinder phorbol-dependent stimulation of aspartate transport. Colchicine, a toxin that disrupts microtubules, also lowered EAAT3 activity without preventing transport stimulation by phorbols, while microtubule stabilization by paclitaxel led to an increase in aspartate transport. It is concluded that, in C6 cells, the PKC-mediated stimulatory effects on EAAT3 are cytoskeleton-independent, while in the absence of phorbols, the transporter is partially inhibited by the disorganization of either actin microfilaments or microtubules. These results suggest that EAAT3 trafficking in C6 cells involves different pools of transporters.

Topics: Actin Cytoskeleton; Animals; Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cytochalasins; Depsipeptides; Enzyme Activation; Enzyme Inhibitors; Excitatory Amino Acid Transporter 3; Glutamic Acid; Microtubules; Neurons; Phorbol Esters; Protein Kinase C; Protein Transport; Rats; Thiazoles; Thiazolidines

The molecular basis for developing epilepsy remains under debate. It is hypothesized that increased excitatory synaptic activity might activate the N-methyl-D-aspartate receptor/Ca(2+) transduction pathway, which induces long-lasting plasticity changes leading to recurrent epileptiform discharges. To determine if these effects are caused by disruption of F-actin in the dendritic spines, we have perfused the hippocampus of conscious rats with the F-actin-depolymerizing agent latrunculin Aand the actin filament stabilizer jasplakinolide. Single perfusions of latrunculin Aand jasplakinolide decrease and increase picrotoxin seizure threshold, respectively. Repeated perfusions of both latrunculin Aand jasplakinolide induce epileptic seizures and a long-term increase in neuronal excitability. These results suggest that actin disruption might not be just a consequence but also a possible cause of epileptic seizures. We propose a new experimental model in rats to study the biochemical changes that might lead to chronic seizures and a method for testing new antiepileptic drugs.

Topics: Actins; Animals; Anticonvulsants; Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Convulsants; Depsipeptides; Hippocampus; Male; Microdialysis; Picrotoxin; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Seizures; Thiazolidines

There exists abundant molecular and ultra-structural evidence to suggest that cytoplasmic actin can physically interact with the nuclear envelope (NE) membrane system. However, this interaction has yet to be characterised in living interphase cells.. Using a fluorescent conjugate of the actin binding drug cytochalasin D (CD-BODIPY) we provide evidence that polymerising actin accumulates in vicinity to the NE. In addition, both transiently expressed fluorescent actin and cytoplasmic micro-injection of fluorescent actin resulted in accumulation of actin at the NE-membrane. Consistent with the idea that the cytoplasmic phase of NE-membranes can support this novel pool of perinuclear actin polymerisation we show that isolated, intact, differentiated primary hepatocyte nuclei support actin polymerisation in vitro. Further this phenomenon was inhibited by treatments hindering steric access to outer-nuclear-membrane proteins (e.g. wheat germ agglutinin, anti-nesprin and anti-nucleoporin antibodies).. We conclude that actin polymerisation occurs around interphase nuclei of living cells at the cytoplasmic phase of NE-membranes.

Topics: Actins; Animals; Binding Sites; Biopolymers; Boron Compounds; Bridged Bicyclo Compounds, Heterocyclic; Circular Dichroism; Cytochalasin D; Depsipeptides; Fluorescent Dyes; HeLa Cells; Humans; Liver; Nuclear Envelope; Rabbits; Rats; Thiazoles; Thiazolidines

Clathrin-mediated endocytosis in mammalian cells is critical for a variety of cellular processes including nutrient uptake and cell surface receptor down-regulation. Despite the findings that numerous endocytic accessory proteins directly or indirectly regulate actin dynamics and that actin assembly is spatially and temporally coordinated with endocytosis, direct functional evidence for a role of actin during clathrin-coated vesicle formation is lacking. Here, we take parallel biochemical and microscopic approaches to address the contribution of actin polymerization/depolymerization dynamics to clathrin-mediated endocytosis. When measured using live-cell fluorescence microscopy, disruption of the F-actin assembly and disassembly cycle with latrunculin A or jasplakinolide results in near complete cessation of all aspects of clathrin-coated structure (CCS) dynamics. Stage-specific biochemical assays and quantitative fluorescence and electron microscopic analyses establish that F-actin dynamics are required for multiple distinct stages of clathrin-coated vesicle formation, including coated pit formation, constriction, and internalization. In addition, F-actin dynamics are required for observed diverse CCS behaviors, including splitting of CCSs from larger CCSs, merging of CCSs, and lateral mobility on the cell surface. Our results demonstrate a key role for actin during clathrin-mediated endocytosis in mammalian cells.

Topics: 3T3 Cells; Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Clathrin; Clathrin-Coated Vesicles; Cytoskeleton; Depsipeptides; Endocytosis; Fibroblasts; Green Fluorescent Proteins; Image Processing, Computer-Assisted; Luminescent Proteins; Mice; Microscopy, Fluorescence; Models, Biological; Red Fluorescent Protein; Thiazoles; Thiazolidines

Precise measurement of the mechanical properties of a cell provides useful information about its structural organization and physiological state. It is interesting to understand the effect of individual components on the mechanical properties of the entire cell. In this study, we investigate the influence of the cytoskeletal actin on the viscoelastic properties of a cell. Actin-specific agents, including latrunculin A and jasplakinolide, are used to alter the organization of the cytoskeletal actin. Brassica oleracea protoplasts are treated with the drugs and deformed under an external electric potential. The relaxation processes of single protoplasts after electrodeformation are measured. The data are analyzed by a model-independent spectrum recovery algorithm. Two distinct characteristic time constants are obtained from the relaxation spectra. Treatment with latrunculin A increases both of the relaxation time constants. The longest relaxation times for control, latrunculin A treated, and jasplakinolide treated cells are determined to be 0.28, 1.0, and 0.21 s, respectively.

Topics: Actins; Algorithms; Biomechanical Phenomena; Brassica; Bridged Bicyclo Compounds, Heterocyclic; Cytoskeletal Proteins; Depsipeptides; Electrodes; Half-Life; Protoplasts; Spectrum Analysis; Thiazoles; Thiazolidines; Viscosity

It has been proposed that cytoskeleton plays a key positive role in the activation of capacitative calcium entry (CCE), which supported the secretion-like hypothesis for the mechanisms underlying this process. However, its role on CCE in native smooth muscle is unknown. Here we demonstrate that CCE in isolated gallbladder myocytes was enhanced by cytochalasin D or latrunculin A treatments (agents that cause actin disassembly) whereas it was reduced by jasplakinolide treatment (which causes actin polymerization), suggesting that actin cytoskeleton acts as a barrier in CCE. In addition, we show for the first time that depletion of intracellular Ca2+ stores by thapsigargin and cholecystokinin in BAPTA-loaded cells induced a decrease in F-actin content that was consistent with a link between CCE and actin reorganization. In conclusion, these data suggest an active participation of actin reorganization in the implementation of CCE and support a conformational coupling model for this process in naive smooth muscle cells.

Topics: Actin Cytoskeleton; Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cytochalasin D; Depsipeptides; Gallbladder; Guinea Pigs; Ion Transport; Male; Muscle, Smooth; Thiazoles; Thiazolidines

We measured actin turnover in lamellipodia and lamellae of migrating cells, using quantitative Fluorescent Speckle Microscopy. Lamellae disassembled at low rates from the front to the back. However, the dominant feature in their turnover was a spatially random pattern of periodic polymerization and depolymerization moving with the retrograde flow. Power spectra contained frequencies between 0.5 and 1 cycle/min. The spectra remained unchanged when applying Latrunculin A and Jasplakinolide in low doses, except that additional frequencies occurred beyond 1 cycle/min. Whereas Latrunculin did not change the rate of mean disassembly, Jasplakinolide halted it completely, indicating that the steady state and the dynamics of actin turnover are differentially affected by pharmacological agents. Lamellipodia assembled in recurring bursts at the leading edge and disassembled approximately 2.5 microm behind. Events of polymerization correlated spatially and temporally with transient formation of Arp2/3 clusters. In lamellae, Arp2/3 accumulation and polymerization correlated only spatially, suggesting an Arp2/3-independent mechanism for filament nucleation. To acquire these data we had to enhance the resolution of quantitative Fluorescent Speckle Microscopy to the submicron level. Several algorithmic advances in speckle image processing are described enabling the analysis of kinetic and kinematic activities of polymer networks at the level of single speckles.

Topics: Actin-Related Protein 2; Actin-Related Protein 3; Actins; Algorithms; Animals; Biophysics; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Cell Movement; Cells, Cultured; Depsipeptides; Epithelial Cells; Image Processing, Computer-Assisted; Kinetics; Microscopy, Confocal; Microscopy, Fluorescence; Models, Molecular; Models, Statistical; Polymers; Potoroidae; Pseudopodia; Thiazoles; Thiazolidines; Time Factors

The highly ordered arrangement of sarcomeric myosin during striated muscle development requires spontaneous calcium (Ca(2+)) transients. Here, we show that blocking transients also compromises patterned assembly of actin thin filaments, titin, and capZ. Because a conserved temporal assembly pattern has been described for these proteins, selective inhibitors of either thick or thin filament formation were used to determine their relative temporal interdependencies. For example, inhibition of myosin light chain kinase (MLCK) by application of a specific inhibitory peptide or phorbol myistate acetate (PMA) disrupts myosin assembly without significantly affecting formation of actin bands. The MLCK inhibitor ML-7, however, disrupted actin as well as myosin. Surprisingly, agents that interfere with actin dynamics, such as cytochalasin D, produced only minor organizational disruptions in actin, capZ, and titin staining. However, cytochalasin D and other actin disrupting compounds significantly perturbed myosin organization. The results indicate that (1) Ca(2+) transients regulate one or more of the earliest steps in sarcomere formation, (2) mature actin filaments can assemble independently of myosin band formation, and (3) myosin thick filament assembly is extremely sensitive to disruption of either the actin or titin filament systems.

Topics: Actin Cytoskeleton; Actins; Animals; Azepines; Bridged Bicyclo Compounds, Heterocyclic; Calcium Signaling; CapZ Actin Capping Protein; Connectin; Cytochalasin D; Depsipeptides; Microfilament Proteins; Muscle Development; Muscle Proteins; Myosin-Light-Chain Kinase; Myosins; Naphthalenes; Peptides, Cyclic; Protein Kinases; Sarcomeres; Tetradecanoylphorbol Acetate; Thiazoles; Thiazolidines; Xenopus

We imaged the interiors of relatively intact Xenopus oocyte nuclei by field emission scanning electron microscopy (feSEM) and visualized a network of filaments that attach to nuclear pore complexes and extend throughout the nucleus. Within the nucleus, these 'pore-linked filaments' (PLFs) were embedded into spherical structures 100 nm to approximately 5 microm in diameter. A subset of spheres was identified as Cajal bodies by immuno-gold labeling; the rest were inferred to be nucleoli and snurposomes both of which are abundant in Xenopus oocyte nuclei. Most PLFs were independent of chromatin. The thickness of a typical PLF was 40 nm (range, approximately 12-100 nm), including the 4 nm chromium coat. PLFs located inside the nucleus merged, bundled and forked, suggesting architectural adaptability. The PLF network collapsed upon treatment with latrunculin A, which depolymerizes actin filaments. Jasplakinolide, which stabilizes actin filaments, produced PLFs with more open substructure including individual filaments with evenly-spaced rows of radially projecting short filaments. Immuno-gold labeling of untreated oocyte nuclei showed that actin and protein 4.1 each localized on PLFs. Protein 4.1-gold epitopes were spaced at approximately 120 nm intervals along filaments, and were often paired ( approximately 70 nm apart) at filament junctions. We suggest that protein 4.1 and actin contribute to the structure of a network of heterogeneous filaments that link nuclear pore complexes to subnuclear organelles, and discuss possible functions for PLFs in nuclear assembly and intranuclear traffic.

Topics: Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Cell Nucleolus; Cell Nucleus; Chromatin; Coiled Bodies; Cytoskeletal Proteins; Depsipeptides; Female; Immunohistochemistry; Membrane Proteins; Microscopy, Electron, Scanning; Nuclear Pore; Oocytes; Protein Binding; Thiazoles; Thiazolidines; Xenopus

A major pathway for stimulated Ca(2+) entry in non-excitable cells is activated following depletion of intracellular Ca(2+) stores. Secretion-like coupling between elements in the plasma membrane (PM) and Ca(2+) stores has been proposed as the most likely mechanism to activate this store-mediated Ca(2+) entry (SMCE) in several cell types. Here we identify two mechanisms for SMCE in human platelets activated by depletion of two independent Ca(2+) pools, which are differentially modulated by the actin cytoskeleton. Ca(2+) entry induced by depletion of a 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ)-sensitive pool is increased by disassembly of the actin cytoskeleton and that induced by a TBHQ-insensitive pool is reduced. Stabilization of the actin cytoskeleton prevented Ca(2+) entry by both mechanisms. We propose that the membrane-associated actin network prevents constitutive Ca(2+) entry via both pathways. Reorganization of the actin cytoskeleton permits the activation of Ca(2+) entry via both mechanisms, but only SMCE activated by the TBHQ-insensitive pool requires new actin polymerization, which may support membrane trafficking toward the PM.

Topics: Actins; Animals; Antineoplastic Agents; Biological Transport; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Calcium-Transporting ATPases; Cytochalasin D; Cytoskeleton; Depsipeptides; Enzyme Inhibitors; Humans; Hydroquinones; Nucleic Acid Synthesis Inhibitors; Peptides, Cyclic; ras Proteins; Signal Transduction; Thiazoles; Thiazolidines

Effects of jasplakinolide (JSP), a stabilizer of F-actin, and latrunculin A (LTA), a destabilizer of F-actin, on a series of events occurring in the execution phase of staurosporine (STS)-induced apoptotic processes were studied using human osteosarcoma 143B cells. Time-dependent apparent increases of the population of cells with collapsed membrane potential of mitochondria (Delta Psi(m)) caused by STS treatment were not due to actual decreases in the Delta Psi(m) per cell, but due to the fragmentation of cells resulting in decreases in the number of active mitochondria per cell. Decreases in the Delta Psi(m) in fragmented cells occurred late in the execution phase. Both JSP and LAT failed to prevent STS-induced release of cytochrome c from mitochondria followed by the activation of caspases 3 and 9, the cleavage of poly (ADP-ribose) polymerase (PARP) and apoptotic nuclear fragmentation. However, both drugs prevented STS-induced apoptotic cell fragmentation and decreases in the Delta Psi(m). These results indicate that physicochemical states of actin filaments play a certain role in the execution phase of STS-induced apoptotic processes.

Topics: Actin Cytoskeleton; Antineoplastic Agents; Apoptosis; Bridged Bicyclo Compounds, Heterocyclic; Caspases; Cell Line, Tumor; Depsipeptides; Enzyme Inhibitors; Humans; Immunoblotting; Membrane Potentials; Microscopy, Confocal; Mitochondria; Staurosporine; Thiazoles; Thiazolidines

Store-mediated Ca2+ entry (SMCE) is one of the main pathways for Ca2+ influx in non-excitable cells. Recent studies favour a secretion-like coupling mechanism to explain SMCE, where Ca2+ entry is mediated by an interaction of the endoplasmic reticulum (ER) with the plasma membrane (PM) and is modulated by the actin cytoskeleton. To explore this possibility further we have now investigated the role of the actin cytoskeleton in the activation and maintenance of SMCE in pancreatic acinar cells, a more specialized secretory cell type which might be an ideal cellular model to investigate further the properties of the secretion-like coupling model. In these cells, the cytoskeletal disrupters cytochalasin D and latrunculin A inhibited both the activation and maintenance of SMCE. In addition, stabilization of a cortical actin barrier by jasplakinolide prevented the activation, but not the maintenance, of SMCE, suggesting that, as for secretion, the actin cytoskeleton plays a double role in SMCE as a negative modulator of the interaction between the ER and PM, but is also required for this mechanism, since the cytoskeleton disrupters impaired Ca2+ entry. Finally, depletion of the intracellular Ca2+ stores induces cytoskeletal association and activation of pp60(src), which is independent on Ca2+ entry. pp60(src) activation requires the integrity of the actin cytoskeleton and participates in the initial phase of the activation of SMCE in pancreatic acinar cells.

Topics: Actins; Animals; Biopolymers; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cytochalasin D; Cytoskeleton; Depsipeptides; Ion Transport; Male; Mice; Pancreas; Peptides, Cyclic; Proto-Oncogene Proteins pp60(c-src); Spectrometry, Fluorescence; Thapsigargin; Thiazoles; Thiazolidines

In cultured chick ciliary neurons, when ATP synthesis is inhibited, ATP depletion is reduced approximately 50% by slowing actin filament turnover with jasplakinolide or latrunculin A. Jasplakinolide inhibits actin disassembly, and latrunculin A prevents actin assembly by sequestering actin monomers. Cytochalasin D, which allows assembly-disassembly, but only at pointed ends, is less effective in conserving ATP. Ouabain, an Na(+)-K(+)-ATPase inhibitor, and jasplakinolide both prevent approximately 50% of the ATP loss. When applied together, they completely prevent ATP loss over a period of 20 min, suggesting that filament stabilization reduces ATP consumption by decreasing actin-ATP hydrolysis directly rather than indirectly by modulating the activity of Na(+)-K(+)-ATPase, a major energy consumer.

Topics: Actin Cytoskeleton; Actins; Adenosine Triphosphate; Animals; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cell Hypoxia; Cells, Cultured; Chick Embryo; Depsipeptides; Energy Metabolism; Enzyme Inhibitors; Hydrolysis; Neurons; Ouabain; Peptides, Cyclic; Sodium; Sodium-Potassium-Exchanging ATPase; Thiazoles; Thiazolidines

We used actin tagged with enhanced green fluorescent protein (EGFP-actin) to characterize the distribution and dynamics of actin in living presynaptic terminals in rat CNS neurons. Actin was preferentially concentrated around--and appeared to surround--the presynaptic vesicle cluster. In resting terminals, approximately 30% of actin was found to be in a polymerized but dynamic state, with a remodeling time scale of approximately 20 s. During electrical activity, actin was further polymerized and recruited from nearby axonal regions to the regions surrounding vesicles. Treatment of terminals with the actin monomer-sequestering agent latrunculin-A completely dispersed the actin network and abolished activity-dependent actin dynamics. We used a variety of methods to examine the role of actin in the presynaptic vesicle cycle. These data rule out a propulsive role for actin, either in maintaining the vesicle cluster or in guiding vesicle recycling. Instead, we propose that actin acts as a scaffolding system for regulatory molecules in the nerve terminal.

Topics: Actins; Animals; Animals, Newborn; Bridged Bicyclo Compounds, Heterocyclic; Cytoskeleton; Depsipeptides; Hippocampus; Kinetics; Organ Culture Techniques; Peptides, Cyclic; Presynaptic Terminals; Protein Transport; Rats; Rats, Sprague-Dawley; Synaptic Transmission; Synaptic Vesicles; Thiazoles; Thiazolidines

One important mechanism cytotoxic T lymphocytes (CTLs) use to kill virus-infected, transplanted or tumour targets is exocytosis of granules that contain cytotoxic agents such as perforin and granzymes. Granule exocytosis-dependent target cell killing is a complex process, involving initial T-cell receptor (TCR)-dependent signalling that includes Ca2+ influx and activation of protein kinase C, shape changes that serve to bind the CTL to the target and, finally, exocytosis of lytic granules at the site of contact with the target cell. Although there is reason to propose that multiple steps in the lytic process could involve the actin cytoskeleton of CTLs, few studies have examined this issue, and those that have do not allow the specific step(s) involved to be determined. We have used the potent membrane-permeant actin cytoskeleton-modifying drugs jasplakinolide and latrunculin A to investigate the actin dependence of defined processes that are expected to be important for granule exocytosis-dependent killing. Our results, obtained using TALL-104 human leukaemic CTLs as a model system, are consistent with the idea that a functional actin cytoskeleton is required for TCR/CD3-dependent signalling, for activation of store-dependent Ca2+ influx and for CTL shape changes. When cells were stimulated with solid-phase anti-CD3 antibodies, treatment with either jasplakinolide or latrunculin A abolished granule exocytosis. However, when cells were stimulated in a manner that bypasses TCR/CD3-dependent signalling, granule exocytosis was not significantly altered, suggesting that the actin cytoskeleton does not function as a barrier to exocytosis.

Topics: Actin Cytoskeleton; Antibodies, Monoclonal; Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Calcium; CD3 Complex; Cell Membrane; Cell Movement; Depsipeptides; Exocytosis; Humans; Leukemia; Lymphoma, B-Cell; Membrane Glycoproteins; Peptides, Cyclic; Perforin; Pore Forming Cytotoxic Proteins; Signal Transduction; T-Lymphocytes, Cytotoxic; Thiazoles; Thiazolidines; Tumor Cells, Cultured

Nicotinic acid adenine dinucleotide phosphate (NAADP) is involved in the Ca2+ response observed at fertilization in several species, including starfish. In this study, we have employed Ca2+ imaging and the single-electrode voltage-clamp technique to investigate whether the NAADP-mediated Ca2+ entry discovered in our laboratory in starfish oocytes was underlain by a membrane current and whether the response to NAADP required an intact cytoskeleton. Uncaging of preinjected NAADP evoked a cortical Ca2+ flash that was followed by the spreading of the wave to the remainder of the cell. No Ca2+ increase was detected in Ca2+-free sea water. Under voltage-clamp conditions, the photoliberation of NAADP activated an inward rectifying membrane current, which reversed at potentials more positive than +50 mV and was abolished by removal of Ca2+ but not of Na+. The current was affected by preincubation with verapamil, SKF 96356, and thapsigargin but not by preinjection of heparin, 8-NH2- cyclic ADP-ribose, or both antagonists. The membrane current and the Ca2+ wave were inhibited by latrunculin-A and jasplakinolide, which depolymerize and stabilize actin cytoskeleton, respectively. These data offer the first demonstration that NAADP initiates a Ca2+ sweep by activating a Ca2+-permeable membrane current that requires an intact F-actin cytoskeleton as other Ca2+-permeable currents, such as ICRAC and IARC.

Topics: Actin Cytoskeleton; Animals; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Calcium Channels; Calcium Signaling; Cell Membrane; Depsipeptides; Electric Conductivity; Models, Biological; NADP; Oocytes; Patch-Clamp Techniques; Peptides, Cyclic; Starfish; Thiazoles; Thiazolidines

The GTPase Rab27A interacts with myosin-VIIa and myosin-Va via MyRIP or melanophilin and mediates melanosome binding to actin. Here we show that Rab27A and MyRIP are associated with secretory granules (SGs) in adrenal chromaffin cells and PC12 cells. Overexpression of Rab27A, GTPase-deficient Rab27A-Q78L, or MyRIP reduced secretory responses of PC12 cells. Amperometric recordings of single adrenal chromaffin cells revealed that Rab27A-Q78L and MyRIP reduced the sustained component of release. Moreover, these effects on secretion were partly suppressed by the actin-depolymerizing drug latrunculin but strengthened by jasplakinolide, which stabilizes the actin cortex. Finally, MyRIP and Rab27A-Q78L restricted the motion of SGs in the subplasmalemmal region of PC12 cells, as measured by evanescent-wave fluorescence microscopy. In contrast, the Rab27A-binding domain of MyRIP and a MyRIP construct that interacts with myosin-Va but not with actin increased the mobility of SGs. We propose that Rab27A and MyRIP link SGs to F-actin and control their motion toward release sites through the actin cortex.

Topics: Actin Cytoskeleton; Actins; Adaptor Proteins, Signal Transducing; Animals; Bridged Bicyclo Compounds, Heterocyclic; Carrier Proteins; Cattle; Chromaffin Cells; Depsipeptides; Exocytosis; Microscopy, Electron; Myosin Heavy Chains; Myosin Type V; PC12 Cells; Peptides, Cyclic; rab GTP-Binding Proteins; rab27 GTP-Binding Proteins; Rats; Secretory Vesicles; Thiazoles; Thiazolidines

The microvilli of the apical membrane of proximal tubule (PT) cells are supported by the underlying actin cytoskeleton. Ischaemic or anoxic ATP-depletion leads to the disruption of the actin cytoskeleton, resulting in microvillar retraction and loss of membrane polarity. Using isolated PT cells, we have previously demonstrated that actin filaments (F-actin) are likely severed during ATP-depletion. A sequential extraction protocol revealed a decrease in actin solubility, resulting in the sequestration of a distinct F-actin pool with the insoluble cellular complex in ATP-depleted PT cells. We demonstrate here that decreased actin solubility is not only a reliable end-marker of ATP-depletion induced injury in freshly isolated PT cells, but also serves as a biochemical marker in the cultured proximal tubular cell line LLC-PK1. In the present studies, we also investigated specific actin-binding drugs to determine if they mimic the effects observed during energy depletion. Jasplakinolide (JP), a compound which binds F-actin and prevents depolymerization, did not effect actin solubility during ATP-depletion. Furthermore, swinholide A (SA), an F-actin severing agent, resulted in decreased actin solubility, mimicking the effects of ATP-depletion. Interestingly, latrunculin A (LA), an agent which depolymerizes F-actin, did not reduce actin solubility, but rather resulted in an increase in digitonin-soluble actin. Taken collectively, our results support previous work and suggest that disruption of the actin cytoskeleton during ATP-depletion is mediated by F-actin severing/fragmentation and not depolymerization. The differential effects of F-actin disrupting agents and the consistencies observed in both models of ischaemic injury will provide a basis for a more detailed understanding of the pathological events of PT-cell dysfunction.

Topics: Actins; Adenosine Triphosphate; Animals; Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Depsipeptides; Female; Kidney Tubules, Proximal; LLC-PK1 Cells; Marine Toxins; Microscopy, Fluorescence; Peptides, Cyclic; Polymers; Rabbits; Solubility; Swine; Thiazoles; Thiazolidines

Simian virus 40 (SV40) utilizes endocytosis through caveolae for infectious entry into host cells. We found that after binding to caveolae, virus particles induced transient breakdown of actin stress fibers. Actin was then recruited to virus-loaded caveolae as actin patches that served as sites for actin "tail" formation. Dynamin II was also transiently recruited. These events depended on the presence of cholesterol and on the activation of tyrosine kinases that phosphorylated proteins in caveolae. They were necessary for formation of caveolae-derived endocytic vesicles and for infection of the cell. Thus, caveolar endocytosis is ligand-triggered and involves extensive rearrangement of the actin cytoskeleton.

Topics: Actin Cytoskeleton; Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Caveolae; Caveolin 1; Caveolins; Cell Line; Cholesterol; Depsipeptides; Dynamins; Endocytosis; GTP Phosphohydrolases; Haplorhini; Peptides, Cyclic; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Recombinant Fusion Proteins; Simian virus 40; Stress Fibers; Thiazoles; Thiazolidines; Transport Vesicles

The diffusion rate of lipids in the cell membrane is reduced by a factor of 5-100 from that in artificial bilayers. This slowing mechanism has puzzled cell biologists for the last 25 yr. Here we address this issue by studying the movement of unsaturated phospholipids in rat kidney fibroblasts at the single molecule level at the temporal resolution of 25 micros. The cell membrane was found to be compartmentalized: phospholipids are confined within 230-nm-diameter (phi) compartments for 11 ms on average before hopping to adjacent compartments. These 230-nm compartments exist within greater 750-nm-phi compartments where these phospholipids are confined for 0.33 s on average. The diffusion rate within 230-nm compartments is 5.4 microm2/s, which is nearly as fast as that in large unilamellar vesicles, indicating that the diffusion in the cell membrane is reduced not because diffusion per se is slow, but because the cell membrane is compartmentalized with regard to lateral diffusion of phospholipids. Such compartmentalization depends on the actin-based membrane skeleton, but not on the extracellular matrix, extracellular domains of membrane proteins, or cholesterol-enriched rafts. We propose that various transmembrane proteins anchored to the actin-based membrane skeleton meshwork act as rows of pickets that temporarily confine phospholipids.

Topics: Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Cell Compartmentation; Cell Line; Cell Membrane; Depsipeptides; Diffusion; Fibroblasts; Gold Colloid; Kidney; Marine Toxins; Models, Biological; Peptides, Cyclic; Phospholipids; Rats; Receptors, Transferrin; Thiazoles; Thiazolidines; Time Factors; Trypsin

Trafficking of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) at synapses has been suggested to play an important role in the expression of synaptic plasticity. Both the regulated and the constitutive trafficking of synaptic AMPARs are thought to involve the insertion and removal of receptors by means of an exocytotic and endocytotic process, respectively. In contrast, N-methyl-d-aspartate (NMDA) receptors (NMDARs), which are colocalized with AMPARs at excitatory synapses, appear to be much less dynamic. Here, we present evidence supporting the idea that synaptic AMPARs turn over through a constitutive endocytotic process and that glutamate application greatly enhances this turnover of AMPARs. The glutamate-induced internalization of AMPARs requires a rise in postsynaptic Ca(2+). The AMPAR internalization is mimicked by latrunculin A, a drug that selectively depolymerizes actin and is blocked by jasplakinolide, a drug which stabilizes actin filaments. The rate of endocytosis is not altered by glutamate application, whereas a clear enhancement is observed with insulin application. We propose a model in which the glutamate-induced dissociation of AMPARs from their anchor on the postsynaptic membrane involves actin depolymerization, which allows the released AMPARs to segregate from the NMDARs and diffuse to a presumably perisynaptic site, where they become available to an endocytotic machinery and are selectively internalized.

Topics: Actins; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Biological Transport; Bridged Bicyclo Compounds, Heterocyclic; Cells, Cultured; Cytoskeleton; Depsipeptides; Endocytosis; Glutamic Acid; Hippocampus; Marine Toxins; Neurons; Peptides, Cyclic; Rats; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Thiazoles; Thiazolidines; Transferrin

Scanning and transmission electron microscopy were used to determine the morphological changes in the egg plasma membrane associated with sperm binding, fusion and incorporation in Xenopus laevis. Sperm incorporation in Xenopus is rapid, occurring within 3-5 min following addition of sperm. Images have been obtained of both early sperm-egg interactions and fertilisation bodies. Additionally, two drugs that specifically alter F-actin dynamics, latrunculin and jasplakinolide, were used to determine whether sperm incorporation is a microfilament-dependent process. Jasplakinolide did not prevent sperm incorporation, cortical granule exocytosis or cortical contraction, suggesting these events can occur without depolymerisation of existing, stabilised filaments. Latrunculin A, which competes with thymosin beta4 in ooplasm for binding actin monomer, did not inhibit cortical granule exocytosis, but blocked cortical contraction in 100% of eggs at a concentration of 5 microM. Although a single penetrating sperm was found on an egg pretreated in latrunculin, fertilisation bodies were never observed. At < 5 microM latrunculin, many eggs did undergo cortical contraction with some exhibiting severe distortions of the plasma membrane and abnormal accumulations of pigment granules. Preincubation of eggs in jasplakinolide before latrunculin mitigated both these effects to some degree. However, eggs incubated in latrunculin either prior to or after insemination never progressed through first cleavage.

Topics: Actin Cytoskeleton; Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Cell Division; Cytoplasmic Granules; Depsipeptides; Female; Larva; Male; Microscopy, Electron; Microscopy, Electron, Scanning; Ovum; Peptides, Cyclic; Sperm-Ovum Interactions; Spermatozoa; Thiazoles; Thiazolidines; Time Factors; Xenopus laevis

Effects of three actin-modifying drugs, cytochalasin D, latrunculin A, and jasplakinolide, on the excystation and metacystic development in vitro of Entamoeba invadens were examined by transfer of the cysts to growth medium with the drugs. Cytochalasin D unexpectedly increased the number of metacystic amoebae of E. invadens strain IP-1 during incubation. Metacystic development, which was determined by the number of nuclei of metacystic amoebae, was faster in the culture with cytochalasin D than in the culture without the drug. These results suggest that cytochalasin D enhances the excystation and metacystic development. In contrast, latrunculin A and jasplakinolide inhibited these process. No excystation occurred in encystation medium even in the presence of cytochalasin D, suggesting that growth medium is essential for excystation. Excystation was further enhanced when the cysts were incubated with cytochalasin D before culture in growth medium with the drug. The enhancing effect of cytochalasin D on the excystation and metacystic development was abrogated by jasplakinolide. Thus, the results indicate that cytochalasin D, unlike latrunculin A and jasplakinolide, caused enhancement of the excystation and metacystic development of this parasite.

Topics: Animals; Bridged Bicyclo Compounds, Heterocyclic; Cytochalasin D; Depsipeptides; Entamoeba; Marine Toxins; Nucleic Acid Synthesis Inhibitors; Peptides, Cyclic; Porifera; Thiazoles; Thiazolidines

The effect of various drugs affecting the integrity of different components of the cytoskeleton on the elasticity of two fibroblast cell lines was investigated by elasticity measurements with an atomic force microscope (AFM). Disaggregation of actin filaments always resulted in a distinct decrease in the cell's average elastic modulus indicating the crucial importance of the actin network for the mechanical stability of living cells. Disruption or chemical stabilization of microtubules did not affect cell elasticity. For the f-actin-disrupting drugs different mechanisms of drug action were observed. Cytochalasins B and D and Latrunculin A disassembled stress fibers. For Cytochalasin D this was accompanied by an aggregation of actin within the cytosol. Jasplakinolide disaggregated actin filaments but did not disassemble stress fibers. Fibrous structures found in AFM images and elasticity maps of fibroblasts could be identified as stress fibers by correlation of AFM data and fluorescence images.

Topics: 3T3 Cells; Actins; Animals; Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Colchicine; Cytochalasin B; Cytochalasin D; Cytoskeleton; Demecolcine; Depsipeptides; Elasticity; Fibroblasts; Kinetics; Marine Toxins; Mice; Microscopy, Atomic Force; Microscopy, Fluorescence; Microtubules; Paclitaxel; Peptides, Cyclic; Rats; Stress, Mechanical; Thiazoles; Thiazolidines

The nature of the mechanism underlying store-mediated Ca(2+) entry has been investigated in human platelets through a combination of cytoskeletal modifications. Inhibition of actin polymerization by cytochalasin D or latrunculin A had a biphasic time-dependent effect on Ca(2+) entry, showing an initial potentiation followed by inhibition of Ca(2+) entry. Moreover, addition of these agents after induction of store-mediated Ca(2+) entry inhibited the Ca(2+) influx mechanism. Jasplakinolide, which reorganizes actin filaments into a tight cortical layer adjacent to the plasma membrane, prevented activation of store-mediated Ca(2+) entry but did not modify this process after its activation. In addition, jasplakinolide prevented cytochalasin D-induced inhibition of store-mediated Ca(2+) entry. Calyculin A, an inhibitor of protein serine/threonine phosphatases 1 and 2 which activates translocation of existing F-actin to the cell periphery without inducing actin polymerization, also prevented activation of store-mediated Ca(2+) entry. Finally, inhibition of vesicular transport with brefeldin A inhibited activation of store-mediated Ca(2+) entry but did not alter this mechanism once initiated. These data suggest that store-mediated Ca(2+) entry in platelets may be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, which shows close parallels to the events mediating secretion.

Topics: Actins; Biological Transport; Blood Platelets; Brefeldin A; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cell Membrane; Cytochalasin D; Cytoskeleton; Depsipeptides; Endoplasmic Reticulum; Humans; Inositol 1,4,5-Trisphosphate; Marine Toxins; Models, Biological; Oxazoles; Peptides, Cyclic; Thiazoles; Thiazolidines

A new method was devised to visualize actin polymerization induced by postsynaptic differentiation signals in cultured muscle cells. This entails masking myofibrillar filamentous (F)-actin with jasplakinolide, a cell-permeant F-actin-binding toxin, before synaptogenic stimulation, and then probing new actin assembly with fluorescent phalloidin. With this procedure, actin polymerization associated with newly induced acetylcholine receptor (AChR) clustering by heparin-binding growth-associated molecule-coated beads and by agrin was observed. The beads induced local F-actin assembly that colocalized with AChR clusters at bead-muscle contacts, whereas both the actin cytoskeleton and AChR clusters induced by bath agrin application were diffuse. By expressing a green fluorescent protein-coupled version of cortactin, a protein that binds to active F-actin, the dynamic nature of the actin cytoskeleton associated with new AChR clusters was revealed. In fact, the motive force generated by actin polymerization propelled the entire bead-induced AChR cluster with its attached bead to move in the plane of the membrane. In addition, actin polymerization is also necessary for the formation of both bead and agrin-induced AChR clusters as well as phosphotyrosine accumulation, as shown by their blockage by latrunculin A, a toxin that sequesters globular (G)-actin and prevents F-actin assembly. These results show that actin polymerization induced by synaptogenic signals is necessary for the movement and formation of AChR clusters and implicate a role of F-actin as a postsynaptic scaffold for the assembly of structural and signaling molecules in neuromuscular junction formation.

Topics: Actins; Animals; Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Cells, Cultured; Cortactin; Depsipeptides; Gene Expression; Genes, Reporter; Green Fluorescent Proteins; Indicators and Reagents; Luminescent Proteins; Microfilament Proteins; Microspheres; Muscles; Neuromuscular Junction; Peptides, Cyclic; Polymers; Receptors, Cholinergic; Sarcolemma; Synaptic Transmission; Thiazoles; Thiazolidines; Xenopus

Depolymerization of actin by latrunculin A transiently promotes neurotransmitter release. The mean rate of mEPSCs increases by a Ca2+-independent process, without a concomitant change in the mean amplitude. The readily releasable vesicle pool size and the rate of refilling of the readily releasable pool remain unaltered by latrunculin treatment. Evoked neurotransmitter release also increases in a manner consistent with an increase in vesicle release probability. The observed enhancement of neurotransmitter release is specific to actin depolymerization mediated by latrunculin A and is not caused by cytochalasin D. Our findings indicate that actin participates in a regulatory mechanism that restrains fusion of synaptic vesicles at the active zone.

Topics: Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cells, Cultured; Cytochalasin D; Depsipeptides; Egtazic Acid; Excitatory Postsynaptic Potentials; Fluorescent Antibody Technique; Green Fluorescent Proteins; Hippocampus; Luminescent Proteins; Marine Toxins; Mice; Nerve Tissue Proteins; Neurotransmitter Agents; Peptides, Cyclic; Presynaptic Terminals; Rats; Recombinant Fusion Proteins; Synapses; Synaptic Transmission; Synaptic Vesicles; Thiazoles; Thiazolidines

Three cell-permeant compounds, cytochalasin D, latrunculin A and jasplakinolide, which perturb intracellular actin dynamics by distinct mechanisms, were used to probe the role of filamentous actin and actin assembly in clathrin-mediated endocytosis in mammalian cells. These compounds had variable effects on receptor-mediated endocytosis of transferrin that depended on both the cell line and the experimental protocol employed. Endocytosis in A431 cells assayed in suspension was inhibited by latrunculin A and jaspiakinolide, but resistant to cytochalasin D, whereas neither compound inhibited endocytosis in adherent A431 cells. In contrast, endocytosis in adherent CHO cells was more sensitive to disruption of the actin cytoskeleton than endocytosis in CHO cells grown or assayed in suspension. Endocytosis in other cell types, including nonadherent K562 human erythroleukemic cells or adherent Cos-7 cells was unaffected by disruption of the actin cytoskeleton. While it remains possible that actin filaments can play an accessory role in receptor-mediated endocytosis, these discordant results indicate that actin assembly does not play an obligatory role in endocytic coated vesicle formation in cultured mammalian cells.

Topics: Actins; Animals; Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Cell Membrane; CHO Cells; Coated Pits, Cell-Membrane; COS Cells; Cricetinae; Cytochalasin D; Cytoskeleton; Depsipeptides; Endocytosis; Freeze Etching; Humans; K562 Cells; Microscopy, Fluorescence; Nucleic Acid Synthesis Inhibitors; Peptides, Cyclic; Thiazoles; Thiazolidines; Time Factors; Transferrin; Tumor Cells, Cultured

Monomeric (G) actin was shown to be involved in inhibiting its own synthesis by an autoregulatory mechanism that includes enhanced degradation of the actin mRNA [Bershadsky et al., 1995; Lyubimova et al., 1997]. We show that the 3'-untranslated region (3'-UTR) of beta-actin mRNA, but not its 5'-untranslated region, is important for this regulation. The level of full-length beta-actin mRNA in cells was reduced when actin filaments were depolymerized by treatment with latrunculin A and elevated when actin polymerization was induced by jasplakinolide. By contrast, the level of actin mRNA lacking the 3'-UTR remained unchanged when these drugs modulated the dynamics of actin assembly in the cell. Moreover, the transfection of cells with a construct encoding the autoregulation-deficient form of beta-actin mRNA led to very high levels of actin expression compared with transfection with the control actin construct and was accompanied by characteristic changes in cell morphology and the structure of the actin cytoskeleton. These results suggest that the autoregulatory mechanism working via the 3'-UTR of actin mRNA is involved in controlling the maintenance of a defined pool of actin monomers that could be necessary for the proper organization of the microfilament system and the cytoskeleton-mediated signaling.

Topics: 3' Untranslated Regions; 3T3 Cells; Actins; Animals; Base Sequence; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Depsipeptides; DNA Primers; Gene Expression Regulation; Mice; Mice, Inbred BALB C; Peptides, Cyclic; RNA, Messenger; Sequence Deletion; Thiazoles; Thiazolidines

Previous studies showed that cAMP-dependent transepithelial Cl- secretion of the intestinal cell line T84 is reduced by the F-actin stabilizer phalloidin, an effect in part attributable to inhibition of basolateral Na-K-2Cl cotransport. However, secretory responses are preserved in cells treated with the microfilament disrupter cytochalasin D. We explored the effects of cytochalasin D and two novel compounds derived from marine sponges on the Cl- secretory apparatus of T84 cells. Jasplakinolide (which stabilizes F-actin inhibited cAMP-dependent secretion and Na-K-2Cl cotransport. Latrunculin A (which sequesters G-actin monomers) profoundly altered the distribution of F-actin and reduced basal transepithelial resistance with minimal effect on secretion. Cytochalasin D, but not latrunculin A, activated Na-K-2Cl cotransport. The results provide further evidence that vectorial ion transport is influenced by the cytoskeleton and support a model in which disassembly of F-actin by specific pharmacological means or in response to secretory agonists favors activation of Na-K-2Cl cotransport.

Topics: Actins; Bridged Bicyclo Compounds, Heterocyclic; Carrier Proteins; Chlorides; Cytochalasin D; Cytoskeleton; Depsipeptides; Drug Stability; Humans; Intestinal Mucosa; Intracellular Membranes; Peptides, Cyclic; Sodium-Potassium-Chloride Symporters; Thiazoles; Thiazolidines