phalloidine and jasplakinolide

Chromosome congression and segregation have been widely known to be coordinated by the function of the dynamic spindle microtubules. But recent work suggests that oocytes may employ a unique actin-dependent mechanism of chromosome delivery to the spindle.

Topics: Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Chromosome Segregation; Depsipeptides; Models, Biological; Nocodazole; Oocytes; Phalloidine; Spindle Apparatus; Starfish; Thiazoles; Thiazolidines

This chapter presents an overview of the most common F-actin influencing substances, used to study actin dynamics in living plant cells for studies on morphogenesis, motility, organelle movement, apoptosis, or abiotic stress. These substances can be divided into two major subclasses-F-actin-stabilizing and F-actin-polymerizing substances like jasplakinolide and chondramides and F-actin-severing compounds like cytochalasins and latrunculins. Jasplakinolide, which may have anti-cancer activities, was originally isolated from a marine sponge and can now be synthesized and has become commercially available, which is responsible for its wide distribution as membrane-permeable F-actin-stabilizing and F-actin-polymerizing agent. Recently an acyclic derivate of jasplakinolide was isolated. Cytochalasins, derived from fungi, show an F-actin-severing function, and many derivatives are commercially available (A, B, C, D, E, H, J), also making it a widely used compound for F-actin disruption. The same can be stated for latrunculins (A, B), derived from Red Sea sponges; however the mode of action is different by binding to G-actin and inhibiting incorporation into the filament. In the case of swinholide, isolated from red algae or the cyanobacterium Nostoc, a stable complex with actin dimers is formed, resulting in severing F-actin.For influencing F-actin dynamics in plant cells, only membrane permeable drugs are useful in a broad range. We, however, introduce also the phallotoxins and synthetic derivatives thereof, as they are widely used to visualize F-actin in fixed cells. A particular uptake mechanism has been shown for hepatocytes but has also been described in siphonal giant algae. The focus is set on F-actin dynamics in plant cells where alterations in cytoplasmic streaming can be particularly well studied; moreover fluorescence methods for phalloidin- and antibody staining as well as techniques for immunoelectron microscopy are explained.

Topics: Actin Cytoskeleton; Actins; Animals; Cytochalasins; Depsipeptides; Phalloidine; Plant Cells; Porifera

Actin undergoes structural transitions during polymerization, ATP hydrolysis, and subsequent release of inorganic phosphate. Several actin-binding proteins sense specific states during this transition and can thus target different regions of the actin filament. Here, we show in atomic detail that phalloidin, a mushroom toxin that is routinely used to stabilize and label actin filaments, suspends the structural changes in actin, likely influencing its interaction with actin-binding proteins. Furthermore, high-resolution cryoelectron microscopy structures reveal structural rearrangements in F-actin upon inorganic phosphate release in phalloidin-stabilized filaments. We find that the effect of the sponge toxin jasplakinolide differs from the one of phalloidin, despite their overlapping binding site and similar interactions with the actin filament. Analysis of structural conformations of F-actin suggests that stabilizing agents trap states within the natural conformational space of actin.

Topics: Actin Cytoskeleton; Antifungal Agents; Binding Sites; Cryoelectron Microscopy; Depsipeptides; Fungal Proteins; Mycotoxins; Phalloidine; Protein Binding

This chapter gives an overview of the most common F-actin-perturbing substances that are used to study actin dynamics in living plant cells in studies on morphogenesis, motility, organelle movement, or when apoptosis has to be induced. These substances can be divided into two major subclasses: F-actin-stabilizing and -polymerizing substances like jasplakinolide and chondramides and F-actin-severing compounds like chytochalasins and latrunculins. Jasplakinolide was originally isolated form a marine sponge, and can now be synthesized and has become commercially available, which is responsible for its wide distribution as membrane-permeable F-actin-stabilizing and -polymerizing agent, which may even have anticancer activities. Cytochalasins, derived from fungi, show an F-actin-severing function and many derivatives are commercially available (A, B, C, D, E, H, J), also making it a widely used compound for F-actin disruption. The same can be stated for latrunculins (A, B), derived from red sea sponges; however the mode of action is different by binding to G-actin and inhibiting incorporation into the filament. In the case of swinholide a stable complex with actin dimers is formed resulting also in severing of F-actin. For influencing F-actin dynamics in plant cells only membrane permeable drugs are useful in a broad range. We however introduce also the phallotoxins and synthetic derivatives, as they are widely used to visualize F-actin in fixed cells. A particular uptake mechanism has been shown for hepatocytes, but has also been described in siphonal giant algae. In the present chapter the focus is set on F-actin dynamics in plant cells where alterations in cytoplasmic streaming can be particularly well studied; however methods by fluorescence applications including phalloidin and antibody staining as well as immunofluorescence-localization of the inhibitor drugs are given.

Topics: Actins; Cytochalasins; Depsipeptides; Microscopy, Immunoelectron; Phalloidine; Plant Cells; Protein Multimerization; Protein Structure, Quaternary; 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

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

In Xenopus oocytes, extremely giant nuclei, termed germinal vesicles, contain a large amount of actin filaments most likely for mechanical integrity. Here, we show that microinjection of phalloidin, an F-actin-stabilizing drug, prevents the germinal vesicle breakdown (GVBD) in oocytes treated with progesterone. These nuclei remained for more 12 h after control oocytes underwent GVBD. Immunostaining showed significant elevation of actin in the remaining nuclei and many actin filament bundles in the cytoplasm. Furthermore, microtubules formed unusual structures in both nuclei and cytoplasm of phalloidin-injected oocytes stimulated by progesterone. Cytoplasmic microtubule arrays and intranuclear microtubules initially formed in phalloidin-injected oocytes as control oocytes exhibited white maturation spots; these structures gradually disappeared and finally converged upon intranuclear short bundles when control oocytes completed maturation. In contrast, treatment of oocytes with jasplakinolide, a cell membrane-permeable actin filament-stabilizing drug, did not affect GVBD. This drug preferentially induced accumulation of actin filaments at the cortex without any increase in cytoplasmic actin staining. Based on these results, intranuclear and cytoplasmic actin filament dynamics appear to be required for the completion of GVBD and critically involved in the regulation of microtubule assembly during oocyte maturation in Xenopus laevis.

Topics: Actin Cytoskeleton; Animals; Antineoplastic Agents; Cell Nucleus; Cytoplasm; Depsipeptides; Female; Immunoblotting; Meiosis; Microinjections; Microscopy, Fluorescence; Microtubules; Oocytes; Oogenesis; Phalloidine; Poisons; Progesterone; Progestins; Xenopus laevis

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

During the polymerization of actin, hydrolysis of bound ATP occurs in two consecutive steps: chemical cleavage of the high-energy nucleotide and slow release of the γ-phosphate. In this study the effect of phalloidin and jasplakinolide on the kinetics of P(i) release was monitored during the formation of actin filaments. An enzyme-linked assay based spectrophotometric technique was used to follow the liberation of inorganic phosphate. It was verified that jasplakinolide reduced the P(i) release in the same way as phalloidin. It was not possible to demonstrate long-range allosteric effects of the toxins by release of P(i) from F-actin. The products of ATP hydrolysis were released by denaturation of the actin filaments. HPLC analysis of the samples revealed that the ATP in the toxin-bound region was completely hydrolysed into ADP and P(i). The effect of both toxins can be sufficiently explained by local and mechanical blockade of P(i) dissociation.

Topics: Actin Cytoskeleton; Actins; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Depsipeptides; Kinetics; Models, Molecular; Phalloidine; Phosphates; Protein Multimerization; Protein Structure, Quaternary; Rabbits

Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility.

Topics: Actin Cytoskeleton; Actins; Amino Acid Substitution; Animals; Binding Sites; Cell Movement; Depsipeptides; Evolution, Molecular; Gene Expression Regulation; Models, Molecular; Parasites; Phalloidine; Phylogeny; Plasmids; Plasmodium; Protein Multimerization; Protozoan Proteins; Toxoplasma

Transient, non-catastrophic brain ischaemia can induce either a protected state against subsequent episodes of ischaemia (ischaemic preconditioning) or delayed, selective neuronal death. Altered glutamatergic signalling and altered Ca(2+) homeostasis have been implicated in both processes. Here we use simultaneous patch-clamp recording and Ca(2+) imaging to monitor early changes in glutamate release and cytoplasmic [Ca(2+)] ([Ca(2+)](c)) in an in vitro slice model of hippocampal ischaemia. In slices loaded with the Ca(2+)-sensitive dye Fura-2, ischaemia leads to an early increase in [Ca(2+)](c) that precedes the severe ischaemic depolarization (ID) associated with pan necrosis. The early increase in [Ca(2+)](c) is mediated by influx through the plasma membrane and release from internal stores, and parallels an early increase in vesicular glutamate release that manifests as a fourfold increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs). However, the increase in mEPSC frequency is not prevented by blocking the increase in [Ca(2+)](c), and the early rise in [Ca(2+)](c) is not affected by blocking ionotropic and metabotropic glutamate receptors. Thus, the increase in [Ca(2+)](c) and the increase in glutamate release are independent of each other. Stabilizing actin filaments with jaspamide or phalloidin prevented vesicle release induced by ischaemia. Our results identify several early cellular cascades triggered by ischaemia: Ca(2+) influx, Ca(2+) release from intracellular stores, actin filament depolymerization, and vesicular release of glutamate that depends on actin dynamics but not [Ca(2+)](c). All of these processes precede the catastrophic ID by several minutes, and thus represent potential target mechanisms to influence the outcome of an ischaemic episode.

Topics: Actin Cytoskeleton; Animals; Brain Ischemia; CA1 Region, Hippocampal; Calcium; Chelating Agents; Depsipeptides; Egtazic Acid; Electrophysiology; Excitatory Postsynaptic Potentials; Fluorescent Dyes; Fura-2; Glutamates; In Vitro Techniques; Patch-Clamp Techniques; Phalloidine; Polymers; Presynaptic Terminals; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Synaptic Vesicles

Jasplakinolide, a cyclo-depsipeptide is a commonly used actin filament polymerizing and stabilizing drug. The substance has originally been isolated from a marine sponge, and can now be synthesized and has become commercially available. This, together with the benefit that jasplakinolide is membrane permeable has made it a commonly used tool in cell biology, when actin filament stabilization or polymerization has to be achieved. This may either be the case in studies on morphogenesis, motility, organelle movement, or when apoptosis has to be induced. Its use as a potent anticancer drug is discussed. The direct action on actin filaments may have further consequences in golgi body and membrane raft protein organization. In this chapter, the visualization of jasplaklinolide effects by different fluorescent and transmission electron microscopic methods is described. As competitive binding capacities of jasplakinolide and phalloidin make the detection of actin filaments by fluorescently labeled phalloidin problematic, alternatives are given here.

Topics: Actin Cytoskeleton; Animals; Biopolymers; Cell Line; Chlorophyta; Depsipeptides; Dictyostelium; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Molecular Structure; Molecular Weight; Phalloidine; T-Lymphocytes; Toxoplasma

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

Dynamic remodeling of the actin cytoskeleton plays an essential role in the migration and proliferation of vascular smooth muscle cells. It has been suggested that actin remodeling may also play an important functional role in nonmigrating, nonproliferating differentiated vascular smooth muscle (dVSM). In the present study, we show that contractile agonists increase the net polymerization of actin in dVSM, as measured by the differential ultracentrifugation of vascular smooth muscle tissue and the costaining of single freshly dissociated cells with fluorescent probes specific for globular and filamentous actin. Furthermore, induced alterations of the actin polymerization state, as well as actin decoy peptides, inhibit contractility in a stimulus-dependent manner. Latrunculin pretreatment or actin decoy peptides significantly inhibit contractility induced by a phorbol ester or an alpha-agonist, but these procedures have no effect on contractions induced by KCl. Aorta dVSM expresses alpha-smooth muscle actin, beta-actin, nonmuscle gamma-actin, and smooth muscle gamma-actin. The incorporation of isoform-specific cell-permeant synthetic actin decoy peptides, as well as isoform-specific probing of cell fractions and two-dimensional gels, demonstrates that actin remodeling during alpha-agonist contractions involves the remodeling of primarily gamma-actin and, to a lesser extent, beta-actin. Taken together, these results show that net isoform- and agonist-dependent increases in actin polymerization regulate vascular contractility.

Topics: Actins; Animals; Cell Differentiation; Cytoskeleton; Depsipeptides; Ferrets; In Vitro Techniques; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Peptide Fragments; Phalloidine; Phenylephrine; Phorbol Esters; Potassium Chloride; Protein Isoforms; Vasoconstriction; Vasoconstrictor Agents

Profilin has been implicated in cell motility and in a variety of cellular processes, such as membrane extension, endocytosis, and formation of focal complexes. In vivo, profilin replenish the pool of ATP-actin monomers by increasing the rate of nucleotide exchange of ADP-actin for ATP-actin, promoting the incorporation of new actin monomers at the barbed end of actin filaments. For this report, we generated a membrane-permeable version of profilin I (PTD4-PfnI) for the alteration of intracellular profilin levels taking advantage of the protein transduction technique. We show that profilin I induces lamellipodia formation independently of growth factor presence in primary bovine trabecular meshwork (BTM) cells. The effects are time- and concentration-dependent and specific to the profilin I isoform. Profilin II, the neuronal isoform, failed to extend lamellipodia in the same degree as profilin I. H133S, a mutation in the polyproline binding domain, showed a reduced ability to induce lamellipodia. H199E, mutation in the actin binding domain failed to induce membrane spreading and inhibit fetal bovine serum (FBS) -induced lamellipodia extension. Incubation with a synthetic polyproline domain peptide (GP5)3, fused to a transduction domain, abolished lamellipodia induction by profilin or FBS. Time-lapse microscopy confirmed the effects of profilin on lamellipodia extension with a higher spreading velocity than FBS. PTD4-Pfn I was found in the inner lamellipodia domain, at the membrane leading edge where it colocalizes with endogenous profilin. While FBS-induced lamellipodia formation activates Rac1, PTD4-Pfn I stimulation did not induce Rac1 activation. We propose a role of profilin I favoring lamellipodia formation by a mechanism downstream of growth factor.

Topics: Actins; Animals; Azepines; Cattle; Cells, Cultured; Depsipeptides; Humans; Intercellular Signaling Peptides and Proteins; Naphthalenes; Peptides; Phalloidine; Phosphatidylinositol 3-Kinases; Profilins; Proto-Oncogene Proteins c-akt; Pseudopodia; rac1 GTP-Binding Protein; Rats; Recombinant Fusion Proteins; Trabecular Meshwork

The stabilisation of magnesium actin filaments by phalloidin and jasplakinolide was studied using the method of differential scanning calorimetry. The results showed that actin could adapt three conformations in the presence of drugs. One conformation was adapted in direct interaction with the drug, while another conformation was identical to that observed in the absence of drugs. A third conformation was induced through allosteric inter-protomer interactions. The effect of both drugs propagated cooperatively along the actin filaments. The number of the cooperative units determined by using a quantitative model was larger for jasplakinolide (15 actin protomers) than for phalloidin (7 protomers).

Topics: Actin Cytoskeleton; Animals; Calorimetry, Differential Scanning; Depsipeptides; Magnesium; Models, Chemical; Molecular Conformation; Phalloidine; Rabbits; Temperature

Knowledge of the phalloidin binding position in F-actin and the relevant understanding of the mechanism of F-actin stabilization would help to define the structural characteristics of the F-actin filament. To determine the position of bound phalloidin experimentally, x-ray fiber diffraction data were obtained from well-oriented sols of F-actin and the phalloidin-F-actin complex. The differences in the layer-line intensity distributions, which were clearly observed even at low resolution (8 A), produced well-resolved peaks corresponding to interphalloidin vectors in the cylindrically averaged difference-Patterson map, from which the radial binding position was determined to be approximately 10 A from the filament axis. Then, the azimuthal and axial positions were determined by single isomorphous replacement phasing and a cross-Patterson map in radial projection to be approximately 84 degrees and 0.5 A relative to the actin mass center. The refined position was close to the position found by prior researchers. The position of rhodamine attached to phalloidin in the rhodamine-phalloidin-F-actin complex was also determined, in which the conjugated Leu(OH)(7) residue was found to face the outside of the filament. The position and orientation of the bound phalloidin so determined explain the increase in the interactions between long-pitch strands of F-actin and would also account for the inhibition of phosphate release, which might also contribute to the F-actin stabilization. The method of analysis developed in this study is applicable for the determination of binding positions of other drugs, such as jasplakinolide and dolastatin 11.

Topics: Actin Cytoskeleton; Actins; Animals; Antineoplastic Agents; Biophysics; Depsipeptides; Electrons; Macromolecular Substances; Models, Molecular; Models, Statistical; Phalloidine; Protein Binding; Protein Conformation; Rhodamines; X-Ray Diffraction

Ahnak, a protein of 5643 amino acids, interacts with the regulatory beta-subunit of cardiac calcium channels and with F-actin. Recently, we defined the binding sites among the protein partners in the carboxyl-terminal domain of ahnak. Here we further narrowed down the beta(2)-interaction sites to the carboxyl-terminal 188 amino acids of ahnak by the recombinant ahnak protein fragments P3 (amino acids 5456-5556) and P4 (amino acids 5556-5643). The effects of these P3 and P4 fragments on the calcium current were investigated under whole-cell patch clamp conditions on rat ventricular cardiomyocytes. P4 but not P3 increased significantly the current amplitude by 22.7 +/- 5% without affecting its voltage dependence. The slow component of calcium current inactivation was slowed down by both P3 and P4, whereas only P3 slowed significantly the fast one. The composite recombinant protein fragment P3-P4 induced similar modifications to the ones induced by each of the ahnak fragments. In the presence of carboxyl-terminal ahnak protein fragments, isoprenaline induced a similar relative increase in current amplitude and shift in current kinetics. The actin-stabilizing agents, phalloidin and jasplakinolide, did not modify the effects of these ahnak protein fragments on calcium current in control conditions nor in the presence of isoprenaline. Hence, our results suggest that the functional effects of P3, P4, and P3-P4 on calcium current are mediated by targeting the ahnak-beta(2)-subunit interaction rather than by targeting the ahnak-F-actin interaction. More specifically they suggest that binding of the beta(2)-subunit to the endogenous subsarcolemmal giant ahnak protein re-primes the alpha(1C)/beta(2)-subunit interaction and that the ahnak-derived proteins relieve the beta(2)-subunit from this inhibition.

Topics: Actins; Animals; Binding Sites; Calcium; Depsipeptides; Electrophysiology; Heart Ventricles; Kinetics; Male; Membrane Proteins; Myocytes, Cardiac; Neoplasm Proteins; Patch-Clamp Techniques; Peptides, Cyclic; Phalloidine; Phosphorylation; Plasmids; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Rats; Rats, Wistar; Recombinant Proteins; Time Factors

In this work the effect of phalloidin and jasplakinolide on the dynamic properties and thermal stability of actin filaments was studied. Temperature dependent fluorescence resonance energy transfer measurements showed that filaments of Ca-actin became more rigid in the presence of phalloidin or jasplakinolide. Differential scanning calorimetric data implied that the stiffer filaments also had greater thermal stability in the presence of phalloidin or jasplakinolide. The fluorescence and calorimetric measurements provided evidences that the extent of stabilization by jasplakinolide was greater than that by phalloidin.

Topics: Actins; Animals; Antineoplastic Agents; Calorimetry; Calorimetry, Differential Scanning; Depsipeptides; Fluorescence Resonance Energy Transfer; Microscopy, Fluorescence; Muscle, Skeletal; Peptides, Cyclic; Phalloidine; Protein Conformation; Rabbits; Temperature; Thermodynamics

Mesangial cells in diverse glomerular diseases become myofibroblast-like, characterized by activation of smooth muscle alpha-actin (alpha-SMA) expression. In cultured mesangial cells, serum-deprivation markedly increases alpha-SMA expression, cell size, and stress fiber formation. Since stress fibers are assembled from actin monomers, we investigated the hypothesis that alterations in stress fiber formation regulate alpha-SMA expression and hypertrophy. Human mesangial cells were treated with agents that disrupt or stabilize actin stress fibers. Depolymerization of actin stress fibers in serum-deprived cells with actin-depolymerizing agents, cytochalasin B (CytB) and latrunculin B (LatB), or with inhibitors of Rho-kinase, Y-27632 and HA-1077 decreased alpha-SMA mRNA as judged by Northern blot analysis. Western blot analysis showed that CytB also reduced alpha-SMA protein levels. In serum-fed cells, agents that stabilized actin stress fibers, jasplakinolide (Jas) and phalloidin, increased alpha-SMA mRNA and protein. Treatment of human or rat mesangial cells with CytB, LatB, or Y-27632 decreased alpha-SMA promoter activity. In contrast, Jas increased promoter activity 5.6-fold in rat mesangial cells. The presence of an RNA polymerase inhibitor blocked degradation of alpha-SMA mRNA in cells treated with CytB suggesting that destabilization of this message is dependent on a newly transcribed or rapidly degraded factor. Inhibition of actin polymerization by CytB, LatB, Y-27623, and HA-1077 inhibited incorporation of (3)[H]-leucine into newly synthesized protein. Additionally, CytB and LatB decreased cell volume as determined by flow cytometry. Collectively, these results indicate that the state of polymerization of the actin cytoskeleton regulates alpha-SMA expression, hypertrophy, and myofibroblast differentiation in mesangial cells.

Topics: Actin Cytoskeleton; Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Cell Size; Cells, Cultured; Cytochalasin B; Depsipeptides; Enzyme Inhibitors; Fibroblasts; Glomerular Mesangium; Humans; Intracellular Signaling Peptides and Proteins; Myocytes, Smooth Muscle; Peptides, Cyclic; Phalloidine; Phenotype; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Rats; rho-Associated Kinases; RNA Stability; RNA, Messenger; Thiazoles; Thiazolidines; Transcriptional Activation

Growth cone motility and guidance depend on the dynamic reorganization of filamentous actin (F-actin). In the growth cone, F-actin undergoes turnover, which is the exchange of actin subunits from existing filaments. However, the function of F-actin turnover is not clear. We used jasplakinolide (jasp), a cell-permeable macrocyclic peptide that inhibits F-actin turnover, to study the role of F-actin turnover in axon extension. Treatment with jasp caused axon retraction, demonstrating that axon extension requires F-actin turnover. The retraction of axons in response to the inhibition of F-actin turnover was dependent on myosin activity and regulated by RhoA and myosin light chain kinase. Significantly, the endogenous myosin-based contractility was sufficient to cause axon retraction, because jasp did not alter myosin activity. Based on these observations, we asked whether guidance cues that cause axon retraction (ephrin-A2) inhibit F-actin turnover. Axon retraction in response to ephrin-A2 correlated with decreased F-actin turnover and required RhoA activity. These observations demonstrate that axon extension depends on an interaction between endogenous myosin-driven contractility and F-actin turnover, and that guidance cues that cause axon retraction inhibit F-actin turnover.

Topics: Actin Cytoskeleton; Actins; Actomyosin; Animals; Antineoplastic Agents; Axons; Cells, Cultured; Chick Embryo; Depsipeptides; Ephrin-A2; Ganglia, Spinal; Growth Cones; Immunoenzyme Techniques; Intracellular Signaling Peptides and Proteins; Microinjections; Microscopy, Video; Microtubules; Myosin-Light-Chain Kinase; Neurons; Peptides, Cyclic; Phalloidine; Protein Binding; Protein Serine-Threonine Kinases; Retina; rho-Associated Kinases; rhoA GTP-Binding Protein

Many critical cellular processes, including proliferation, vesicle trafficking, and secretion, are regulated by both phospholipase D (PLD) and the actin microfilament system. Stimulation of human PLD1 results in its association with the detergent-insoluble actin cytoskeleton, but the molecular mechanisms and functional consequences of PLD-actin interactions remain incompletely defined. Biochemical and pharmacologic modulation of actin polymerization resulted in complex bidirectional effects on PLD activity, both in vitro and in vivo. Highly purified G-actin inhibited basal and stimulated PLD activity, whereas F-actin produced the opposite effects. Actin-induced modulation of PLD activity was independent of the activating stimulus. The efficacy and potency of the effects of actin were isoform-specific but broadly conserved among actin family members. Human betagamma-actin was only 45% as potent and 40% as efficacious as rabbit skeletal muscle alpha-actin, whereas its inhibitory profile was similar to the single actin species from the yeast, Saccharomyces cerevisiae. Use of actin polymerization-specific reagents indicated that PLD1 binds both monomeric G-actin, as well as actin filaments. These data are consistent with a model in which the physical state of the actin cytoskeleton is a critical determinant of its regulation of PLD activity.

Topics: Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Cell Membrane; Cytosol; Deoxyribonuclease I; Depsipeptides; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Kinetics; Macromolecular Substances; Magnesium Chloride; Mammals; Marine Toxins; Peptides, Cyclic; Phalloidine; Phospholipase D; Protein Isoforms; Tetradecanoylphorbol Acetate; Thiazoles; Thiazolidines; U937 Cells

The successful synthesis of dolastatin 11, a depsipeptide originally isolated from the mollusk Dolabella auricularia, permitted us to study its effects on cells. The compound arrested cells at cytokinesis by causing a rapid and massive rearrangement of the cellular actin filament network. In a dose-and time-dependent manner, F-actin was rearranged into aggregates, and subsequently the cells displayed dramatic cytoplasmic retraction. The effects of dolastatin 11 were most similar to those of the sponge-derived depsipeptide jasplakinolide, but dolastatin 11 was about 3-fold more cytotoxic than jasplakinolide in the cells studied. Like jasplakinolide, dolastatin 11 induced the hyperassembly of purified actin into filaments of apparently normal morphology. Dolastatin 11 was qualitatively more active than jasplakinolide and, in a quantitative assay we developed, dolastatin 11 was twice as active as jasplakinolide and 4-fold more active than phalloidin. However, in contrast to jasplakinolide and phalloidin, dolastatin 11 did not inhibit the binding of a fluorescent phalloidin derivative to actin polymer nor was it able to displace the phalloidin derivative from polymer. Thus, despite its structural similarity to other agents that induce actin assembly (all are peptides or depsipeptides), dolastatin 11 may interact with actin polymers at a distinct drug binding site.

Topics: Actin Cytoskeleton; Actins; Animals; Antineoplastic Agents; Bacterial Proteins; Cell Division; Cells, Cultured; Depsipeptides; Dipodomys; Fluorescent Dyes; Isothiocyanates; Oligopeptides; Peptides; Peptides, Cyclic; Phalloidine

The retinal pigment epithelium (RPE) of teleosts contains pigment granules that migrate in response to changes in light condition. Dissociated, cultured RPE cells in vitro can be triggered to aggregate or disperse pigment granules by the application of cAMP or dopamine, respectively. Previous research using the actin-disrupting drug, cytochalasin D, suggested that pigment granule motility is actin dependent. To further examine the role of actin in pigment granule motility, we tested the effects of the actin-stabilizing drug, jasplakinolide, on pigment granule motility. Pigment granules in previously dispersed RPE cells remained dispersed after jasplakinolide exposure (0.1-1 microM), but the drug halted movement of most pigment granules and stimulated rapid bi-directional movements in a small subset of granules. Jasplakinolide also blocked net pigment granule aggregation and interfered with the maintenance of full aggregation. Although jasplakinolide did not block pigment granule dispersion, it did alter the motility of dispersing granules compared to control cells; rather than the normal saltatory, primarily centrifugal movements, granules of jasplakinolide-treated cells demonstrated slow, creeping centrifugal movements and more rapid bi-directional movements. Jasplakinolide also altered cell morphology; the length and thickness of apical projections increased, and enlarged, paddle-like structures, which contained F-actin appeared at the tips of projections. Actin antibody labeling of jasplakinolide-treated cells revealed a more reticulated network of actin compared to antibody-labeled control cells. These results indicate that jasplakinolide-induced disruption of the actin network compromises normal pigment granule dispersion and aggregation in isolated RPE cells, thus providing further evidence that these movements are actin dependent.

Topics: Actins; Animals; Antineoplastic Agents; Cardiotonic Agents; Cell Movement; Cells, Cultured; Cyclic AMP; Cytochalasin D; Depsipeptides; Dopamine; Dose-Response Relationship, Drug; Fluorescent Dyes; Microscopy, Video; Nucleic Acid Synthesis Inhibitors; Peptides, Cyclic; Perciformes; Phalloidine; Pigment Epithelium of Eye; Rhodamines; Time Factors

Plasma membrane disruption is a common form of cell injury in many normal biological environments, including many mammalian tissues. Survival depends on the initiation of a rapid resealing response that is mounted only in the presence of physiological levels of extracellular Ca(2+). Vesicle-vesicle and vesicle-plasma membrane fusion events occurring in cortical cytoplasm surrounding the defect are thought to be a crucial element of the resealing mechanism. However, in mammalian cells, the vesicles used in this fusion reaction (endosomes/lysosomes) are not present in a 'pre-docked' configuration and so must be brought into physical contact with one another and with the plasma membrane. We propose that a requisite prelude to fusion is the disassembly in local cell cortex of the physical barrier constituted by filamentous actin. Consistent with this hypothesis, we found that rat gastric epithelial (RGM1) cell cortical staining with phalloidin was apparently reduced at presumptive disruption sites. Moreover, flow cytofluorometric analysis of wounded RGM1 populations revealed a small, but significant, Ca(2+)-dependent reduction in whole cell phalloidin staining. The functional significance of this disruption-induced depolymerization response was confirmed in several independent tests. Introduction into RGM1 cells of the filamentous actin-depolymerizing agent, DNase1, enhanced resealing, although cytochalasin treatment, by itself, had no effect. By contrast, when the filamentous actin cytoskeleton was stabilized experimentally, using phalloidin or jasplakinolide, resealing was strongly inhibited. Cells in wounded cultures displayed an enhanced cortical array of filamentous actin, and resealing by such cells was enhanced strongly by both cytochalasin and DNase 1, demonstrating the specific reversibility of a biologically mediated, polymerization-induced inhibition of resealing. We conclude that localized filamentous actin disassembly removes a cortical barrier standing in the way of membrane-membrane contacts leading to resealing-requisite homotypic and exocytotic fusion events.

Topics: Actins; Animals; Antineoplastic Agents; Calcium; Cell Line; Cell Membrane; Cytochalasin B; Cytoplasm; Cytoskeleton; Deoxyribonuclease I; Depsipeptides; Epithelial Cells; Exocytosis; Gastric Mucosa; Microscopy, Electron; Peptides, Cyclic; Phalloidine; Polymers; Rats

Jasplakinolide paradoxically stabilizes actin filaments in vitro, but in vivo it can disrupt actin filaments and induce polymerization of monomeric actin into amorphous masses. A detailed analysis of the effects of jasplakinolide on the kinetics of actin polymerization suggests a resolution to this paradox. Jasplakinolide markedly enhances the rate of actin filament nucleation. This increase corresponds to a change in the size of actin oligomer capable of nucleating filament growth from four to approximately three subunits, which is mechanistically consistent with the localization of the jasplakinolide-binding site at an interface of three actin subunits. Because jasplakinolide both decreases the amount of sequestered actin (by lowering the critical concentration of actin) and augments nucleation, the enhancement of polymerization by jasplakinolide is amplified in the presence of actin-monomer sequestering proteins such as thymosin beta(4). Overall, the kinetic parameters in vitro define the mechanism by which jasplakinolide induces polymerization of monomeric actin in vivo. Expected consequences of jasplakinolide function are consistent with the experimental observations and include de novo nucleation resulting in disordered polymeric actin and in insufficient monomeric actin to allow for remodeling of stress fibers.

Topics: Actins; Animals; Biopolymers; Cell Line; Depsipeptides; Kinetics; Peptides, Cyclic; Phalloidine; Rabbits; Rats; Thymosin

In the present study, we have characterized the action of the natural cyclodepsipeptide jasplakinolide (JAS) on the cytoplasmic architecture, actin-based cytoplasmic motility, and the organization of the actin cytoskeleton in selected examples of green algae (Acetabularia, Pseudobryopsis and Nitella) and higher plant cells (Allium bulb scale cells and Sinapis root hairs). JAS was capable of influencing the actin cytoskeleton and inhibiting cytoplasmic streaming in a differential, cell type-specific manner. With the exception of Nitella, two consecutive responses were observed upon incubation with 2.5 microM JAS: In the first phase cytoplasmic streaming increased transiently alongside with minor modifications of the actin cytoskeleton in the form of adventitious actin spots and spikes appearing throughout the cell cortex in addition to the normal actin bundle system typical for each cell type. In the second phase, cytoplasmic streaming stopped and the actin cytoskeleton became heavily reorganized into shorter, straight, more and more randomly oriented bundle segments. JAS exerted severe long-term effects on the actin cytoskeleton when treatments exceeded 30min at a concentration of 2.5 microM. An in situ competition assay using equimolar concentrations of JAS and FITC-phalloidin suggested that JAS has a phalloidin-like action. Effects of JAS were significantly different from those of cytochalasin D with respect to the resulting degree of perturbance of cytoplasmic organization, the distribution of actin filaments and the speed of reversibility.

Topics: Actins; Animals; Biological Transport; Chlorophyta; Cytochalasin D; Cytoplasm; Cytoskeleton; Depsipeptides; Fluorescein-5-isothiocyanate; Growth Inhibitors; Mice; Mustard Plant; Onions; Organelles; Peptides, Cyclic; Phalloidine; Plants, Medicinal

Arabidopsis thaliana trichomes provide an attractive model system to dissect molecular processes involved in the generation of shape and form in single cell morphogenesis in plants. We have used transgenic Arabidopsis plants carrying a GFP-talin chimeric gene to analyze the role of the actin cytoskeleton in trichome cell morphogenesis. We found that during trichome cell development the actin microfilaments assumed an increasing degree of complexity from fine filaments to thick, longitudinally stretched cables. Disruption of the F-actin cytoskeleton by actin antagonists produced distorted but branched trichomes which phenocopied trichomes of mutants belonging to the 'distorted' class. Subsequent analysis of the actin cytoskeleton in trichomes of the distorted mutants, alien, crooked, distorted1, gnarled, klunker and wurm uncovered actin organization defects in each case. Treatments of wild-type seedlings with microtubule-interacting drugs elicited a radically different trichome phenotype characterized by isotropic growth and a severe inhibition of branch formation; these trichomes did not show defects in actin cytoskeleton organization. A normal actin cytoskeleton was also observed in trichomes of the zwichel mutant which have reduced branching. ZWICHEL, which was previously shown to encode a kinesin-like protein is thought to be involved in microtubule-linked processes. Based on our results we propose that microtubules establish the spatial patterning of trichome branches whilst actin microfilaments elaborate and maintain the overall trichome pattern during development.

Topics: Actins; Arabidopsis; Bridged Bicyclo Compounds, Heterocyclic; Cytoskeleton; Depsipeptides; Microtubules; Morphogenesis; Mutagenesis; Peptides, Cyclic; Phalloidine; Phenotype; Plants, Genetically Modified; Thiazoles; Thiazolidines

We have electroporated Dictyostelium amoebae with fluorescent phalloidin in order to visualize the localization and behavior of F-actin filaments in living cells. Immediately after electroporation with phalloidin, cells became round and showed bright staining in the cortical region. Over time, the cortical staining disappeared and was replaced by a large aggregate of actin filaments. The aggregates were predominantly localized to the apical posterior of actively moving cells and in the middle of dividing cells or stationary AX4 cells. Mutants lacking myosin II or ABP-120 also formed actin aggregates; however, the rate of formation of aggregates was slower in myosin II mutant cells. In order to investigate this phenomenon further, we have used jasplakinolide, a membrane-permeable drug that also stabilizes F-actin filaments. Cells treated with jasplakinolide formed actin aggregates in a concentration-dependent manner. Drug treatment led to an increase in the proportion of actin associated with the cytoskeleton. Jasplakinolide-treated cells were still motile; however, their rate of movement was less than that of untreated cells. Cytochalasin B and nocodazole had inhibitory effects on aggregate formation, while azide blocked the process completely. We hypothesize that aggregates are formed from the cortical flow of F-actin filaments. These filaments would normally be depolymerized but are artificially stabilized by phalloidin or jasplakinolide binding. The localization of the aggregate is likely to be an indication of the direction of cortical flow.

Topics: Actins; Animals; Cytochalasin B; Cytoskeleton; Depsipeptides; Dictyostelium; Kinetics; Nocodazole; Peptides, Cyclic; Phalloidine

Jasplakinolide, a naturally occurring cyclic peptide from the marine sponge, Jaspis johnstoni, has both fungicidal and antiproliferative activity. We now report that this peptide is a potent inducer of actin polymerization in vitro. The peptide has a much greater effect on Mg(2+)-actin than on Ca(2+)-actin. Competitive binding studies using rhodamine-phalloidin suggest that jasplakinolide binds to F-actin competitively with phalloidin with a dissociation constant of approximately 15 nM. This compares favorably to the previously reported IC50 of 35 nM for the antiproliferative effect of jasplakinolide on PC3 prostate carcinoma cells. The binding curve suggests that nearest neighbor positive cooperativity influences the binding of jasplakinolide (and perhaps also phalloidin) to F-actin. These results imply that jasplakinolide may exert its cytotoxic effect in vivo by inducing actin polymerization and/or stabilizing pre-existing actin filaments.

Topics: Actins; Animals; Binding, Competitive; Biopolymers; Cytotoxins; Depsipeptides; Kinetics; Peptides, Cyclic; Phalloidine; Porifera; Rabbits; Spectrometry, Fluorescence