phalloidine and thymosin-beta(4)

The beta-thymosins are intracellular monomeric (G-)actin sequestering proteins forming 1:1 complexes with G-actin. Here, we analysed the interaction of thymosin beta(4) with F-actin. Thymosin beta(4) at 200 microM was chemically cross-linked to F-actin. In the presence of phalloidin, the chemically cross-linked actin:thymosin beta(4) complex was incorporated into F-actin. These mixed filaments were of normal appearance when inspected by conventional transmission electron microscopy after negative staining. We purified the chemically cross-linked actin:thymosin beta(4) complex, which polymerised only when phalloidin and the gelsolin:2-actin complex were present simultaneously. Using scanning transmission electron microscopy, the mass-per-length of control and actin:thymosin beta(4) filaments was found to be 16.0(+/-0.8) kDa/nm and 18.0(+/-0.9) kDa/nm, respectively, indicating an increase in subunit mass of 5.4 kDa. Analysis of the helical parameters revealed an increase of the crossover spacing of the two right-handed long-pitch helical strands from 36.0 to 40.5 nm. Difference map analysis of 3-D helical reconstruction of control and actin:thymosin beta(4) filaments yielded an elongated extra mass. Qualitatively, the overall size and shape of the difference mass were compatible with published data of the atomic structure of thymosin beta(4). The deduced binding sites of thymosin beta(4) to actin were in agreement with those identified previously. However, parts of the difference map might represent subtle conformational changes of both proteins occurring upon complex formation.

Topics: Actins; Animals; Biopolymers; Cross-Linking Reagents; Gelsolin; Humans; Kinetics; Microscopy, Electron; Microscopy, Electron, Scanning Transmission; Models, Molecular; Molecular Weight; Muscle, Skeletal; Phalloidine; Protein Binding; Protein Structure, Quaternary; Protein Structure, Secondary; Protein Subunits; Rabbits; Thymosin

Protein kinase C-epsilon coordinately regulates changes in cell growth and shape. Cells overproducing protein kinase C-epsilon spontaneously acquire a polarized morphology and extend long cellular membrane protrusions that are reminiscent of the morphology observed in ras-transformed fibroblasts. Here we report that the regulatory C1 domain contains an actin binding hexapeptide motif that is essential for the morphogenic effects of protein kinase C-epsilon in cultured NIH3T3 murine fibroblasts. The extension of elongate processes by protein kinase C-epsilon transformed fibroblasts appeared to be driven by a kinase-independent mechanism that required organized networks of both actin and microtubules. Flow cytometry of phalloidin-stained cells demonstrated that protein kinase C-epsilon significantly increased the cellular content of polymerized actin in NIH3T3 cells. Studies with a cell-free system suggest that protein kinase C-epsilon inhibits the in vitro disassembly of actin filaments, is capable of desequestering actin monomers from physiologically relevant concentrations of thymosin beta4, and increases the rate of actin filament elongation by decreasing the critical concentration of actin. Based on these and other observations, it is proposed that protein kinase C-epsilon may function as a terminal downstream effector in at least one of the signaling pathways that mitogens engage to initiate outgrowth of cellular protrusions.

Topics: 3T3 Cells; Actins; Animals; Binding Sites; Cell Line, Transformed; Cell Size; Gene Deletion; Isoenzymes; Mice; Microfilament Proteins; Microtubules; Phalloidine; Polymers; Protein Kinase C; Protein Kinase C-epsilon; Protein Structure, Tertiary; Rabbits; Staining and Labeling; Thymosin

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

To address the mechanisms through which agonists stimulate actin polymerization, we examined the roles of monomer sequestering proteins and free barbed ends on actin polymerization induced by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) in neutrophils permeabilized with streptolysin O. Addition of profilin (without GTP gamma S) caused a net decrease in F-actin. Thus, merely making profilin available in the cell was not sufficient to induce actin polymerization. On the other hand, addition of profilin hardly affected the polymerization induced by GTP gamma S, while thymosin beta 4 or DNase I decreased this polymerization. These data suggested that GTP gamma S induced polymerization by increasing the availability of barbed ends. In the presence of cytochalasin B, profilin did inhibit polymerization induced by GTP gamma S, demonstrating that GTP gamma S did not inhibit profilin's monomer sequestering ability. The F-actin induced by GTP gamma S was not limited by a time-dependent loss of G-actin or G-proteins from permeabilized cells since, following stimulation with suboptimal concentrations of GTP gamma S, addition of more GTP gamma S induced further polymerization. Barbed ends remained free after F-actin reached plateau since (a) cytochalasin B caused depolymerization of induced F-actin and (b) profilin did not depolymerize induced F-actin unless the cells were first treated with cytochalasin to cap barbed ends. The data indicate that GTP gamma S maintains an increased level of F-actin by keeping at least a few barbed ends available for polymerization.

Topics: Actins; Animals; Bacterial Proteins; Cell Membrane Permeability; Cytochalasin B; Deoxyribonuclease I; Fluorescent Dyes; Guanosine 5'-O-(3-Thiotriphosphate); In Vitro Techniques; Kinetics; Mathematics; Neutrophils; Phalloidine; Rabbits; Rhodamines; Streptolysins; Thymosin; Time Factors

The thermodynamic basis for actin-based motility of Listeria monocytogenes has been investigated using cytoplasmic extracts of Xenopus eggs, initially developed by Theriot et al. (Theriot, J. A., J. Rosenblatt, D. A. Portnoy, P. J. Goldschmidt-Clermont, and T. J. Mitchison. 1994. Cell. 76:505-517) as an in vitro cell-free system. A large proportion (75%) of actin was found unpolymerized in the extracts. The amount of unassembled actin (12 microM) is accounted for by the sequestering functions of T beta 4Xen (20 microM) and profilin (5 microM), the barbed ends being capped. Movement of Listeria was not abolished by depletion of over 99% of the endogenous profilin. The proline-rich sequences of ActA are unlikely to be the target of profilin. All data support the view that actin assembly at the rear of Listeria results from a local shift in steady state due to a factor, keeping filaments uncapped, bound to the surface of the bacterium, while barbed ends are capped in the bulk cytoplasm. Movement is controlled by the energetic difference (i.e., the difference in critical concentration) between the two ends of the filaments, hence a constant ATP supply and the presence of barbed end capped F-actin in the medium are required to buffer free G-actin at a high concentration. The role of membrane components is demonstrated by the facts that: (a) Listeria movement can be reconstituted in the resuspended pellets of high speed-centrifuged extracts that are enriched in membranes; (b) Actin-based motility of endogenous vesicles, exhibiting the same rocketing movement as Listeria, can be observed in the extracts.

Topics: Actins; Adenosine Triphosphate; Animals; Cell Extracts; Cell Movement; Contractile Proteins; Deoxyribonuclease I; Female; Listeria monocytogenes; Microfilament Proteins; Ovum; Peptides; Phalloidine; Profilins; Thymosin; Xenopus

Thymosin beta 4 forms a 1:1 complex with actin and thereby prevents polymerization. Rapid formation of filaments from this complex was observed, however, when actin trimers were added. Polymerization can likewise be initiated by the addition of one equivalent of phalloidin or, less effectively, cytochalasin B. Since both toxins, which reportedly support nucleation, have similar effects as the covalently linked actin trimers, it appears that the formation of filaments from the actin-thymosin beta 4 complex depends on the availability of stable actin nuclei. Remarkably, rapid polymerization was also observed if small amounts of myosin S1 were added, suggesting that also myosin, a protein functionally connected with polymeric actin, can serve as a nucleation center. Considering the existence of thymosin beta 4 and related peptides in numerous mammalian tissues, our data suggest that spontaneous formation of microfilaments in non-muscle cells may be regulated at the level of nucleation. Uncontrolled polymerization induced by the formation of phalloidin-stabilized nuclei may explain the acute toxic effects of phalloidin in hepatocytes.

Topics: Actins; Cytochalasin B; Fluorescence; Kinetics; Macromolecular Substances; Muscles; Myosin Subfragments; Phalloidine; Polymers; Potassium Chloride; Thymosin; Viscosity

Thymosin beta 4 is able to form 1:1 complexes with monomeric (G) actin, thereby stabilizing the intracellular pool of unpolymerized actin. We have searched for factors that are able to induce the polymerization of actin from the actin:thymosin beta 4 complex. Phalloidin, subfragment 1 isolated from rabbit skeletal muscle myosin and chicken intestinal myosin I are demonstrated to be able to polymerize the actin from this complex in the presence of 1 mM MgCl2. Polymerization of actin was verified by the DNase I inhibition assay, by cosedimentation and from the fluorescence increase of pyrene-labelled actin. Actin filaments formed under the influence of subfragment 1 or phalloidin were visualized under the electron microscope after negative staining. Polymerization of skeletal muscle actin from the complex with thymosin beta 4 by phalloidin is accompanied by the hydrolysis of the actin-bound ATP to ADP. Polymerization was also induced by sonicated F-actin which possessed a high concentration of free filament ends. F-actin was severed by 0.01 M human cytoplasmic gelsolin, which is known to possess blocked+ends. Free, slowly growing-ends were unable to induce polymerization of actin from the thymosin beta 4 complex. However, when gelsolin on its own or in complex with two actin molecules was added to actin:thymosin beta 4 under nucleating conditions, it was found to be able to promote actin repolymerization provided that its concentration was close to the dissociation constant (Kd) of actin:thymosin beta 4. This Kd was found to be 0.4 microM in the presence of 1 mM MgCl2 and the absence of KCl and, thus, close to the critical concentration of actin polymerization under these conditions. The source of actin did not influence its polymerization from the thymosin beta 4 complex; rabbit skeletal muscle actin and porcine brain actin were polymerized with equal efficiency from their complexes with thymosin beta 4 by both phalloidin and myosin subfragment 1. Skeletal muscle, but not cytoplasmic actin, was found to be also polymerized in the presence of increased CaCl2 concentrations to values above 1 mM.

Topics: Actins; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Binding Sites; Calcium Chloride; Chickens; Hydrolysis; Magnesium Chloride; Microscopy, Electron; Muscles; Myosin Subfragments; Myosins; Phalloidine; Polymers; Potassium Chloride; Rabbits; Thymosin