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PubMed1980

Chicken-gizzard actin: polymerization and stability.

Preparations of chicken gizzard actin obtained from acetone-dried muscle powders prepared with various methods developed for skeletal muscle contain variable amounts of a beta-actinin-like protein. This contamination is minimized if the procedure of muscle powder preparation includes washing with EDTA solution, and can be completely removed by gel filtration of G-actin on Sephadex G-100. The presence of beta-actinin activity manifests itself in an increased rate of actin polymerization, low filament lengths resulting in low reduced viscosity and enhanced ATP-splitting activity of actin polymer, and instability of the polymer in the absence of free ATP. Gizzard actin purified on a Sephadex G-100 column does not differ from rabbit skeletal muscle actin in its polymerization properties. The distinct property of gizzard actin is the instability of its G form in the absence of added Ca2+, indicating that the affinity of this cation for the single high-affinity site in gizzard actin is lower than in skeletal muscle actin.

Actins↗

PubMed2001

[The effect of medium viscosity on actin polymerization].

A comparative study of the influence of two factors--excluded volume effect, and medium viscosity--on actin polymerization was carried out. Dextrane--500 (1%), poly(ethylene)glycol--6000 (2.7%), Ficol (1.8%), methyl cellulose (0.04%), saccharose (10%), and glycerine (10%) were used for creating a viscous medium. The concentrations of these agents in actin solution made approximately the same viscosity commensurable with the cytoplasm viscosity. By this it was possible to bring conditions of actin polymerization in vitro closer to those in vivo. It is shown that the medium viscosity in contrast to the excluded volume effect, prolongs the nucleation phase during actin polymerization. A conclusion is made that in the cell both factors can be involved in the regulation of actin filament formation.

Actins↗

PubMed1985

A model for actin polymerization and the kinetic effects of ATP hydrolysis.

A model for actin polymerization is proposed in which the rate of elongation of actin filaments depends on whether adenosine 5'-triphosphate or adenosine 5'-diphosphate is bound to the two terminal subunits of the filament. This model accounts quantitatively for the experimental data on the kinetics of filament elongation and explains the effect of ATP hydrolysis on actin polymerization.

Actin Cytoskeleton↗

PubMed1999

Actin polymerization induces shedding of FcgammaRIIIb (CD16) from human neutrophils.

FcgammaRIIIb (CD16) is a glycosyl phosphatidylinositol (GPI)-anchored low-affinity IgG receptor, exclusively expressed on human neutrophils. FcgammaRIIIb associates with complement receptor 3 (CR3, Mac-1, CD11b/CD18), which may indirectly link FcgammaRIIIb to the actin cytoskeleton. Upon neutrophil activation, apoptosis, or chemotaxis, FcgammaRIIIb is shed from the cell surface. In all of these events, actin rearrangements play an important role. To establish a role for the actin cytoskeleton in the control of FcgammaRIIIb shedding, we treated human neutrophils with jasplakinolide, an actin-polymerizing peptide. We show that enhanced actin polymerization induces time- and dose-dependent shedding of FcgammaRIIIb. This effect was not restricted to FcgammaRIIIb, because the cell surface expression of CD43, CD44, and L-selectin was also downregulated after induction of actin polymerization. This actin-dependent pathway is staurosporine sensitive but does not appear to involve activation of PKC or CR3. These data show that the actin cytoskeleton can regulate protein ectodomain shedding from human neutrophils.

Actins↗

PubMed2004

Stretch of the vascular wall induces smooth muscle differentiation by promoting actin polymerization.

Stretch of the vascular wall by the intraluminal blood pressure stimulates protein synthesis and contributes to the maintenance of the smooth muscle contractile phenotype. The expression of most smooth muscle specific genes has been shown to be regulated by serum response factor and stimulated by increased actin polymerization. Hence we hypothesized that stretch-induced differentiation is promoted by actin polymerization. Intact mouse portal veins were cultured under longitudinal stress and compared with unstretched controls. In unstretched veins the rates of synthesis of several proteins associated with the contractile/cytoskeletal system (alpha-actin, calponin, SM22alpha, tropomyosin, and desmin) were dramatically lower than in stretched veins, whereas other proteins (beta-actin and heat shock proteins) were synthesized at similar rates. The cytoskeletal proteins gamma-actin and vimentin were weakly stretch-sensitive. Inhibition of Rho-associated kinase by culture of stretched veins with Y-27632 produced similar but weaker effects compared with the absence of mechanical stress. Induction of actin polymerization by jasplakinolide increased SM22alpha synthesis in unstretched veins to the level in stretched veins. Stretch stimulated Rho activity and phosphorylation of the actin-severing protein cofilin-2, although both effects were slow in onset (Rho-GTP, >15 min; cofilin-P, >1 h). Cofilin-2 phosphorylation of stretched veins was inhibited by Y-27632. The F/G-actin ratio after 24 h of culture was significantly greater in stretched than in unstretched veins, as shown by both ultracentrifugation and confocal imaging with phalloidin/DNase I labeling. The results show that stretch of the vascular wall stimulates increased actin polymerization, activating synthesis of smooth muscle-specific proteins. The effect is partially, but probably not completely, mediated via Rho-associated kinase and cofilin downstream of Rho.

Actin Depolymerizing Factors↗

PubMed1999

Actin polymerization: Where the WASP stings.

How do extracellular signals induce actin polymerization, as required for many cellular responses? Key signal transducers, such as the small GTPases Cdc42 and Rac, have now been shown to link via proteins of the WASP family to the Arp2/3 complex, which nucleates actin polymerization.

Actin-Related Protein 2↗

PubMed1990

Thyroxine-dependent modulation of actin polymerization in cultured astrocytes. A novel, extranuclear action of thyroid hormone.

Actin depolymerization specifically blocks the rapid thyroid hormone-dependent inactivation of type II iodothyronine 5'-deiodinase. Thyroid hormone appears to regulate enzyme inactivation by modulating actin-mediated internalization of this plasma membrane-bound protein. In this study, we examined the interrelationships between thyroxine-dependent enzyme inactivation and the organization of the actin cytoskeleton in cultured astrocytes. Steady-state enzyme levels were inversely related to actin content in dibutyryl cAMP-stimulated astrocytes, and increases in filamentous actin resulted in progressively shorter enzyme half-lives without affecting enzyme synthesis. In the absence of thyroxine, filamentous actin decreased by approximately 40% and soluble actin correspondingly increased; thyroxine normalized filamentous actin levels without changing total cell actin. Thyroxine treatment for only 10 min resulted in an approximately 50% loss of enzyme and increased filamentous actin 2-fold. Neither cycloheximide nor actinomycin D affected the thyroxine-induced actin polymerization. Astrocytes grown without thyroxine also showed a disorganized actin cytoskeleton, and 10 nM thyroxine or 10 nM reverse triiodothyronine normalized the actin cytoskeleton appearance within 20 min; 10 nM 3,3',5-triiodothyronine had no effect. These data show that thyroxine modulates the organization of the actin cytoskeleton in astrocytes and suggest that regulation of actin polymerization may contribute to thyroid hormone's influence on arborization, axonal transport, and cell-cell contact in the developing brain.

Actins↗

PubMed1988

Binding of phosphate to F-ADP-actin and role of F-ADP-Pi-actin in ATP-actin polymerization.

Our previous work (Carlier, M.-F., and Pantaloni, D. (1986) Biochemistry 25, 7789-7792) had shown that F-ADP-Pi-actin is a major intermediate in ATP-actin polymerization, due to the slow rate of Pi release following ATP cleavage on filaments. To understand the mechanism of ATP-actin polymerization, we have prepared F-ADP-Pi-actin and characterized its kinetic parameters. 32Pi binds to F-ADP-actin with a stoichiometry of 1 mol/mol of F-actin subunit and an equilibrium dissociation constant Kpi of 1.5 mM at pH 7.0 Kpi increases with pH, indicating that the H2PO-4 species binds to F-actin. ADP-Pi-actin subunits dissociate much more slowly from filament ends than ADP-actin subunits; therefore, the stability of filaments in ATP is due to terminal ADP-Pi subunits. The slow rate of dissociation of ADP-Pi-actin also explains the decrease in critical concentration of ADP-actin in the presence of Pi reported by Rickard and Sheterline (Richard, J. E., and Sheterline, P. (1986) J. Mol. Biol. 191, 273-280). The effect of Pi on the rate of actin dissociation from filaments is much more pronounced at the barbed end than at the pointed end. Using gelsolin to block the barbed end, we have shown that the two ends are energetically different in the presence of ATP and saturating Pi, but less different than in the absence of Pi. The results are interpreted within a new model for actin polymerization. It is possible that phosphate binding to F-actin can regulate motile events in muscle and nonmuscle cells.

Actins↗

PubMed1995

Actin polymerization and intracellular solvent flow in cell surface blebbing.

The cortical actin gel of eukaryotic cells is postulated to control cell surface activity. One type of protrusion that may offer clues to this regulation are the spherical aneurysms of the surface membrane known as blebs. Blebs occur normally in cells during spreading and alternate with other protrusions, such as ruffles, suggesting similar protrusive machinery is involved. We recently reported that human melanoma cell lines deficient in the actin filament cross-linking protein, ABP-280, show prolonged blebbing, thus allowing close study of blebs and their dynamics. Blebs expand at different rates of volume increase that directly predict the final size achieved by each bleb. These rates decrease as the F-actin concentration of the cells increase over time after plating on a surface, but do so at lower concentrations in ABP-280 expressing cells. Fluorescently labeled actin and phalloidin injections of blebbing cells indicate that a polymerized actin structure is not present initially, but appears later and is responsible for stopping further bleb expansion. Therefore, it is postulated that blebs occur when the fluid-driven expansion of the cell membrane is sufficiently rapid to initially outpace the local rate of actin polymerization. In this model, the rate of intracellular solvent flow driving this expansion decreases as cortical gelation is achieved, whether by factors such as ABP-280, or by concentrated actin polymers alone, thereby leading to decreased size and occurrence of blebs. Since the forces driving bleb extension would always be present in a cell, this process may influence other cell protrusions as well.

Actins↗

PubMed2006

Mechanism of actin polymerization by myosin subfragment-1 probed by dynamic light scattering.

Monomeric actin (G-actin) polymerizes spontaneously into helical filaments in the presence of inorganic salts. The slowest, rate-limiting step of the polymerization process is formation of actin trimers, the smallest oligomers that serve as nuclei for fast filament growth (filament elongation) by monomer addition at the filament ends. In low ionic-strength solutions, actin can be polymerized by myosin subfragment-1 (S1). In early works it has been suggested that G-actin-S1 1:1 complexes (GS) assemble into filaments according to the nucleation-filament elongation scheme. Subsequent studies indicated that one S1 molecule can bind two actin monomers, and that oligomerization of the initial complexes is a fast reaction. This has led to suggest an alternative mechanism, with a ternary G(2)S complex and its oligomers being predominant intermediates of S1-induced assembly of G-actin into filaments. We used dynamic light scattering to analyze the initial steps of S1-induced polymerization of actin. Our results suggest formation of GS complexes and their oligomers in the presence of S1 equimolar to or in excess over actin. We confirm formation of G(2)S complexes as intermediates of S1-induced polymerization in the presence of actin in excess over S1.

Actins↗

PubMed1995

Calponin induces actin polymerization at low ionic strength and inhibits depolymerization of actin filaments.

Calponin from chicken gizzard induced polymerization of actin in the presence of 10 mM KCl. Only 2 min after the addition of KCl in the presence of a 0.0625-0.25:1 molar ratio of calponin to actin, a Poisson-type length distribution (with an average length of approx. 0.7 micron) was observed with formed actin filaments. This result suggests that calponin-actin complexes served as nuclei for rapid elongation. Calponin caused a rapid polymerization of actin even in G-buffer (2 mM Tris/HCl, pH 8.0) which is usually used for depolymerization of actin filaments. Binding of calponin at a level of up to 1.25 mol per mol of actin was observed in the actin filaments formed in the presence of calponin at very low ionic strengths. When actin filaments were exposed to 3.3 mM KCl, by dilution with G-buffer, a rapid depolymerization occurred. Addition of calponin greatly retarded the depolymerization process and, in the presence of an equimolar ratio of calponin to actin, depolymerization hardly occurred. In the presence of calmodulin, this inhibitory effect on depolymerization was reversed by Ca2+, releasing calponin from actin filaments.

Actins↗

PubMed1996

Fluid shear stress induces actin polymerization in human neutrophils.

We have previously reported that a physiological range of shear stress induces neutrophil homotypic aggregation mediated by lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-3 (ICAM-3) interactions. To further characterized the homotypic aggregation, actin polymerization was investigated in neutrophils stimulated by shear stress in comparison with formyl-methionyl-leucyl-phenylalanine (fMLP). In fMLP-stimulated neutrophils, actin polymerization was localized in the pseudopods, and this reaction was not mediated by a cytosolic level of Ca2+. In contrast to fMLP stimulation, the actin polymerization induced by shear stress in a cone-plate viscometer was localized in cell-cell contact regions, and this polymerization required the increase of intracellular Ca2+. This shear stress-induced actin polymerization was not observed when neutrophils were pretreated with anti-LFA-1 or anti-ICAM-3 antibody. In conclusion, LFA-1 and ICAM-3 interaction mediated by the increase of [Ca2+]i generated the intercellular signal in order to accumulate F-actin in the cell-cell contact regions.

Actins↗

PubMed1987

Effect of phosphatidyl-L-serine and vinculin on actin polymerization.

The effects of phosphatidyl-L-serine (PS) and/or vinculin on actin polymerization are examined by spectrophotometry, viscometry and electrophoresis. Actin polymerization is inhibited by PS alone and stimulated by PS and vinculin. The results suggest that actin does not directly adhere to cell membrane and that vinculin is a protein which is involved in structures connecting actin microfilaments to cell membranes.

Actins↗

PubMed1985

pH control of actin polymerization by cofilin.

Cofilin, a 21,000 molecular weight actin-regulatory protein (Nishida, E., Maekawa, S., and Sakai, H. (1984) Biochemistry 23, 5307-5313), was here shown to be capable of reversibly controlling actin polymerization and depolymerization in a pH-sensitive manner. When cofilin was reacted with F-actin at different pH, the depolymerized actin concentration (= monomeric actin concentration) was higher at elevated pH. At pH less than 7.3, the monomeric actin concentrations did not exceed approximately 1 microM even in the presence of excess amounts of cofilin, whereas at pH greater than 7.3 it increased in proportion to the concentration of cofilin added, and complete depolymerization of F-actin occurred by the addition of an excess amount of cofilin. Moreover, in the presence of cofilin, rapid interconversion of monomeric and polymeric forms of actin can be induced by simply changing the pH of the medium. Thus, this study provides a new possible mechanism regulating actin polymerization, pH control.

Actin Depolymerizing Factors↗

PubMed1996

Yeast actin: polymerization kinetic studies of wild type and a poorly polymerizing mutant.

Wild-type actin and a mutant actin were isolated from yeast (Saccharomyces cerevisiae) and the polymerization properties were examined at pH 8.0 and 20 degrees C. The polymerization reaction was followed either by an increase in pyrene-labeled actin fluorescence or by a decrease in intrinsic fluorescence in the absence of pyrene-labeled actin. While similar to the properties of skeletal muscle actin, there are several important differences between the wild-type yeast and muscle actins. First, yeast actin polymerizes more rapidly than muscle actin under the same experimental conditions. The difference in rates may result from a difference in the steps involving formation of the nucleating species. Second, as measured with pyrene-labeled yeast actin, but not with intrinsic fluorescence, there is an overshoot in the fluorescence that has not been observed with skeletal muscle actin under the same conditions. Third, in order to simulate the polymerization process of wild-type yeast actin it is necessary to assume some fragmentation of the filaments. Finally, gelsolin inhibits polymerization of yeast actin but is known to accelerate the polymerization of muscle actin. A mutant actin (R177A/D179A) has also been isolated and studied. The mutations are at a region of contact between monomers across the long axis of the actin filament. This mutant polymerizes more slowly than wild type and filaments do not appear to fragment during polymerization. Elongation rates of the wild type and the mutant differ by only about 3-fold, and the slower polymerization of the mutant appears to result primarily from poorer nucleation.

Actins↗

PubMed1986

Direct evidence for ADP-Pi-F-actin as the major intermediate in ATP-actin polymerization. Rate of dissociation of Pi from actin filaments.

The sequence of reactions involved in the polymerization of ATP-actin and accompanying hydrolysis of ATP has been investigated by using a new glass-fiber filter assay. The assay allows the rapid separation of filaments from monomeric actin, and therefore the straightforward identification of the nucleotide bound to F-actin in the time course of polymerization, using double-labeled [gamma-32P,3H]ATP. The data bring a direct confirmation of the existence of the previously proposed ATP-F-actin intermediate in the time course of polymerization. Moreover, comparison of the hydrolyzed ATP (i.e., acid-labile [32P]Pi) and of 32P bound to F-actin provides direct evidence for the second intermediate ADP-Pi-F-actin in the polymerization process. This latter species is the major transient in the polymerization of ATP-actin, its lifetime being of the order of minutes.

Actins↗

PubMed1983

Identification and purification of calcium ion dependent modulators of actin polymerization from bovine thyroid.

We describe the purification of Ca2+-dependent actin modulator proteins from bovine thyroid using DNase I affinity chromatography and diethylaminoethylcellulose chromatography. The 40K actin modulator has been purified to 98% homogeneity. It is a single polypeptide chain with a molecular weight of approximately 40 000 and an isoelectric point of 8.1. Its amino acid composition is different from previously described actin-associated proteins and thyroid actin. On the basis of the centrifugation assay and the DNase I inhibition assay, the actin complexed with the 40K protein is G-actin in its conformation rather than F-actin oligomers. Substoichiometric concentrations of the 40K protein rapidly inhibit actin polymerization in the presence of physiological concentrations of Ca2+ and Mg2+. An 80K actin modulator also has been purified to 98% homogeneity. It is a single polypeptide chain with a molecular weight of approximately 80 000 and an isoelectric point of 6.35-7.0. Its amino acid composition is different from those of villin, gelsolin, and leukocyte actin polymerization inhibitor. On the basis of the DNase inhibition assay and the centrifugation assay, the nonprecipitable actin associated with the 80K protein was F-actin in its conformation. The 80K protein acts very efficiently as a Ca2+-dependent nucleator for actin assembly and reduces its viscosity. In addition to the 40K and 80K actin modulators, 91K and 95K actin-associated proteins were partially purified. The 91K-95K fraction has similar activity to the 80K protein regarding precipitation of F-actin. The 125I-G-actin polyacrylamide gel overlay technique [Snabes, M. C., Boyd, A.E., & Bryan, J. (1981) J. Cell Biol. 90, 809-812] revealed that both the 91K and 95K proteins bind 125I-actin after sodium dodecyl sulfate (NaDodSO4) electrophoresis while the 80K and 40K proteins do not. Thyroid 91K protein comigrated with a human platelet 91K actin binding protein on NaDodSO4 gels and may be similar to macrophage gelsolin. The 95K protein may be similar to villin, the intestinal cytoskeletal protein.

Actins↗

PubMed2001

Cryptosporidium parvum infection requires host cell actin polymerization.

The intracellular protozoan parasite Cryptosporidium parvum accumulates host cell actin at the interface between the parasite and the host cell cytoplasm. Here we show that the actin polymerizing proteins Arp2/3, vasodilator-stimulated phosphoprotein (VASP), and neural Wiskott Aldrich syndrome protein (N-WASP) are present at this interface and that host cell actin polymerization is necessary for parasite infection.

Actin-Related Protein 2↗