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PubMed2005

Stimulation of actin polymerization by filament severing.

The extent and dynamics of actin polymerization in solution are calculated as functions of the filament severing rate, using a simple model of in vitro polymerization. The model is solved by both analytic theory and stochastic-growth simulation. The results show that severing essentially always enhances actin polymerization by freeing up barbed ends, if barbed-end cappers are present. Severing has much weaker effects if only pointed-end cappers are present. In the early stages of polymerization, the polymerized-actin concentration grows exponentially as a function of time. The exponential growth rate is given in terms of the severing rate, and the latter is given in terms of the maximum slope in a polymerization time course. Severing and branching are found to act synergistically.

Actin Depolymerizing Factors↗

PubMed1986

N-ethylmaleimide cannot inhibit actin polymerization in platelets.

We investigated the effects of the N-ethylmaleimide (NEM), a sulfhydryl(SH) radical blocker, on platelet activation. Platelet aggregation and ATP release was suppressed by 0.2 mM NEM during ADP (20 microM) stimulation and by 0.5 mM NEM during A23187 (4 microM) stimulation. However the agent had no effect on actin polymerization in stimulated platelets. In the absence of a stimulant, NEM (over 1 mM) induced shape changes and slight (5%) actin polymerization, but not aggregation or ATP release. Although platelet aggregation and ATP release were suppressed by the addition of 1 mM NEM during the process of both reactions, the amount of polymerized actin was not influenced by the addition. The reconstructed system consisting of actin and partially purified regulatory proteins without myosin showed a dose-dependent increase in turbidity by the addition of NEM. From these findings, we concluded that NEM enhances actin polymerization, although actin molecules contain SH-radicals, and that actin polymerization has little affect on aggregation and release reaction.

Actins↗

PubMed1985

[Cytochrome c-induced acceleration of the reaction of muscle actin polymerization].

The reaction of rabbit skeletal muscle actin polymerization initiated by the addition of neutral salts is accelerated in the presence of mitochondrial cytochrome c. The observed effect is specific, since the addition of serum albumin does not change the initial velocity of this process. The dependence of the rate of actin polymerization and exogenous cytochrome c concentration correlates within the molar protein ratios from 43:1 to 9:1, respectively. The increase in the polymerization rate occurs immediately after addition of cytochrome c to the reaction mixture; however, the maximal effect is observed only after 5 min coincubation of the proteins. The ability of cytochrome c to stimulate this process is abolished at alkaline values of pH (8.5), which points to the significant role of the molecule positive charge which, in all probability, serves as a primer of the muscle actin polymerization reaction.

Actins↗

PubMed1995

Chronic myeloid leukemia granulocytes exhibit reduced actin polymerization after chemotactic peptide stimulation.

Polymorphonuclear leukocytes (PMNL) from chronic myeloid leukemia (CML) patients are defective for chemotaxis in response to the synthetic chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (fMLP) as compared to normal PMNL. The present study investigated whether the defective chemotactic response was mediated through altered actin polymerization induced with fMLP. Granulocytes isolated from seven normal subjects and seven CML patients were stimulated with fMLP and lysed with Triton containing buffer at time points of 0, 30 seconds, and 1, 2, and 10 minutes. The Triton insoluble cytoskeleton containing polymerized actin was analyzed by SDS-PAGE and densitometry. The CML PMNL polymerized significantly lesser actin than normal PMNL on stimulation with 10 nM (p > 0.05) and 1 nM (p > 0.01) fMLP. This lower actin polymerization observed in fMLP-stimulated CML PMNL may be responsible for the defective chemotaxis seen in these cells.

Actins↗

PubMed1985

Effect of capping protein on the kinetics of actin polymerization.

Acanthamoeba capping protein increased the rate of actin polymerization from monomers with and without calcium. In the absence of calcium, capping protein also increased the critical concentration for polymerization. Various models were evaluated for their ability to predict the effect of capping protein on kinetic curves for actin polymerization under conditions where the critical concentration was not changed. Several models, which might explain the increased rate of polymerization from monomers, were tested. Two models which predicted the experimental data poorly were (1) capping protein was similar to an actin filament, bypassing nucleation, and (2) capping protein fragmented filaments. Three models in which capping protein accelerated, but did not bypass, nucleation predicted the data well. In the best one, capping protein resembled a nondissociable actin dimer. Several lines of evidence have supported the idea that capping protein blocks the barbed end of actin filaments, preventing the addition and loss of monomers [Cooper, J. A., Blum, J. D., & Pollard, T. D. (1984) J. Cell Biol. 99, 217-225; Isenberg, G. A., Aebi, U., & Pollard, T. D. (1980) Nature (London) 288, 455-459]. This mechanism was also supported here by the effect of capping protein on the kinetics of actin polymerization which was nucleated by preformed actin filaments. Low capping protein concentrations slowed nucleated polymerization, presumably because capping protein blocked elongation at barbed ends of filaments. High capping protein concentrations accelerated nucleated polymerization because of capping protein's ability to interact with monomers and accelerate nucleation.

Actin Depolymerizing Factors↗

PubMed1993

Both isoforms of skeletal muscle subfragment 1 (S1A1 and S1A2) can induce actin polymerization with equal speed in the absence of ATP.

The ability of myosin subfragment 1 to induce actin polymerization was reinvestigated using the DNase I inhibition assay, by electron microscopy after negative staining, cosedimentation, measurement of viscosity and the fluorescence increase of pyrenyl-labeled actin. Using these techniques we demonstrate that rabbit skeletal muscle myosin subfragment 1 containing either the alkali light chain 1 (S1A1) or the alkali light chain 2 (S1A2) is able to promote actin polymerization even in the absence of divalent cations or salt. In the presence of ATP the rate of induction of actin polymerization by S1A2 is slower than by A1A1. In contrast, in the absence of free ATP, both subfragment 1 variants exhibit equal ability to induce actin polymerization. Evidence is given that the slower rate of induction of actin polymerization by S1A2 in the presence of free ATP is due to a slower rate of ATP-hydrolysis by S1A2 and thus to a slower rate of ATP depletion. We therefore assume that the formation of rigor type complexes involving the subfragment 1 heavy chain is necessary for the induction of actin polymerization. The ability of subfragment 1 to induce actin polymerization is retarded by a synthetic heavy chain mimetic peptide which inhibits its actin binding or after proteolytic cleavage of the subfragment 1 heavy chain by trypsin.

Actins↗

PubMed1992

Stimulus-dependent actin polymerization in bovine neutrophils.

Polymorphonuclear leukocytes (PMNs) are responsible for much of the first wave of leukocyte-mediated host defense against microbial pathogens. In order to migrate through the endothelium of vessel walls, undergo chemotaxis, and phagocytize microbes, PMNs must modulate their cytoskeletal elements and undergo change of cellular shape. We have used fluorescence flow cytometric analysis and cellular microscopic observations to demonstrate actin polymerization in bovine PMNs and to examine the kinetics of PMN actin polymerization utilizing different PMN stimuli. In addition, we compared temporal relationships between cellular shape and actin polymerization. Actin polymerization occurred rapidly, and the kinetics of actin polymerization were similar for each of the three PMN agonists used, ZAS (10%), PAF (10(-6) M), and rhC5a (10(-7) M). Actin polymerization was near-maximal by 10 sec poststimulation (95.4% of maximal F-actin content attained by 10 sec poststimulation with ZAS stimulation), and reached peak values by 30 sec. The maximal increase in F-actin content of agonist-stimulated cells as compared to resting cells was 2.8-fold with ZAS; 2.3-fold with PAF; and 2.3-fold with rhC5a. PMN shape change (pseudopodia, membrane ruffles) was not as rapid, with only 22.4% of cells attaining visible membrane deformation by 10 sec and requiring 120 sec to reach peak shape-change values. After attaining peak values, the two events also differed. Whereas the percent of shape-changed PMNs remained plateaued up to 5 min poststimulation, the F-actin content gradually decreased after 30 sec, approaching F-actin values of unstimulated PMNs.

Actins↗

PubMed1991

Control of actin polymerization in live and permeabilized fibroblasts.

We have investigated the spatial control of actin polymerization in fibroblasts using rhodamine-labeled muscle actin in; (a) microinjection experiments to follow actin dynamics in intact cells, and (b) incubation with permeabilized cells to study incorporation sites. Rhodamine-actin was microinjected into NIH-3T3 cells which were then fixed and stained with fluorescein-phalloidin to visualize total actin filaments. The incorporation of newly polymerized actin was assayed using rhodamine/fluorescein ratio-imaging. The results indicated initial incorporation of the injected actin near the tip and subsequent transport towards the base of lamellipodia at rates greater than 4.5 microns/min. Furthermore, both fluorescein- and rhodamine-intensity profiles across lamellipodia revealed a decreasing density of actin filaments from tip to base. From this observation and the presence of centripetal flux of polymerized actin we infer that the actin cytoskeleton partially disassembles before it reaches the base of the lamellipodium. In permeabilized cells we found that, in agreement with the injection studies, rhodamine-actin incorporated predominantly in a narrow strip of less than 1-microns wide, located at the tip of lamellipodia. The critical concentration for the rhodamine-actin incorporation (0.15 microM) and its inhibition by CapZ, a barbed-end capping protein, indicated that the nucleation sites for actin polymerization most likely consist of free barbed ends of actin filaments. Because any potential monomer-sequestering system is bypassed by addition of exogenous rhodamine-actin to the permeabilized cells, these observations indicate that the localization of actin incorporation in intact cells is determined, at least in part, by the presence of specific elongation and/or nucleation sites at the tips of lamellipodia and not solely by localized desequestration of subunits. We propose that the availability of the incorporation sites at the tips of lamellipodia is because of capping activities which preferentially inhibit barbed-end incorporation elsewhere in the cell, but leave barbed ends at the tips of lamellipodia free to add subunits.

Actins↗

PubMed1994

Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin.

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.

Actins↗

PubMed1988

Regulation of actin polymerization by membrane fraction of platelets.

We studied the interaction between the purified membrane fraction of human platelets and the polymerization of skeletal actin. The viscosity of actin was measured by the falling ball method. The fraction suppressed the polymerization of actin in the presence of 20 mM KCl and 0.4 mM EGTA. The addition of calcium ion or thrombin to the fraction did not cause suppression. A DNase I affinity column bound the membrane fraction in the presence of calcium ion. The frozen membrane fraction and the vesicles reconstituted with lipids from the platelet membrane enhanced the polymerization of actin. Trypsinized membrane fraction and the membrane fraction treated with phospolipase A2 enhanced the polymerization of actin, but membrane fraction treated with phospholipase C had no effect. The reconstituted membrane vesicles mentioned above lowered the critical concentration for actin polymerization. These findings suggested that the polymerization of intracellular actin is enhanced not only by the mobilization of calcium ion, but also by biochemical changes in the membrane lipids.

Actins↗

PubMed1988

Lack of correlation between induction of chemotactic peptide receptors and stimulus-induced actin polymerization in HL-60 cells treated with dibutyryl cyclic adenosine monophosphate or retinoic acid.

We used the promyelocytic leukemic cell line HL-60 to explore the molecular mechanisms regulating stimulus-induced actin polymerization in myeloid cells. HL-60 cells express very few chemotactic peptide receptors in their undifferentiated state and fail to undergo actin polymerization when stimulated with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). However, when the cells were induced to differentiate with dibutyryl cyclic AMP (dbcAMP) or retinoic acid, they acquired the ability to undergo actin polymerization on stimulation with FMLP or phorbol myristate acetate. Kinetic experiments revealed that in the first 48 h of retinoic acid treatment there was no increase in the chemotactic peptide receptors on HL-60 cells, but the cells were capable of undergoing actin polymerization on stimulation with FMLP. Similarly, treatment with dbcAMP showed no increase in chemotactic peptide receptors until 24 h but stimulus-induced actin polymerization was demonstrable as early as 4 h after the treatment. In addition, with dbcAMP-treated cells the magnitude of stimulus-induced actin polymerization showed large variation depending on the duration of exposure to the drug. Dual-label studies using propidium iodide to measure DNA content and NBD-phallacidin to measure the F-actin content revealed that these variations were not related to the stages of cell cycle. Cells in all stages of the cell cycle responded to stimulus-induced actin polymerization, but the magnitude of the response appeared to be more in cells in G2/M phase. The observations reported here indicate that the small number of chemotactic peptide receptors present on HL-60 cells are adequate to mount an actin polymerization response, provided the required intracellular mechanisms exist. Differentiation-inducing agents, therefore, must cause changes within the cell, such as induction of actin-binding proteins, to cause actin polymerization following FMLP stimulation. The HL-60 system serves as a useful model for studying the molecular mechanisms regulating stimulus-induced actin polymerization in human neutrophils.

Actins↗

PubMedDate not supplied

Modulation of actin polymerization by an exogenous protein, lysozyme.

Several methods (fluorescence, high- and low-shear viscosity, and electron microscopy) have been applied to measure the effects of lysozyme on actin polymerization. Under our conditions, at pH 8.0 and 20 degrees C, lysozyme is predominantly dimeric and its major effect is to inhibit the steady-state polymerization of actin. Those actin filaments formed in the presence of lysozyme are significantly shortened with recurrent amorphous densities along the filament length. However, at pH 6.4 and 37 degrees C, lysozyme is monomeric and actin filament cross-linking is observed. We reasoned that in hen egg white lysozyme the tripeptide L-arginyl-glycyl-aspartate (RGD), a sequence capable of mimicking a portion of the receptor sites of extracellular matrix proteins, might be important in lysozyme self-association and, therefore, actin-lysozyme interaction. The presence of RGD in the lysozyme-actin polymerizing solutions at pH 8.0 and 20 degrees C caused an inhibition of the dimeric lysozyme effects, while RGD alone had no effects on actin polymerization. Therefore, RGD most likely binds to a complementary RGD sequence on lysozyme and alters its ability to interact with actin and modify polymerization.

Actin Cytoskeleton↗

PubMed1990

Phalloidin reduces the release of inorganic phosphate during actin polymerization.

Phalloidin, an actin-filament stabilizing peptide from Amanita phalloides, did not inhibit ATP hydrolysis during actin polymerization but strongly retarded the release of the hydrolysis product Pi. Thus, the lifetime of the intermediate F-actin-ADP-Pi is significantly increased by phalloidin. The results suggest a close correlation between filament stability and F-actin-ADP-Pi intermediates.

Actins↗

PubMed1990

Ketotifen inhibits PAF-induced actin polymerization in a human eosinophilic leukaemia cell line, EoL-1.

The inhibitory effect of ketotifen on platelet activating factor (PAF)-induced actin polymerization in a human eosinophilic leukaemia cell line, EoL-1, was examined by flow cytometry with the use of reagents specific for the filamentous form of actin (F-actin). Actin polymerization has been considered to be essential for locomotion of cells, chemotaxis and chemokinesis, and thus it reflects the chemotactic reaction of EoL-1 cells stimulated by PAF. Unstimulated EoL-1 cells showed little PAF-induced actin polymerization, whereas EoL-1 cells cultured for 9 days with the supernatant of a human ATL cell line, HIL-3 (HIL-3 sup), showed marked actin polymerization when stimulated with PAF. The actin polymerization in EoL-1 cells induced by PAF was seen in a dose-dependent manner at concentrations of 10(-10) M to 10(-6) M of PAF, and the maximum effect was seen at 10(-7) M of PAF. CV-3988, a specific antagonist of PAF, inhibited 80% of the actin polymerization in EoL-1 cells induced by PAF at a concentration of 10(-5) M. Ketotifen inhibited up to 40% of the PAF-induced actin polymerization of EoL-1 cells in a dose-dependent manner at concentrations of 10(-9) M to 10(-5) M. These results suggest that ketotifen may play an important role in the prevention of eosinophil-induced inflammation in allergic disorders by inhibiting PAF-induced chemotaxis of eosinophils.

Actins↗

PubMed1983

Kinetic evidence for a monomer activation step in actin polymerization.

We measured the time course of skeletal muscle actin polymerization at different actin concentrations. In 0.1 M KCl with 1 mM Mg2+, log/log plots of the rate of the early, slow phase of polymerization vs. actin concentration were linear with slopes from 1.0 to 1.3. Computer-assisted calculations of similar curves from theoretical models with different sizes for the nucleus showed that no simple model gave a log/log plot with a slope less than 1.5. Addition of a first-order, monomer activation step before nucleation allowed models of any reasonable nucleus size to have a slope of 1. This is the first evidence that such a step is part of the kinetic pathway for actin polymerization. In 0.1 M KCl with 0.2 mM Ca2+, log/log plots of the rate of the slow phase vs. actin concentration were linear with slopes from 2.0 to 2.5. Monomer activation was not necessary to account for this slope. However, fits of kinetic curves calculated from theoretical models to experimental kinetic curves showed that filament fragmentation was important to achieve a good fit, confirming the finding of Wegner and Savko [Wegner, A., & Savko, P. (1982) Biochemistry 21, 1909-1913]. Our fit procedure also allowed us to estimate the size of the nucleus and the rate constants for activation, nucleation, and fragmentation. In 0.1 M KCl with 1 mM Mg2+, the nucleus was a dimer or trimer, and nucleation was fast. In 0.1 M KCl with 2.0 mM Ca2+, the nucleus was a trimer, and nucleation was slow.

Actins↗

PubMed1992

Actin-actin contact: inhibition of actin-polymerization by subdomain 4 peptide fragments.

F-Actin was digested with alpha-chymotrypsin in 6 M urea, and two peptide fragments from subdomain 4 of actin molecule [Kabsch, W., Mannherz, H.G., Suck, D., Pai, E.F., & Holmes K.C. (1990) Nature 347, 37-44] were purified by reverse-phase HPLC and Sephadex G-50 gel filtration. The peptide fragments were identified as segments from Arg-177 to Tyr-198 (2.6-kDa peptide) and from Ser-199 to Tyr-279 (9.1-kDa peptide). Their effects on actin polymerization induced by 50 or 100 mM KCl were studied by measuring the increase in viscosity by the falling ball method. The 2.6-kDa peptide decreased the rate of actin polymerization and increased the critical concentration for the polymerization. Based on the atomic model of the actin filament [Holmes, K.C., Popp, D., Gebhard, W., & Kabsch, W. (1990) Nature 347, 44-49], the peptide is presumed to bind to the barbed end of the actin filament and inhibit the polymerization. By assuming that the peptide affected the rate of association of the actin monomer to the end of the actin filament, well-fitting curves for the polymerization kinetics were calculated. Computer-assisted results indicated that the dissociation constant of the 2.6-kDa peptide for F-actin is 200 to 260 microM. In contrast, the 9.1-kDa peptide only slightly inhibited actin polymerization. These results suggest that the actin-actin interface in the region between Arg-177 and Tyr-198 has a stronger interaction than those between Ser-199 and Tyr-279. The amino acid sequence L-T-D-Y-L present in the 2.6-kDa segment is homologous to a common sequence in the F-actin capping domain of various actin-binding proteins.

Actins↗

PubMed2005

LIM kinase 1 coordinates microtubule stability and actin polymerization in human endothelial cells.

Microtubule (MT) destabilization promotes the formation of actin stress fibers and enhances the contractility of cells; however, the mechanism involved in the coordinated regulation of MTs and the actin cytoskeleton is poorly understood. LIM kinase 1 (LIMK1) regulates actin polymerization by phosphorylating the actin depolymerization factor, cofilin. Here we report that LIMK1 is also involved in the MT destabilization. In endothelial cells endogenous LIMK1 co-localizes with MTs and forms a complex with tubulin via the PDZ domain. MT destabilization induced by thrombin or nocodazole resulted in a decrease of LIMK1 colocalization with MTs. Overexpression of wild type LIMK1 resulted in MT destabilization, whereas the kinase-dead mutant of LIMK1 (KD) did not affect MT stability. Importantly, down-regulation of endogenous LIMK1 by small interference RNA resulted in abrogation of the thrombin-induced MTs destabilization and the inhibition of thrombin-induced actin polymerization. Expression of Rho kinase 2, which phosphorylates and activates LIMK1, dramatically decreases the interaction of LIMK1 with tubulin but increases its interaction with actin. Interestingly, expression of KD-LIMK1 or small interference RNA-LIMK1 prevents thrombin-induced microtubule destabilization and F-actin formation, suggesting that LIMK1 activity is required for thrombin-induced modulation of microtubule destabilization and actin polymerization. Our findings indicate that LIMK1 may coordinate microtubules and actin cytoskeleton.

Actins↗

PubMed1999

Cooperative symmetry-breaking by actin polymerization in a model for cell motility.

Polymerizing networks of actin filaments are capable of exerting significant mechanical forces, used by eukaryotic cells and their prokaryotic pathogens to change shape or to move. Here we show that small beads coated uniformly with a protein that catalyses actin polymerization are initially surrounded by symmetrical clouds of actin filaments. This symmetry is broken spontaneously, after which the beads undergo directional motion. We have developed a stochastic theory, in which each actin filament is modelled as an elastic brownian ratchet, that quantitatively accounts for the observed emergent symmetry-breaking behaviour. Symmetry-breaking can only occur for polymers that have a significant subunit off-rate, such as the biopolymers actin and tubulin.

Actins↗