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Results for “actin polymerization”

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PubMed1980

A platelet inhibitor protein with cytochalasin-like activity against actin polymerization in vitro.

We have obtained an inhibitor fraction containing cytochalasin-like activity from human platelets. Using a procedure involving DEAE-cellulose, hydroxyapatite and gel filtration column chromatography, we obtained a fraction from human platelets which apparently can compete with 3H--cytochalasin B for binding to spectrin-actin complexes from human erythrocytes. The inhibitor activity is nondialyzable, sensitive to heat and to trypsin and has a Stoke's radius of 40 A. This fraction stops nuclei-induced actin polymerization in 0.4 mM MgCl2 and reduces the viscosity of F actin to that of G actin, which suggests depolymerization of the filaments. These results suggest that the inhibitor fraction contains a protein which interacts with actin filaments and nuclei in a manner similar to that of cytochalasin B. It is possible that such a protein is involved in the control of cell motility by affecting assembly and disassembly of actin-containing microfilaments in vivo.

Actins↗

PubMed2006

Polyamines, PI(4,5)P2, and actin polymerization.

Spermine (SPM) and spermidine (SPD) activate isolated phosphatidylinositol-4-phosphate 5-kinases (PI(4)P5K), enzymes that convert phosphatidylinositol-4-phosphate to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). PI(4,5)P2 formation is known to be involved in cellular actin reorganization and motility, functions that are also influenced by polyamines. It has not been proven that endogenous polyamines can control inositol phospholipid metabolism. We evoked large decreases in SPD and putrescine (PUT) contents in HL60 cells, using the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine (DFMO), which resulted in decreases in PI(4,5)P2 content per cell and inositol phosphate formation to 76.9 +/- 3.5% and 81.5 +/- 4.0% of control, respectively. Accurately reversing DFMO-evoked decreases in SPD content by incubating cells with exogenous SPD for 20 min rescued these decreases. DFMO treatment and SPD rescues also changed the ratio of total cellular PI(4,5)P2 to PIP suggesting involvement of a SPD-sensitive PI(4)P5K. PUT and SPM were not involved in DFMO-evoked changes in cellular PI(4,5)P2 contents. In DFMO-treated HL60 cells, the percent of total actin content that was filamentous was decreased to 59.1 +/- 5.8% of that measured in paired control HL60 cells, a finding that was rescued following reversal of DFMO-evoked decreases in SPD and PI(4,5)P2 contents. In slowly proliferating DMSO-differentiated HL60 cells, inositol phospholipid metabolism was uncoupled from SPD control. We conclude: in rapidly proliferating HL60 cells, but not in slowly proliferating differentiated HL60 cells, there are endogenous SPD-sensitive PI(4,5)P2 pools, probably formed via SPD-sensitive PI(4)P5K, that likely control actin polymerization.

Actins↗

PubMed1990

The effects of new cytochalasins from Phomopsis sp. and the derivatives on cellular structure and actin polymerization.

The effects of ten 10-phenyl-[11]cytochalasins produced by Phomopsis sp. including novel compounds having 5,7- or 6,7-glycol structures and their derivatives, on the cell morphology and actin distribution in C3H-2K cells, as well as on lymphocyte capping and actin polymerization, were examined. The structure-activity relationship reported in the previous papers has been confirmed. The novel glycol type compounds showed little or no activity, suggesting the importance of the perhydroisoindol-1-one nucleus for the manifestation of the cytochalasin actions.

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PubMed2004

Connecting the dots: from actin polymerization to synapse formation.

Protrusive behavior of dendritic spines on developing neurons has been previously suggested to mediate the formation of new axodendritic synaptic contacts. A study by Zito et al. in this issue of Neuron links actin polymerization in dendritic spines with the motility that the spines exhibit and the synapses that they form.

Actins↗

PubMed1992

Comparative actin polymerization in neonatal and adult bovine neutrophils in vitro.

Neutrophils rely on active reorganization of the cytoskeleton during movement, and functional deficiencies in the cytoskeletal elements may result in impaired neutrophil-mediated host defense. We have compared and quantitated actin polymerization in neonatal (< or = 48 h old) and adult bovine peripheral-blood polymorphonuclear leukocytes (PMN) using fluorescence flow cytometry. Baseline filamentous actin (F-actin) content of neonatal and adult PMN at time zero differed slightly but were not statistically different (p > 0.05). F-actin content of recombinant human C5a (10(-7) M)-stimulated neonatal PMN increased rapidly within 10 s of stimulation to 59.0% over baseline, then declined. F-actin in adult recombinant human C5a-stimulated PMN continued to increase for 30 s and was elevated 87.3% over baseline before subsequently declining. When stimulated with zymosan-activated bovine serum (10%), neonatal (120.7% increase) and adult PMN (115.1% increase) had similar profiles with no significant differences, and both groups reached peak F-actin levels at 30 s after stimulation. Neonatal PMN stimulated with platelet-activating factor (10(-6) M) attained peak F-actin values at 10 s (72.0% increase over baseline), but actin rapidly depolymerized by 30 s poststimulation (reduced to 29.0% increase). Adult PMN stimulated by platelet-activating factor also attained peak values by 10 s (97.6% increase over baseline), but in contrast to neonatal PMN the F-actin remained elevated at 30 s in the adult PMN (still increased 79.5%; p < 0.0.5 compared to neonatal F-actin).(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1987

Modulation of actin polymerization by 47,000-dalton protein of human platelets.

We purified 47,000-dalton proteins from both thrombin-stimulated and unstimulated human platelets. The purity of the protein was almost 80% on SDS-polyacrylamide gel electrophoresis. The protein obtained from unstimulated platelets strongly inhibited actin gelation when its molar ratio to actin was 1:200 or higher. The protein obtained from thrombin-stimulated platelets had no inhibitory activity. The results suggest that the 47,000-dalton protein modulates actin polymerization through phosphorylation.

Actins↗

PubMed1977

Phalloidin-induced actin polymerization in the cytoplasm of cultured cells interferes with cell locomotion and growth.

Phalloidin, the toxic drug from the mushroom Amanita phalloides, was injected into the cytoplasm of tissue culture cells and the changes in intracellular actin distribution were followed by immunofluorescence microscopy with actin antibody. At low concentrations, phalloidin recruits the non- or less highly polymerized forms of cytoplasmic actin into stable "islands" of aggregated actin polymers and does not interfere with the preexisting thick bundles of microfilaments (stress fibers). Differential focusing shows that these islands of phalloidin-induced actin polymers occur at a level in the cytoplasm that is above the submembranous bundles of microfilaments present on the adhesive side of the cells. The pattern of cytoplasmic microtubules remains unaffected by the injection of phalloidin; however, filamin, a protein usually associated with actin in the cytoplasm, is also recruited into the islands. At higher phalloidin concentrations, contraction of the cell is observed. These results are discussed in the light of previous biochemical studies by Wieland and Faulstich and their coworkers [for a review see Wieland, T. (1977) Naturwissenschaften 64, 303-309] on the in vitro interaction of phalloidin with muscle actin, which have documented that phalloidin reacts stoichiometrically with actin, promotes actin polymerization, and stabilizes actin polymers. In addition, we show that microinjection of phalloidin interferes in a concentration-dependent manner with cell locomotion and cell growth. These results indicate that a well-balanced controlled reversible equilibrium between different polymerization states of actin may be a necessary requirement for cell locomotion and may also influence other cellular functions such as growth.

Actins↗

PubMed1999

Actin polymerization enhances Pasteurella haemolytica leukotoxicity.

Pasteurella haemolytica leukotoxin is cytotoxic to bovine leukocytes, causing increased cell membrane permeability, osmotic swelling, release of cytosolic proteins and cell lysis. These studies were designed to test if leukotoxin causes release of the cytoskeletal protein, actin, from bovine leukemia cells and if purified actin-influenced bacterial growth or leukotoxin production. Culture supernatants caused a 7-fold decrease in viability of bovine leukemia cells and increased cell permeability that was accompanied by release of beta-actin into the cell culture supernatant. Exposing P. haemolytica to purified actin solutions induced the conversion of monomeric G-actin to polymerized F-actin. This conversion was partially inhibited by bovine P. haemolytica immune, but not pre-immune, serum. Loss of streptomycin resistance following treatment of the organism with acridine orange ablated the polymerizing activity. Incubation of P. haemolytica in the presence of purified F-actin did not affect growth but resulted in culture supernatant that had 3.0-3.9-fold greater leukotoxicity compared to medium alone or medium containing G-actin, heat-denatured actin or albumin. The effect of actin on leukotoxicity was concentration-dependent and directly associated with increases in secreted leukotoxin. The interaction between P. haemolytica and actin is potentially detrimental to the host by inducing polymerization of actin into insoluble filaments and by enhancing leukotoxicity.

Actins↗

PubMed2007

Abeta1-42 stimulates actin polymerization in hippocampal neurons through Rac1 and Cdc42 Rho GTPases.

A number of psychiatric and neurodegenerative disorders, such as Alzheimer's disease, are characterized by abnormalities in the neuronal cytoskeleton. Here, we find that the enhancement in actin polymerization induced by fibrillar amyloid-beta peptide (Abeta) is associated with increased activity of Rac1/Cdc42 Rho GTPases. Rac1 upregulation involves the participation of Tiam1, a Rac guanine-nucleotide exchange factor, where Abeta exposure leads to Tiam1 activation by a Ca(2+)-dependent mechanism. These results point to Rho GTPases as one of the targets in Abeta-induced neurodegeneration in Alzheimer's disease pathology, with a role in mediating changes in the actin cytoskeletal dynamics.

Actins↗

PubMed1978

Correlation between actin polymerization and surface receptor segregation in neuroblastoma cells treated with concanavalin A.

In response to concanavalin A (Con A), neuroblastoma cells undergo marked morphological changes which involve the retraction of neurites and the induction of broad and extensive lamellar regions around the cell periphery. From the use of FITC-Con A it was shown that the membrane formed on the induced lamellar regions lacked receptors to Con A from the onset of lamella formation. These receptors were confined to the cell body; they initially showed a uniform distribution and were subsequently collected into patches and finally into aggregates or caps. When the aggregates occurred on the cell periphery their position coincided with areas free of lamellae. Investigations of the lamellar regions in Triton-extracted cell monolayers showed them to consist of a meshwork of actin filaments containing radiating thin filament bundles or microspikes. With increasing time in the presence of Con A there was a progressive increase in the number of radiating microspikes. Previous studies have shown the actin in these lamellar regions to be singly polarized with respect to the cell body. From the segregation of Con A receptors away from areas of actin polymerization in the lamellae it is concluded that actin is involved in some indirect way in surface receptor movement.

Actins↗

PubMed1994

Nucleation of actin polymerization by gelsolin.

The time-course of assembly of actin with gelsolin was measured by the fluorescence increase of a fluorescent label covalently linked to actin. The actin concentrations ranged from values far below the critical concentration to values above the critical concentration of the pointed ends of actin filaments. If the concentration of actin was in the range of the critical monomer concentration (0.64 microM), the time-course of the concentration of actin assembled with gelsolin revealed a sigmoidal shape. At higher actin concentrations the time-course of association of actin with gelsolin approximated an exponential curve. The measured time-courses of assembly were quantitatively interpreted by kinetic rate equations. A poor fit was obtained if two actin molecules were assumed to bind to gelsolin to form a 1:2 gelsolin-actin complex and subsequently further actin molecules were assumed to polymerize onto the 1:2 gelsolin-actin complex toward the pointed end. A considerably better agreement between calculated and measured time-courses was achieved if additional creation of actin filaments by fast fragmentation of newly formed actin filaments by not yet consumed gelsolin was assumed to occur. This suggests that both polymerization of actin onto gelsolin and fragmentation of actin filaments contribute to formation of new actin filaments by gelsolin. Furthermore it could be demonstrated that below the critical monomer concentration appreciable amounts of actin are incorporated into gelsolin-actin oligomers.

Actins↗

PubMed1993

Beta 2 integrin engagement triggers actin polymerization and phosphatidylinositol trisphosphate formation in non-adherent human neutrophils.

Beta 2 integrins are involved in the adhesion of leukocytes to other cells and surfaces. Although adhesion is required for cell locomotion, little is known regarding the way beta 2 integrin-receptors affect the actin network in leukocytes. In the present study filamentous actin (F-actin) levels in non-adherent human neutrophils have been measured by phalloidin staining after antibody cross-linking of beta 2 integrins. Antibody engagement of beta 2 integrins resulted in a rapid and sustained (146 and 131% after 30 and 300 s, respectively) increase in the neutrophil F-actin content. This is in contrast to stimulation with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP), which causes a prompt and pronounced but rapidly declining rise in F-actin (214 and 127% after 15 and 300 s, respectively). Priming neutrophils with 1 nM PMA, a low concentration that did not influence the F-actin content per se, increased the magnitude of the beta 2 integrin-induced response but had no effect on the kinetics (199% after 30 s and 169% after 300 s). Removal of extracellular Ca2+ only marginally affected the beta 2 integrin-induced F-actin response for cells that were pretreated with PMA whereas the response for nonprimed cells was reduced by half. This suggests that even though extracellular Ca2+ has a modulatory effect it is not an absolute requirement for beta 2 integrin-induced actin polymerization. beta 2 integrin engagement did not affect the resting cellular level of cAMP arguing against a role of cAMP in beta 2 integrin-induced actin assembly.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗

PubMed1981

Visualization of actin polymerization and depolymerization cycles during polyamine-induced cytokinesis in living Amoeba proteus.

Microinjection of spermine induces cytokinesis of Amoeba proteus. Within 30--60 s after spermine injection cells form one, or less commonly, two cleavage furrows and within the following 4--10 min the constrictions are completed. The resulting nucleated cell parts show normal streaming and locomotion, whereas the non-nucleated cell parts remain stationary and later degenerate. The intracellular distribution of fully polymerization-competent fluorescently labelled muscle actin was followed by image intensification. Double injection experiments initially using labelled action and 30 min later spermine revealed a ring-like structure of enhanced fluorescence corresponding to the constricting cleavage furrow. Immediately after cleavage was completed, the ring disappeared. Electron microscopy of cells fixed during spermine-induced cytokinesis showed numerous well aligned actin and myosin filaments in the developing cleavage furrow. These filaments are a specialized manifestation of the cell cortex. The results demonstrate that cycles of actin and myosin polymerization and depolymerization and the parallel alignment of preexisting filaments (crosslinking) represent a basic mechanism in the generation of the motive force during cytokinesis.

Actins↗

PubMed1991

Quantitation of actin polymerization in two human fibroblast sub-types responding to mechanical stretching.

To study early reorganization of the cytoskeleton in response to physical forces, human gingival and periodontal ligament fibroblasts were cultured on flexible plastic substrata and stretched by mechanical deformation of the substratum. F-actin was measured by quantitative spectrofluorimetry of FITC-phalloidin-stained cells. Fluorescence due to FITC-phalloidin was reduced stoichiometrically by co-incubation with unlabeled phalloidin. Increases in stretch-induced fluorescence were blocked by preincubation of cells with cytochalasin D. Compared to baseline values, F-actin in gingival fibroblasts was reduced by 50% at 10 s after stretching but was increased more than 100% at 50 s after stretching. Increases of F-actin were also dependent on the amount of deformation of membrane: 3.3% stretching produced the largest increase and 9.2% stretching produced the least. Stretch-activated actin polymerization was blocked by pre-incubation of cells with 500 ng ml-1 pertussis toxin or 10 mM EGTA. Compared to gingival cells, periodontal ligament fibroblasts exhibited twice the total actin per cell and three times the baseline F-actin per mm2, however, there was no increase in fluorescence after stretching. In both cell types, stretching caused increased ruffling activity and no detectable shape changes at 60 s, but at 180 s the cells shortened and pseudopods retracted. The microfilament system of fibroblasts exhibits rapid dynamic responses to mechanical deformation prior to cell shape changes and appears to be dependent on the cell type.

Actins↗

PubMed1992

Effects of phenobarbital on leukocyte activation: membrane potential, actin polymerization, chemotaxis, respiratory burst, cytokine production, and lymphocyte proliferation.

Leukocyte activation is known to involve cell membrane potential changes. Phenobarbital, an anesthetic and anticonvulsant that can inhibit neuronal membrane depolarization, may also affect leukocyte activation. Measuring membrane potential, actin polymerization, chemotaxis, superoxide production, lymphocyte proliferation, intracellular calcium concentration, and cytokine production, we found that phenobarbital at a concentration of 15-30 micrograms/ml, which is considered a therapeutic serum level for controlling seizures, did not affect polymorphonuclear neutrophil (PMN) activation. At levels higher than 100 micrograms/ml, phenobarbital significantly suppressed formylmethionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis. Concentrations greater than 300 micrograms/ml also inhibited phorbol myristate acetate-stimulated membrane potential change. In contrast, 30 micrograms/ml phenobarbital significantly inhibited lymphocyte proliferation stimulated by phytohemagglutinin (PHA) and pokeweed mitogen. This concentration of phenobarbital also suppressed the increase of intracellular free calcium induced by PHA. However, only a higher concentration of phenobarbital (300 micrograms/ml) was able to inhibit PHA-induced interleukin-2 (IL-2) production and suppress the proliferation of PHA-induced IL-2 receptor-bearing lymphocytes. These results suggest that concentrations of phenobarbital associated with anticonvulsive levels do not affect PMN activation but suppress lymphocyte activation, possibly by affecting intracellular signal transduction.

Actins↗

PubMed2005

Antibody against the carboxyl terminus of intimin alpha reduces enteropathogenic Escherichia coli adherence to tissue culture cells and subsequent induction of actin polymerization.

The C-terminal third of intimin binds to its translocated receptor (Tir) to promote attaching and effacing lesion formation during infection with enteropathogenic Escherichia coli (EPEC). We observed that the adherence of EPEC strains to HEp-2 cells was reduced and that actin polymerization was blocked by antibody raised against the C-terminal third of intimin alpha.

Actins↗

PubMed1986

Ornithine decarboxylase activity and actin polymerization in testosterone--stimulated mouse kidney.

Renal hypertrophy was induced in mice by injection of testosterone. Associated with an increase in renal tissue mass were increases in the concentrations of spermine and spermidine and in the activity of ornithine decarboxylase (ODC), the rate limiting enzyme in their synthesis. The increased activity of ODC was shown to be due to an increase in tissue ODC content. Total tissue actin was not altered by testosterone treatment although the proportion of unpolymerized (soluble) actin was increased significantly. These data are discussed in relation to the postulated mechanism of polyamine stimulation of actin polymerization.

Actins↗

PubMed1986

Effect of temperature on the mechanism of actin polymerization.

The rate of the Mg2+-induced polymerization of rabbit skeletal muscle G-actin has been measured as as function of temperature at pH 8 by using various concentrations of Mg2+, Ca2+, and G-actin. A polymerization mechanism similar to that proposed at this pH [Frieden, C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6513-6517] was found to fit the data from 10 to 35 degrees C. From the kinetic data, no evidence for actin filament fragmentation was found at any temperature. Dimer formation is the most temperature-sensitive step, with the ratio of forward and reverse rate constants changing 4 orders of magnitude from 10 to 35 degrees C. Over this temperature change, all other ratios of forward and reverse rate constants change 7-fold or less, and the critical concentration remains nearly constant. The reversible Mg2+-induced isomerization of G-actin monomer occurs to a greater extent with increasing temperature, measured either by using N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled actin or by simulation of the full-time course of the polymerization reaction. This is partially due to Mg2+ binding becoming tighter, and Ca2+ binding becoming weaker, with increasing temperature. Elongation rates from the filament-pointed end, determined by using actin nucleated by plasma gelsolin, show a temperature dependence slightly larger than that expected for a diffusion-limited reaction.

Actins↗