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Results for “actin polymerization”

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PubMed2006

Phosphorylation of WAVE1 regulates actin polymerization and dendritic spine morphology.

WAVE1--the Wiskott-Aldrich syndrome protein (WASP)--family verprolin homologous protein 1--is a key regulator of actin-dependent morphological processes in mammals, through its ability to activate the actin-related protein (Arp2/3) complex. Here we show that WAVE1 is phosphorylated at multiple sites by cyclin-dependent kinase 5 (Cdk5) both in vitro and in intact mouse neurons. Phosphorylation of WAVE1 by Cdk5 inhibits its ability to regulate Arp2/3 complex-dependent actin polymerization. Loss of WAVE1 function in vivo or in cultured neurons results in a decrease in mature dendritic spines. Expression of a dephosphorylation-mimic mutant of WAVE1 reverses this loss of WAVE1 function in spine morphology, but expression of a phosphorylation-mimic mutant does not. Cyclic AMP (cAMP) signalling reduces phosphorylation of the Cdk5 sites in WAVE1, and increases spine density in a WAVE1-dependent manner. Our data suggest that phosphorylation/dephosphorylation of WAVE1 in neurons has an important role in the formation of the filamentous actin cytoskeleton, and thus in the regulation of dendritic spine morphology.

Actins↗

PubMed2002

Phosphorylation of tyrosine 291 enhances the ability of WASp to stimulate actin polymerization and filopodium formation. Wiskott-Aldrich Syndrome protein.

Wiskott-Aldrich Syndrome protein (WASp) is a key regulator of the Arp2/3 complex and the actin cytoskeleton in hematopoietic cells. WASp is capable of forming an auto-inhibited conformation, which can be disrupted by binding of Cdc42 and phosphatidylinositol 4,5-bisphosphate, leading to its activation. Stimulation of the collagen receptor on platelets and crosslinking the B-cell receptor induce tyrosine phosphorylation of WASp. Here we show that the Src family kinase Hck induces phosphorylation of WASp-Tyr(291) independently of Cdc42 and that this causes a shift in the mobility of WASp upon SDS-PAGE. A phospho-mimicking mutant, WASp-Y291E, exhibited an enhanced ability to stimulate actin polymerization in a cell-free system and when microinjected into primary macrophages induced extensive filopodium formation with greater efficiency than wild-type WASp or a Y291F mutant. We propose that phosphorylation of Tyr(291) directly regulates WASp function.

Actins↗

PubMed2006

Pasteurella multocida toxin (PMT) activates RhoGTPases, induces actin polymerization and inhibits migration of human dendritic cells, but does not influence macropinocytosis.

Dendritic cells (DCs) are considered as one of the principal initiators of immune responses. In their immature state, they migrate into peripheral tissue in order to uptake antigen and to patrol for danger signals. Upon maturation, they acquire the ability to migrate to the lymph nodes and present the captured antigens to T cells in order to direct the development of specific immune responses. There is evidence that microbial compounds interfere with proper functions of DCs in order to block innate and specific immunity. Here we characterized the influence of Pasteurella multocida toxin (PMT) on monocyte-derived DCs. Using pull-down assays with recombinant rhotekin or p21-activated kinase, we demonstrated the activation of RhoGTPases by PMT in DCs. Moreover, PMT induced changes in DC morphology and actin polymerization, impaired chemotaxin-induced actin re-organization and inhibited their migration response. However, macropinocytosis was not influenced by PMT. In summary, these data indicate that PMT inhibits proper function of the motility machinery in DCs, which might limit the development of adaptive immune surveillance during infection with Pasteurella multocida.

Actins↗

PubMed2006

Dynamics and morphology of microvilli driven by actin polymerization.

Many different cell types have dynamic protrusions, called microvilli, on their surface. We model these structures as arising from the balance between the force of actin polymerization and the restoring force of the membrane. From this simple model we calculate the distribution function of microvilli heights for several cells. We further describe the phase diagram and the resulting morphology of the microvilli aggregates on the cell surface.

Actins↗

PubMed2001

Nck and phosphatidylinositol 4,5-bisphosphate synergistically activate actin polymerization through the N-WASP-Arp2/3 pathway.

The Wiskott-Aldrich syndrome protein (WASP) and its relative neural WASP (N-WASP) regulate the nucleation of actin filaments through their interaction with the Arp2/3 complex and are regulated in turn by binding to GTP-bound Cdc42 and phosphatidylinositol 4,5-bisphosphate. The Nck Src homology (SH) 2/3 adaptor binds via its SH3 domains to a proline-rich region on WASP and N-WASP and has been implicated in recruitment of these proteins to sites of tyrosine phosphorylation. We show here that Nck SH3 domains dramatically stimulate the rate of nucleation of actin filaments by purified N-WASP in the presence of Arp2/3 in vitro. All three Nck SH3 domains are required for maximal activation. Nck-stimulated actin nucleation by N-WASP.Arp2/3 complexes is further stimulated by phosphatidylinositol 4,5-bisphosphate, but not by GTP-Cdc42, suggesting that Nck and Cdc42 activate N-WASP by redundant mechanisms. These results suggest the existence of an Nck-dependent, Cdc42-independent mechanism to induce actin polymerization at tyrosine-phosphorylated Nck binding sites.

Actin-Related Protein 2↗

PubMed1993

Location of profilin at presynaptic sites in the cerebellar cortex; implication for the regulation of the actin-polymerization state during axonal elongation and synaptogenesis.

Profilin is a 15 kDa protein that binds actin monomers and inhibits their polymerization in vitro. The actin-profilin complex can be rapidly dissociated in vitro by phosphatidylinositol-4,5-bis-phosphate, providing a mechanism for regulating actin assembly-disassembly cycles during cell motile events. We have used a polyclonal antibody to calf spleen profilin to analyse the developmental expression and cellular distribution of profilin in the rat cerebellum and cultured cortical neurons. Immature neurons contain large amount of profilin both in vivo and in vitro. Immunofluorescence showed it to be present in developing neurites and growth cones but not in the filopodia of cortical neurons in culture. Profilin immunoreactivity was intense in the parallel fibres, the granule cell axons of the cerebellar cortex, at the time when they are elongating. Purkinje cell dendrites were not labelled. Profilin immunostaining was present in presynaptic varicosities, but not in dendritic spines within the molecular layer of juvenile and adult rats. The profilin concentration was higher in synaptosomes than in the total cerebellum during the second and third postnatal weeks, a period of intense synaptogenesis. Thus, profilin may help regulate actin polymerization and depolymerization during axonal elongation and synaptogenesis. Its restriction to the presynaptic site in the adult suggests that it may also be involved in the regulation of the release of synaptic vesicles.

Actins↗

PubMed1994

Two-step mechanism for actin polymerization in human erythroleukemia cells induced by phorbol ester.

Human erythroleukemia (HEL) cells grow in suspension, but after treatment with nM PMA the cells adhere and spread on glass or fibronectin [Jarvinen et al., 1987: Eur. J. Cell Biol. 44:238-246]. We observed an early (20-30 min) stage of spreading in which F-actin was organized into peripheral arcs near the spreading margin and vinculin was localised to the cell's periphery at the ends of these arcs. By 1 h the cells were well spread with straight actin bundles many of which ended at more central sites terminating on patches containing vinculin and talin; thus the cells assemble typical stress fibers but do not appear to polarize. The cells also spread on RGD polymer. DiC8 (1,2-dioctanoyl-sn-glycerol, C8:0, Sigma Chemical Co., St. Louis, MO) induced spreading but only if DAG kinase inhibitor and A-23187 were also present; in their absence cells adhered but did not spread. Spreading was approximately 85% inhibited by 100 nM staurosporine. PKC-beta was shown to be present in the cells by immunoblotting. In cells spread for 1 h with PMA, F-actin increased to 180% of control levels as measured by RP binding and the actin sequestering complex of G-actin-thymosin beta 4 decreased significantly. To determine whether the F-actin increase required adhesion, we inhibited cell attachment to the substratum by adding RGDS, by coating glass surfaces with hemoglobin, or by a combined treatment. Under these conditions PMA-treated suspended cells still increased their F-actin to 126-137% of controls, a significant increase over control levels. Staurosporine inhibited F-actin increases under all the conditions studied. Permeabilized cell suspensions, incubated with rhodamine labelled G-actin, incorporated the labelled actin along cell membranes at a low level. A few minutes preincubation with either diC8 plus DAG kinase inhibitor or with PMA strongly increased the incorporation. This increased incorporation was reduced to below control levels by either staurosporine (100 nM) or cytochalasin D (1 microM). We conclude that both suspended and spreading HEL cells can be stimulated to polymerize actin by a mechanism dependent on PKC or a PKC-like molecule. In suspended cells, the polymerization occurs along the membrane. When cells spread, F-actin increased to a significantly greater extent. This second step could involve additional polymerization, perhaps at the observed adhesion sites, decreased turnover of the actin bundles, or a combined effect of both mechanisms.

Actins↗

PubMed2006

Phosphoinositides and Rho proteins spatially regulate actin polymerization to initiate and maintain directed movement in a one-dimensional model of a motile cell.

Gradient sensing, polarization, and chemotaxis of motile cells involve the actin cytoskeleton, and regulatory modules, including the phosphoinositides (PIs), their kinases/phosphatases, and small GTPases (Rho proteins). Here we model their individual components (PIP1, PIP2, PIP3; PTEN, PI3K, PI5K; Cdc42, Rac, Rho; Arp2/3, and actin), their interconversions, interactions, and modular functions in the context of a one-dimensional dynamic model for protrusive cell motility, with parameter values derived from in vitro and in vivo studies. In response to a spatially graded stimulus, the model produces stable amplified internal profiles of regulatory components, and initiates persistent motility (consistent with experimental observations). By connecting the modules, we find that Rho GTPases work as a spatial switch, and that the PIs filter noise, and define the front versus back. Relatively fast PI diffusion also leads to selection of a unique pattern of Rho distribution from a collection of possible patterns. We use the model to explore the importance of specific hypothesized interactions, to explore mutant phenotypes, and to study the role of actin polymerization in the maintenance of the PI asymmetry. We also suggest a mechanism to explain the spatial exclusion of Cdc42 and PTEN and the inability of cells lacking active Cdc42 to properly detect chemoattractant gradients.

Actins↗

PubMed2006

A steric antagonism of actin polymerization by a salmonella virulence protein.

Salmonella spp. require the ADP-ribosyltransferase activity of the SpvB protein for intracellular growth and systemic virulence. SpvB covalently modifies actin, causing cytoskeletal disruption and apoptosis. We report here the crystal structure of the catalytic domain of SpvB, and we show by mass spectrometric analysis that SpvB modifies actin at Arg177, inhibiting its ATPase activity. We also describe two crystal structures of SpvB-modified, polymerization-deficient actin. These structures reveal that ADP-ribosylation does not lead to dramatic conformational changes in actin, suggesting a model in which this large family of toxins inhibits actin polymerization primarily through steric disruption of intrafilament contacts.

ADP Ribose Transferases↗

PubMed1981

Cell beta-actinin, an accelerator of actin polymerization, isolated from rat kidney cytosol.

A motility-regulatory protein called cell beta-actinin was isolated from rat kidney cytosol. Although its molecular weight (approximately 80,000) is different from that of beta-actinin from skeletal muscle (heterodimer of 34,000 and 37,000), the action on actin is the same for both. Cell beta-actinin accelerates polymerization of actin by enhanced nucleation resulting in shorter actin filaments, inhibits the reassociation of sonicated F-actin fragments, and forms a Mg polymer in the presence of Mg ions. Cell beta-actinin appears to be present in various tissues of rat, rabbit, chicken, and pig. Cell beta-actinin is thought to function as an accelerator of actin polymerization in nonmuscle cells, in which actin monomers are abundantly present.

Actinin↗

PubMed1996

Phosphatidylserine directs differential phosphorylation of actin and glyceraldehyde-3-phosphate dehydrogenase by protein kinase C: possible implications for regulation of actin polymerization.

The phospholipid-dependent protein kinase C is implicated in the regulation of cellular motility and energy metabolism. Phosphatidylserine, a main cofactor of protein kinase C, is involved in the regulation of glyceraldhehyde-3-phosphate dehydrogenase, which as actin, was shown to be phosphorylated by purified protein kinase C. Here, we study the effect of phosphatidylserine on the enzyme-substrate interaction of protein kinase C with glyceraldhehyde-3-phosphate dehydrogenase and actin. The stoichiometry of glyceraldhehyde-3-phosphate dehydrogenase phosphorylation is not affected by varying the level of phosphatidylserine. However, actin phosphorylation is dependent on phosphatidylserine level, peaking at high phosphatidylserine concentration. Moreover, if actin and glyceraldhehyde-3-phosphate dehydrogenase are cophosphorylated at high phosphatidylserine concentration, actin phosphorylation is favored, despite lower affinity for protein kinase C. Hence, phosphatidylserine directs differential phosphorylation of these key proteins of glycolysis and cellular motility and might be capable of recruiting protein kinase C for preferential actin phosphorylation. The sedimentation of phosphorylated actin is increased 3.8 fold and total actin 1.7 fold, suggesting that phosphorylation promotes actin polymerization.

Actins↗

PubMed1993

B-50/GAP-43 binds to actin filaments without affecting actin polymerization and filament organization.

To investigate a possible function of the nervous tissue-specific protein kinase C substrate B-50/GAP-43 in regulation of the dynamics of the submembranous cytoskeleton, we studied the interaction between purified B-50 and actin. Both the phosphorylated and dephosphorylated forms of B-50 cosedimented with filamentous actin (F-actin) in a Ca(2+)-independent manner. Neither B-50 nor phospho-B-50 had any effect on the kinetics of actin polymerization and on the critical concentration at steady state, as measured using pyrenylated actin. Light scattering of F-actin samples was not increased in the presence of B-50, suggesting that B-50 does not bundle actin filaments. The number of actin filaments, determined by [3H]cytochalasin B binding, was not affected by either phospho- or dephospho-B-50, indicating that B-50 has neither a severing nor a capping effect. These observations were confirmed by electron microscopic evaluation of negatively stained F-actin samples, which did not reveal any structural changes in the actin meshwork on addition of B-50. We conclude that B-50 is an actin-binding protein that does not directly affect actin dynamics.

Actins↗

PubMed2002

Signaling through integrin LFA-1 leads to filamentous actin polymerization and remodeling, resulting in enhanced T cell adhesion.

The integrins can activate signaling pathways, but the final downstream outcome of these pathways is often unclear. This study analyzes the consequences of signaling events initiated by the interaction of the leukocyte integrin LFA-1 with its ligand, dimeric ICAM-1. We show that the active form of LFA-1 regulates its own function on primary human T cells by directing the remodeling of the F-actin cytoskeleton to strengthen T cell adhesion to ICAM-1. Confocal microscopy revealed that both F-actin bundling and overall levels of F-actin are increased in the ICAM-1-adhering T cells. This increase in F-actin levels and change in F-actin distribution was quantitated for large numbers of T cells using the technique of laser scanning cytometry and was found to be significant. The study went on to show that clustering of conformationally altered LFA-1 is essential for the changes in F-actin, and a model is proposed in which clustered, high-avidity T cell LFA-1, interacting with multivalent ICAM-1, causes LFA-1 signaling, which results in F-actin polymerization and higher-order F-actin bundling. The findings demonstrate that LFA-1 acts not only as an adhesion receptor but also as a signaling receptor by actively initiating the F-actin reorganization that is essential for many T cell-dependent processes.

Actin Cytoskeleton↗

PubMed1992

Thymosin beta 4 (Fx peptide) is a potent regulator of actin polymerization in living cells.

Thymosin beta 4 (beta 4) is a 5-kDa polypeptide originally identified in calf thymus. Although numerous activities have been attributed to beta 4, its physiological role remains elusive. Recently, beta 4 was found to bind actin in human platelet extracts and to inhibit actin polymerization in vitro, raising the possibility that it may be a physiological regulator of actin assembly. To examine this potential function, we have increased the cellular beta 4 concentration by microinjecting synthetic beta 4 into living epithelial cells and fibroblasts. The injection induced a diminution of stress fibers and a dose-dependent depolymerization of actin filaments as indicated by quantitative image analysis of phalloidin binding. Our results show that beta 4 is a potent regulator of actin assembly in living cells.

3T3 Cells↗

PubMed1991

The regulation of actin polymerization in differentiating U937 cells correlates with increased membrane levels of the pertussis-toxin-sensitive G-protein Gi2.

Undifferentiated U937 cells appear to lack a capacity of increase cellular F-actin. However, electropermeabilized cells gain the ability to respond in this way to a guanine nucleotide analogue, guanosine 5'-[gamma-thio]trisphosphate (GTP[S]) after 1 h of treatment with dibutyryl cyclic AMP (db-cAMP). The results reported here show that the levels of membrane association of the G-protein Gi2 alpha increase with a time course identical with that of the GTP[S]-sensitivity of electropermeabilized cells. These results suggest that Gi2 alpha may be involved in the signal-transduction pathway leading to actin polymerization in db-cAMP-differentiated U937 cells.

Actins↗

PubMed1984

Fluorescence energy transfer measurements between the nucleotide binding site and Cys-373 in actin and their application to the kinetics of actin polymerization.

Intramonomer fluorescence energy transfer between the donor epsilon-ATP bound to the nucleotide-binding site and the acceptor 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole bound to Cys-373 in G-actin was measured by steady-state fluorimetry. Assuming for the orientation factor its dynamic limit K2 = 2/3, the donor and acceptor distance in a G-actin molecule was calculated to be about 3 nm. The intermonomer energy transfer in F-actin occurring between the donor bound to an actin monomer and the acceptor bound to the nearest-neighbour actin monomer was also measured and the distance was calculated to be about 4 nm. The kinetics of the actin polymerization process was studied by following the decrease in fluorescence intensity upon addition of salts to G-actin solution. The initial velocity of the fluorescence intensity change was proportional to the square of the initial G-actin concentration. The temperature dependence of the velocity was proportional to the square of the initial G-actin concentration. The temperature dependence of the velocity was proportional to exp(-10/RT). These results indicated that the initial fluorescence intensity change corresponds to monomer-dimer transformation and its activation enthalpy was 10 kcal/mol.

4-Chloro-7-nitrobenzofurazan↗

PubMed1992

Actin-dependent myoid elongation in teleost rod inner/outer segments occurs in the absence of net actin polymerization.

In the retinas of teleost fish, rod photoreceptors elongate in response to light. Light-activated elongation is mediated by the myoid of the rod inner segment and is actin-dependent. Inner segment F-actin filaments form bundles running parallel to the cell's long axis. We examined the mechanism of rod elongation using mechanically-detached rod fragments, consisting of the motile inner segment and sensory outer segment (RIS-ROS). When RIS-ROS are isolated from dark-adapted green sunfish and cultured in the light, they elongate 15 microns at 0.3-0.6 microns/min. Elongation was inhibited 65% by 0.1 microM Cytochalasin D, suggesting a requirement for actin assembly. To determine the extent of assembly during elongation, we used three approaches to measure the F-actin content in RIS-ROS: detection of pelletable actin by SDS-PAGE after detergent-extraction of RIS-ROS; quantification of fluorescein-phalloidin binding by fluorimetry, fluorescence-activated cell sorting and image analysis; estimation of total F-actin filament length by electron microscopy. All three assays indicated that no net assembly of RIS-ROS F-actin accompanied myoid elongation. An increase in F-actin content within the elongated myoid was counterbalanced by a decrease in F-actin content within the 13 microvillus-like calycal processes located at the end of the inner segment opposite to the growing myoid. O'Connor and Burnside (Journal of Cell Biology 89:517-524, 1981) showed that minus-ends of rod F-actin filaments are oriented towards the elongating myoid while plus-ends are oriented towards the shortening calycal processes. Our observations suggest that RIS-ROS elongation entails actin polymerization at the minus-ends of filaments coupled with depolymerization at the filament plus-ends.

Actin Cytoskeleton↗

PubMed1997

Evidence for genetic control of changes in f-actin polymerization caused by pathogenic microorganisms: in vitro assessment using gingival fibroblasts from human twins.

Attachment to and migration upon a substratum, as well as other functions of connective tissue cells, are regulated mainly by cytoplasmic structural proteins, particularly filamentous actin (f-actin). Pathogenic microorganisms exert negative effects on cytoskeletal proteins. In the present study, normal gingival fibroblasts from 10 sets of human twins (6 fraternal, DZ; 4 identical, MZ) were exposed to soluble extracts from Porphyromonas gingivalis or Fusobacterium nucleatum, then f-actin was stained using FITC-labeled phalloidin. Cells were examined under fluorescence, and a computer-assisted image analyzer quantitated f-actin polymerization as fluorescence intensity on a per-cell basis. Intraclass correlation coefficients for f-actin in MZ/MZ vis-a-vis DZ/DZ paired cell cultures were determined to assess the possible heritability of responses to the microorganism preparations. F-actin labeling was significantly different between control cultures and those exposed to the extracts. Both F. nucleatum and P. gingivalis effected f-actin and fibroblast morphology. When the data were adjusted for gender and age effects, and for differences in control f-actin levels, fibroblasts from MZ twin pairs were moderately similar in both absolute and relative responses to bacterial challenges; cells from DZ twins showed little similarity when response was measured on the absolute scale, and moderate similarity using the relative scale.

Actins↗