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PubMed2003

Phosphorylation of the WASP-VCA domain increases its affinity for the Arp2/3 complex and enhances actin polymerization by WASP.

Wiskott-Aldrich syndrome protein (WASP) and neural (N)-WASP regulate dynamic actin structures through the ability of their VCA domains to bind to and stimulate the actin nucleating activity of the Arp2/3 complex. Here we identify two phosphorylation sites in the VCA domain of WASP at serines 483 and 484. S483 and S484 are substrates for casein kinase 2 in vitro and in vivo. Phosphorylation of these residues increases the affinity of the VCA domain for the Arp2/3 complex 7-fold and is required for efficient in vitro actin polymerization by the full-length WASP molecule. We propose that constitutive VCA domain phosphorylation is required for optimal stimulation of the Arp2/3 complex by WASP.

3T3 Cells↗

PubMed2002

The diaphanous-related formin mDia1 controls serum response factor activity through its effects on actin polymerization.

SRF-dependent transcription is regulated by the small GTPase RhoA via its effects on actin dynamics. The diaphanous-related formin (DRF) proteins have been identified as candidate RhoA effectors mediating signaling to SRF. Here we investigate the relationship between SRF activation and actin polymerization by the DRF mDia1. We show that the ability of mDia1 to potentiate SRF activity is strictly correlated with its ability to promote F-actin assembly. Both processes can occur independently of the mDia1 FH1 domain but require sequences in an extended C-terminal region encompassing the conserved FH2 domain. mDia-mediated SRF activation, but not F-actin assembly, can be blocked by a nonpolymerizable actin mutant, placing actin downstream of mDia in the signal pathway. The SRF activation assay was used to identify inactive mDia1 derivatives that inhibit serum- and LPA-induced signaling to SRF. We show that these interfering mutants also block F-actin assembly, whether induced by mDia proteins or extracellular signals. These results identify novel functional elements of mDia1 and show that it regulates SRF activity by inducing depletion of the cellular pool of G-actin.

3T3 Cells↗

PubMed1999

Influence of the C terminus of Wiskott-Aldrich syndrome protein (WASp) and the Arp2/3 complex on actin polymerization.

The 70 C-terminal amino acids of Wiskott-Aldrich syndrome protein (WASp WA) activate the actin nucleation activity of the Arp2/3 complex. WASp WA binds both the Arp2/3 complex and actin monomers, but the mechanism by which it activates the Arp2/3 complex is not known. We characterized the effect of WASp WA on actin polymerization in the absence and presence of the human Arp2/3 complex. WASp WA binds actin monomers with an apparent K(d) of 0.4 microM, inhibiting spontaneous nucleation and subunit addition to pointed ends, but not addition to barbed ends. A peptide containing only the WASp homology 2 motif behaves similarly but with a 10-fold lower affinity. In contrast to previously published results, neither WASp WA nor a similar region of the protein Scar1 significantly depolymerizes actin filaments under a variety of conditions. WASp WA and the Arp2/3 complex nucleate actin filaments, and the rate of this nucleation is a function of the concentrations of both WASp WA and the Arp2/3 complex. With excess WASp WA and <10 nM Arp2/3 complex, there is a 1:1 correspondence between the Arp2/3 complex and the concentration of filaments produced, but the filament concentration plateaus at an Arp2/3 complex concentration far below the cellular concentration determined to be 9.7 microM in human neutrophils. Preformed filaments increase the rate of nucleation by WASp WA and the Arp2/3 complex but not the number of filaments that are generated. We propose that filament side binding by the Arp2/3 complex enhances its activation by WASp WA.

Actin Cytoskeleton↗

PubMed1999

GCS1, an Arf guanosine triphosphatase-activating protein in Saccharomyces cerevisiae, is required for normal actin cytoskeletal organization in vivo and stimulates actin polymerization in vitro.

Recent cloning of a rat brain phosphatidylinositol 3,4, 5-trisphosphate binding protein, centaurin alpha, identified a novel gene family based on homology to an amino-terminal zinc-binding domain. In Saccharomyces cerevisiae, the protein with the highest homology to centaurin alpha is Gcs1p, the product of the GCS1 gene. GCS1 was originally identified as a gene conditionally required for the reentry of cells into the cell cycle after stationary phase growth. Gcs1p was previously characterized as a guanosine triphosphatase-activating protein for the small guanosine triphosphatase Arf1, and gcs1 mutants displayed vesicle-trafficking defects. Here, we have shown that similar to centaurin alpha, recombinant Gcs1p bound phosphoinositide-based affinity resins with high affinity and specificity. A novel GCS1 disruption strain (gcs1Delta) exhibited morphological defects, as well as mislocalization of cortical actin patches. gcs1Delta was hypersensitive to the actin monomer-sequestering drug, latrunculin-B. Synthetic lethality was observed between null alleles of GCS1 and SLA2, the gene encoding a protein involved in stabilization of the actin cytoskeleton. In addition, synthetic growth defects were observed between null alleles of GCS1 and SAC6, the gene encoding the yeast fimbrin homologue. Recombinant Gcs1p bound to actin filaments, stimulated actin polymerization, and inhibited actin depolymerization in vitro. These data provide in vivo and in vitro evidence that Gcs1p interacts directly with the actin cytoskeleton in S. cerevisiae.

ADP-Ribosylation Factor 1↗

PubMed1987

Nucleation of actin polymerization by villin and elongation at subcritical monomer concentration.

We have obtained a quantitative description of villin-nucleated actin polymerization in physiological salt by determining the concentrations of free villin (V), villin-actin monomer (VA), villin-actin dimer (VA2), and villin-actin oligomer (VAn). Over a range of actin-villin ratios from 0.1 to 20 we determined the concentration of actin-bound villin by measuring the low-intensity pyrenylactin fluorescence of the two terminal actins in each villin-actin polymer. (To this end we first showed that each villin-actin oligomer and polymer contains two low-intensity pyrenylactin molecules.) We determined the concentration of free villin using a calibrated cutting activity assay. The pattern of increase in bound villin together with the pattern of increase in high-intensity pyrenylactin fluorescence with increasing G-actin concentration indicated, first, that villin-actin monomers were not formed at detectable levels even at a 12-fold villin excess over actin. Second, there was no stoichiometric villin-actin dimer formation at actin-villin ratios of 2. Instead there was an equilibrium between free villin, VA2, and VAn. Defining K1 = [VA]/[V][A] and K2 = [VA2]/[VA][A], a good fit of the data was obtained with K1 much less than K2 and a value of K1K2 = Kv = 10(12)-10(13) M-2 = [VA2]/[V][A]2, i.e., 1/Kv1/2 = (0.3-1) X 10(-6) M. We have assumed here that the monomer binding constant of VA2 to form VA3 was equal to the monomer binding constant of pointed filament ends, K infinity = 1/c infinity, obtained as described below.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed2000

Directed actin polymerization is the driving force for epithelial cell-cell adhesion.

We have found that epithelial cells engage in a process of cadherin-mediated intercellular adhesion that utilizes calcium and actin polymerization in unexpected ways. Calcium stimulates filopodia, which penetrate and embed into neighboring cells. E-cadherin complexes cluster at filopodia tips, generating a two-rowed zipper of embedded puncta. Opposing cell surfaces are clamped by desmosomes, while vinculin, zyxin, VASP, and Mena are recruited to adhesion zippers by a mechanism that requires alpha-catenin. Actin reorganizes and polymerizes to merge puncta into a single row and seal cell borders. In keratinocytes either null for alpha-catenin or blocked in VASP/Mena function, filopodia embed, but actin reorganization/polymerization is prevented, and membranes cannot seal. Taken together, a dynamic mechanism for intercellular adhesion is unveiled involving calcium-activated filopodia penetration and VASP/Mena-dependent actin reorganization/polymerization.

Actins↗

PubMed2003

Salmonella SipA polymerizes actin by stapling filaments with nonglobular protein arms.

Like many bacterial pathogens, Salmonella spp. use a type III secretion system to inject virulence proteins into host cells. The Salmonella invasion protein A (SipA) binds host actin, enhances its polymerization near adherent extracellular bacteria, and contributes to cytoskeletal rearrangements that internalize the pathogen. By combining x-ray crystallography of SipA with electron microscopy and image analysis of SipA-actin filaments, we show that SipA functions as a "molecular staple," in which a globular domain and two nonglobular "arms" mechanically stabilize the filament by tethering actin subunits in opposing strands. Deletion analysis of the tethering arms provides strong support for this model.

Actin Cytoskeleton↗

PubMed1990

Involvement of GTP-binding proteins in actin polymerization in human neutrophils.

The motility of human neutrophils, which is of vital importance for the role of these cells in host defense, is based on rapid and dynamic changes of the filamentous actin F-actin) network. Consequently, to understand how neutrophils move and ingest particles, we need to know how polymerization and depolymerization of actin are regulated. Previous studies by several investigators have, based on indirect evidence obtained with pertussis toxin, suggested a role for GTP-binding protein(s) (G protein) in chemotaxis-induced, but not phagocytosis-induced, reorganization of the F-actin network. The aim of the present investigation was to study the effects of directly activated G proteins (i.e., without prior ligand-receptor complex formation) on the F-actin content in human neutrophils. AlF4- induced a pronounced and sustained increase in F-actin in intact neutrophils. This effect coincided with an increase in cytosolic free Ca2+, indicating that phospholipase C and the subsequent transduction mechanism were also activated. Inhibition of phospholipase C activity by extensive depression of the cytosolic free Ca2+ level (less than 20 nM) only marginally affected the AlF4(-)-induced rise in F-actin content. The major part of the AlF4(-)-induced rise in F-actin content was also resistant to pertussis toxin, suggesting that pertussis toxin-insensitive G proteins in neutrophils are also able to trigger actin polymerization. The specificity of AlF4- in activating G proteins was also tested in permeabilized cells. In this case the effect was more rapid and could be totally abolished by guanosine 5'-[beta-thio]diphosphate. In analogy, in permeabilized cells guanosine 5'-[gamma-thio]triphosphate mimicked the effect of AlF4- on actin polymerization, and the effect induced by this nonhydrolyzable GTP analogue could also be totally abolished by guanosine 5'-[beta-thio]diphosphate. In summary, the present data support our previous hypothesis that G proteins are intimately linked to actin polymerization in human neutrophils.

Actins↗

PubMed1994

Actin polymerization and leukocyte function.

The coordinated remodeling of the filamentous actin-based microfilamentous cytoskeleton via regulated polymerization and depolymerization of globular and filamentous actin is required for polymorphonuclear leukocyte motile functions including locomotion, shape change, phagocytosis, and adhesion. Significant new observations on the structure and function of distinct filamentous actin pools in polymorphonuclear leukocytes, the mechanisms of chemotactic peptide-mediated actin polymerization, the role of filamentous actin in polymorphonuclear shape change in suspension and on a surface, the identification and characterization of rare patients with polymorphonuclear motile defects and actin dysfunctions, and regulation of actin reorganizations by actin regulatory proteins, the phosphorylation or dephosphorylation states of proteins, and the second messengers--the phosphoinositides--were reported in the past year. These observations form the basis for an improved understanding of the cellular and molecular role of actin assembly in polymorphonuclear function and are the subject of this review.

Actins↗

PubMed1983

Effect of Acanthamoeba profilin on the pre-steady state kinetics of actin polymerization and on the concentration of F-actin at steady state.

The interaction of Acanthamoeba actin and Acanthamoeba profilin was evaluated as a function of ionic conditions. In the presence of 2 mM MgCl2 or 1 mM MgCl2 and 50 mM KCl, profilin decreased the concentration of F-actin at steady state, and inhibited the rates of filament elongation and spontaneous nucleation and polymerization. All of the experimental data were quantitatively accounted for on the basis of a 1:1 complex between profilin and monomeric actin with a Kr between 4 and 9 microM, the same value obtained previously in the absence of MgCl2. Therefore, the Mg2+ concentration did not affect the KD of the profilin-actin complex in these experiments. On the other hand, profilin did greatly amplify the decrease in concentration of F-actin at steady state caused by lowering the Mg2+ concentration. This results from the effect of Mg2+ on the critical concentration of the actin monomer with which the profilin-actin complex is in equilibrium. When the Mg2+ concentration is lowered, the critical concentration of actin monomer increases so that more profilin-actin complex is formed. Thus, appreciably more F-actin depolymerizes than in the absence of profilin. In this way, profilin could function intracellularly to convert small changes in critical concentration into large changes in the concentration of F-actin.

Actins↗

PubMed1997

Phosphorylation-dependent effects of synapsin IIa on actin polymerization and network formation.

The synapsins are a family of synaptic vesicle phosphoproteins which play a key role in the regulation of neurotransmitter release and synapse formation. In the case of synapsin I, these biological properties have been attributed to its ability to interact with both synaptic vesicles and the actin-based cytoskeleton. Although synapsin II shares some of the biological properties of synapsin I, much less is known of its molecular properties. We have investigated the interactions of recombinant rat synapsin Ila with monomeric and filamentous actin and the sensitivity of those interactions to phosphorylation, and found that: i) dephosphorylated synapsin II stimulates actin polymerization by binding to actin monomers and forming actively elongating nuclei and by facilitating the spontaneous nucleation/elongation processes; ii) dephosphorylated synapsin II induces the formation of thick and ordered bundles of actin filaments with greater potency than synapsin I; iii) phosphorylation by protein kinase A markedly inhibits the ability of synapsin II to interact with both actin monomers and filaments. The results indicate that the interactions of synapsin II with actin are similar but not identical to those of synapsin I and suggest that synapsin II may play a major structural role in mature and developing nerve terminals, which is only partially overlapping with the role played by synapsin I.

Actins↗

PubMed2002

Two verprolin homology domains increase the Arp2/3 complex-mediated actin polymerization activities of N-WASP and WAVE1 C-terminal regions.

WASP family proteins induce actin polymerization through a C-terminal verprolin homology, cofilin homology, and acidic (VCA) region by activating the Arp2/3 complex. The N-WASP VCA region is the most potent activator of the Arp2/3 complex. In addition, full-length WAVE1 and a WAVE1 VCA fragment show differential activity. The mechanisms underlying these differences are poorly understood. We examined the activities of various N-WASP and WAVE1 VCA mutant proteins with several types of fusion moieties. When fused to GST, maltose-binding protein, or the WAVE1 proline-rich domain, N-WASP VCA and WAVE1 VCA mutant proteins with two V motifs showed stronger activities than wild-type WAVE1 VCA with one V motif, demonstrating the importance of two V motifs for strong VCA activity. A WAVE1 VCA fragment tagged with six histidines (His) showed markedly reduced activity compared to GST-fused VCA, whereas His-tagged N-WASP VCA showed similar activity to GST-fused VCA. An additional V motif failed to enhance WAVE1 VCA activity in the His-tagged form. Thus, the WAVE1 VCA fragment may exist in an unfavorable conformation to activate the Arp2/3 complex, implying the existence of a structural difference between WAVE1 and N-WASP VCAs in addition to the number of V motifs.

Actin-Related Protein 2↗

PubMed1996

Dynamic changes in the state of actin polymerization in human alveolar cells exposed to the oxidant agent paraquat.

To investigate the role of dynamic changes in actin cytoskeleton in cellular response to oxidative stress, we have analyzed the state of actin polymerization and synthesis in human alveolar cells exposed to paraquat, an oxidant agent. Cellular content of monomeric actin was assayed by DNase I inhibition. It decreased significantly in treated cells and depended on paraquat concentration. Paraquat treatment of cells caused an increase of the filamentous pool of actin and a parallel decrease of the monomeric one. Such shift was shown to be irreversible. SDS-PAGE of cytoskeletal fractions was performed under reducing and non-reducing conditions. No cross-linking of actin monomers to form large aggregates appeared to be related to the observed paraquat-induced increase of the filamentous actin pool. Morphological analyses by indirect immunofluorescence and ultrastructural examination confirmed the presence of microfilaments in treated cells. Conventional bundles of filaments were not observed, but numerous single filaments appeared dispersed within the cytoplasm. Pulse-chase experiments showed a strong increase of de novo synthesis of actin in treated cells, whereas actin degradation rate remained unaffected. In conclusion, the different approaches lead to a concordant picture of cellular response to oxidant stress at the level of the actin filament system. Actin pools are modified: the overall number of filaments increases, whereas the monomeric species decreases. As a result of the shift of actin from the monomeric pool to the filamentous one, the de novo synthesis of actin is increased.

Actins↗

PubMed1986

The distribution of polymerized actin in the rat egg and its sensitivity to cytochalasin B during fertilization.

The distribution of polymerized actin in rat eggs fertilized in vitro was determined using NBD-phallacidin (NBD-ph). Unfertilized and fertilized eggs exhibited a 3-5-micron-thick band of fluorescence that encompassed the entire cortical cytoplasm. There was no dramatic increase in the staining of the cortex in association with any component of the fertilizing sperm during its incorporation into the egg. Unfertilized eggs and fertilized eggs obtained at intervals after sperm-egg fusion were treated with cytochalasin B (CB; 5 micrograms/ml) and subsequently stained with NBD-ph. Unfertilized eggs treated with CB exhibited a continuous ring of cortical staining identical to that seen in untreated eggs. Eggs treated with CB 15 min after sperm-egg fusion exhibited small gaps in the cortical staining pattern, whereas those exposed to CB 1 hr after fusion exhibited larger gaps and the staining pattern appeared punctate. This pattern could be seen throughout the remainder of the 7 hr period of sperm incorporation and for at least 13 hr thereafter. CB-treated fertilized eggs that were washed to remove the drug again exhibited uninterrupted cortical staining on treatment with NBD-ph. CB also induced the resorption of surface elevations that are normally seen on the eggs during sperm incorporation, but it did not affect the morphology of unfertilized eggs. The sensitivity to CB during fertilization coincides with the onset of a variety of egg shape changes that occur during the period of sperm incorporation (Battaglia and Gaddum-Rosse, Gamete Res., 10:107-118, 1984a).(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1996

The end of a polymerizing actin filament contains numerous ATP-subunit segments that are disconnected by ADP-subunits resulting from ATP hydrolysis.

ATP hydrolysis by copolymers of ATP-actin and ADP-actin was investigated in order to analyze the effect of interfaces between ATP-subunits and ADP-subunits on hydrolysis of actin-bound ATP. Copolymers of ATP- and ADP-subunits were formed by polymerization of ATP- and ADP-actin monomers onto filaments. By changing the ratio of polymerizing ATP-actin monomers to ADP-actin monomers, the number of interfaces between ATP- and ADP-subunits and of ATP-subunits only surrounded by further ATP-subunits was varied. The rate of actin polymerization and of ATP hydrolysis was measured simultaneously on the same samples. The lag time between incorporation of actin monomers into filaments and subsequent ATP hydrolysis was found to be similar both for polymerized ATP-actin and for copolymers formed by various ratios of ATP- to ADP-actin. The experiments were performed in the presence of 1 mM MgCl2, 0.05 mM CaCl2, and 100 mM KCl or of 1 mM MgCl2, and 0.4 mM EGTA. The type of cations was found to have no major effect on the rate of ATP hydrolysis. A quantitative evaluation of the experimental data suggests that ATP at interfaces between ATP- and ADP-subunits is hydrolyzed not more than 10 times faster than ATP of subunits surrounded by further ATP-subunits. On the basis of these results, one can conclude that an actin filament onto which ATP-actin monoMers polymerize contains numerous segments of ATP-subunits that are disconnected by ADP-subunits resulting from ATP hydrolysis. The average length of the numerous ATP segments of a steadily polymerizing filament is in the range of 10 ATP-subunits or below.

Actins↗

PubMed1999

Effects of chlorpromazine on actin polymerization: slackening of filament elongation and filament annealing.

We have analyzed the effect of chlorpromazine (CPZ) on pure actin. We have found that CPZ quenches Trp-79 and Trp-86 fluorescence and, in agreement with an earlier report on conventional actin, inhibits actin polymerization, lowering the extent of polymerization. Moreover, novel polymerization data are presented indicating that CPZ decreases the maximum polymerization rate in a dose-dependent manner. The assembly inhibition results from the slackening of oligomer formation during the early stages of polymerisation, of filament elongation and of filament annealing. Finally, CPZ strongly inhibits actin filament network formation.

Actins↗

PubMed2006

Actin polymerization upon processive capping by formin: a model for slowing and acceleration.

Formin family proteins act as processive cappers of actin filaments, and determine the dynamics of a number of intracellular processes that are based on actin polymerization. The rate of filament growth upon processive capping varies within a broad range depending on the formin type and presence of profilin. While FH2 domains of various formins slow down polymerization by different extents, the FH1-FH2 domains in conjunction with profilin accelerate the reaction. Study of the physical mechanism of processive capping is vital for understanding the intracellular actin dynamics. We propose a model predicting that variation of a single physical parameter-the effective elastic energy of the formin-capped barbed end-results in the observed diversity of the polymerization rates. The model accounts for the whole range of the experimental results including the drastic slowing down of polymerization by FH2 of Cdc12 formin and the 4.5-fold acceleration of the reaction by FH1-FH2 of mDai1 formin in the presence of profilin. Fitting the theoretical predictions to the experimental curves provides the values of the effective elastic energies of different formin-barbed end complexes.

Actin Cytoskeleton↗

PubMed2002

Osmotic stress activates Rac and Cdc42 in neutrophils: role in hypertonicity-induced actin polymerization.

Hypertonicity inhibits a variety of neutrophil functions through poorly defined mechanisms. Our earlier studies suggest that osmotically induced actin polymerization and cytoskeleton remodeling is a key component in the hypertonic block of exocytosis and cell movement. To gain insight into the signaling mechanisms underlying the hyperosmotic F-actin response, we investigated whether hypertonicity stimulates Rac and Cdc42 and, if so, whether their activation contributes to the hypertonic rise in F-actin. Using a recently developed pull-down assay that specifically captures the active forms of these small GTPases, we found that hypertonicity caused an ~2.5- and ~7.2-fold activation of Rac and Cdc42, respectively. This response was rapid and sustained. Small GTPase activation was not mediated by the osmotic stimulation of Src kinases, heterotrimeric G proteins, or phosphatidylinositol 3-kinase. Interestingly, an increase in intracellular ionic strength was sufficient to activate Rac even in the absence of cell shrinkage. Inhibition of Rac and Cdc42 by Clostridium difficile toxin B substantially reduced but did not abolish the hypertonicity-induced F-actin response. Thus hypertonicity is a potent activator of Rac and Cdc42, and this effect seems to play an important but not exclusive role in the hyperosmolarity-triggered cytoskeleton remodeling.

Actins↗