Interaction of cortactin and N-WASp with Arp2/3 complex

BACKGROUND: Dynamic actin assembly is required for diverse cellular processes and often involves activation of Arp2/3 complex. Cortactin and N-WASp activate Arp2/3 complex, alone or in concert. Both cortactin and N-WASp contain an acidic (A) domain that is required for Arp2/3 complex binding. RESULTS: We investigated how cortactin and the constitutively active VCA domain of N-WASp interact with Arp2/3 complex. Structural studies showed that cortactin is a thin, elongated monomer. Chemical crosslinking studies demonstrated selective interaction of the Arp2/3 binding NTA domain of cortactin (cortactin NTA) with the Arp3 subunit and VCA with Arp3, Arp2, and ARPC1/p40. Cortactin NTA and VCA crosslinking to the Arp3 subunit were mutually exclusive; however, cortactin NTA did not inhibit VCA crosslinking to Arp2 or ARPC1/p40, nor did it inhibit activation of Arp2/3 complex by VCA. We conducted an experiment in which a saturating concentration of cortactin NTA modestly lowered the binding affinity of VCA for Arp2/3; the results of this experiment provided further evidence for ternary complex formation. Consistent with a common binding site on Arp3, a saturating concentration of VCA abolished binding of cortactin to Arp2/3 complex. CONCLUSIONS: Under certain circumstances, cortactin and N-WASp can bind simultaneously to Arp2/3 complex, accounting for their synergy in activation of actin assembly. The interaction of cortactin NTA with Arp2/3 complex does not inhibit Arp2/3 activation by N-WASp, despite competition for a common binding site located on the Arp3 subunit. These results suggest a model in which cortactin may bridge Arp2/3 complex to actin filaments via Arp3 and N-WASp activates Arp2/3 complex by binding Arp2 and/or ARPC1/p40.     

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.     

Sequential interaction of actin-related proteins 2 and 3 (Arp2/3) complex with neural Wiscott-Aldrich syndrome protein (N-WASP) and cortactin during branched actin filament network formation

The WASP and cortactin families constitute two distinct classes of Arp2/3 modulators in mammalian cells. Physical and functional interactions among the Arp2/3 complex, VCA (a functional domain of N-WASP), and cortactin were examined under conditions that were with or without actin polymerization. In the absence of actin, cortactin binds significantly weaker to the Arp2/3 complex than VCA. At concentrations of VCA 20-fold lower than cortactin, the association of cortactin with the Arp2/3 complex was nearly abolished. Analysis of the cells infected with Shigella demonstrated that N-WASP located at the tip of the bacterium, whereas cortactin accumulated in the comet tail. Interestingly, cortactin promotes Arp2/3 complex-mediated actin polymerization and actin branching in the presence of VCA at a saturating concentration, and cortactin acquired 20 nm affinity for the Arp2/3 complex during actin polymerization. The interaction of VCA with the Arp2/3 complex was reduced in the presence of both cortactin and actin. Moreover, VCA reduced its affinity for Arp2/3 complex at branching sites that were stabilized by phalloidin. These data imply a novel mechanism for the de novo assembly of a branched actin network that involves a coordinated sequential interaction of N-WASP and cortactin with the Arp2/3 complex.     

Identification of another actin-related protein (Arp) 2/3 complex binding site in neural Wiskott-Aldrich syndrome protein (N-WASP) that complements actin polymerization induced by the Arp2/3 complex activating (VCA) domain of N-WASP.

Neural Wiskott-Aldrich syndrome protein (N-WASP) is an essential regulator of actin cytoskeleton formation via its association with the actin-related protein (Arp) 2/3 complex. It is believed that the C-terminal Arp2/3 complex-activating domain (verprolin homology, cofilin homology, and acidic (VCA) or C-terminal region of WASP family proteins domain) of N-WASP is usually kept masked (autoinhibition) but is opened upon cooperative binding of upstream regulators such as Cdc42 and phosphatidylinositol 4,5-bisphosphate (PIP2). However, the mechanisms of autoinhibition and association with Arp2/3 complex are still unclear. We focused on the acidic region of N-WASP because it is thought to interact with Arp2/3 complex and may be involved in autoinhibition. Partial deletion of acidic residues from the VCA portion alone greatly reduced actin polymerization activity, demonstrating that the acidic region contributes to Arp2/3 complex-mediated actin polymerization. Surprisingly, the same partial deletion of the acidic region in full-length N-WASP led to constitutive activity comparable with the activity seen with the VCA portion. Therefore, the acidic region in full-length N-WASP plays an indispensable role in the formation of the autoinhibited structure. This mutant contains WASP-homology (WH) 1 domain with weak affinity to the Arp2/3 complex, leading to activity in the absence of part of the acidic region. Furthermore, the actin comet formed by the DeltaWH1 mutant of N-WASP was much smaller than that of wild-type N-WASP. Partial deletion of acidic residues did not affect actin comet size, indicating the importance of the WH1 domain in actin structure formation. Collectively, the acidic region of N-WASP plays an essential role in Arp2/3 complex activation as well as in the formation of the autoinhibited structure, whereas the WH1 domain complements the activation of the Arp2/3 complex achieved through the VCA portion.     

Requirement of the basic region of N-WASP/WAVE2 for actin-based motility

WASP family proteins activate nucleation by the Arp2/3 complex, inducing rapid actin polymerization in vitro. Although the C-terminal portion of WASP family proteins (VCA) activates nucleation by the Arp2/3 complex in pure systems, we find that this fragment lacks activity in cell extracts. Thus, polystyrene beads coated with VCA did not move in brain cytosol, while beads coated with N-WASP or WAVE2 did move. The basic clusters between the WH1 domain and the CRIB domain of N-WASP were critical for movement since beads coated with N-WASP or WAVE2 constructs missing the basic clusters (Delta basic) also did not move. Furthermore, VCA and N-WASP/WAVE2 Delta basic constructs were much less able than wild-type N-WASP and WAVE2 to induce actin polymerization in cytosol. All of the proteins, with or without the basic domain, were potent activators of nucleation by purified Arp2/3 complex.     

A conserved amphipathic helix in WASP/Scar proteins is essential for activation of Arp2/3 complex

Members of the Wiskott-Aldrich syndrome protein (WASP) family link Rho GTPase signaling pathways to the cytoskeleton through a multiprotein assembly called Arp2/3 complex. The C-terminal VCA regions (verprolin-homology, central hydrophobic, and acidic regions) of WASP and its relatives stimulate Arp2/3 complex to nucleate actin filament branches. Here we show by differential line broadening in NMR spectra that the C (central) and A (acidic) segments of VCA domains from WASP, N-WASP and Scar bind Arp2/3 complex. The C regions of these proteins have a conserved sequence motif consisting of hydrophobic residues and an arginine residue. Point mutations in this conserved sequence motif suggest that it forms an amphipathic helix that is required in biochemical assays for activation of Arp2/3 complex. Key residues in this motif are buried through contacts with the GTPase binding domain in the autoinhibited structure of WASP and N-WASP, indicating that sequestration of these residues is an important aspect of autoinhibition.     

X-ray scattering study of activated Arp2/3 complex with bound actin-WCA

Previous structures of Arp2/3 complex, determined in the absence of a nucleation-promoting factor and actin, reveal its inactive conformation. The study of the activated structure has been hampered by uncontrollable polymerization. We have engineered a stable activated complex consisting of Arp2/3 complex, the WCA activator region of N-WASP, and one actin monomer, and studied its structure in solution by small angle X-ray scattering (SAXS). The scattering data support a model in which the first actin subunit binds at the barbed end of Arp2, and disqualify an alternative model that places the first actin subunit at the barbed end of Arp3. This location of the first actin and bound W motif constrains the binding site of the C motif to subunits Arp2 and ARPC1, from where the A motif can reach subunits Arp3 and ARPC3. The results support a model of activation that is consistent with most of the biochemical observations.     

The p40/ARPC1 Subunit of Arp2/3 Complex Performs Multiple Essential Roles in WASp-regulated Actin Nucleation

The Arp2/3 complex is a conserved seven-subunit actin-nucleating machine activated by WASp (Wiskott Aldrich syndrome protein). Despite its central importance in a broad range of cellular processes, many critical aspects of the mechanism of the Arp2/3 complex have yet to be resolved. In particular, some of the individual subunits in the complex have not been assigned clear functional roles, including p40/ARPC1. Here, we dissected the structure and function of Saccharomyces cerevisiae p40/ARPC1, which is encoded by the essential ARC40 gene, by analyzing 39 integrated alleles that target its conserved surfaces. We identified three distinct sites on p40/ARPC1 required for function in vivo: one site contacts p19/ARPC4, one contacts p15/ARPC5, and one site resides in an extended structural “arm” of p40/ARPC1. Using a novel strategy, we purified the corresponding lethal mutant Arp2/3 complexes from yeast and compared their actin nucleation activities. Lethal mutations at the contact with p19/ARPC4 specifically impaired WASp-induced nucleation. In contrast, lethal mutations at the contact with p15/ARPC5 led to unregulated (“leaky”) nucleation in the absence of WASp. Lethal mutations in the extended arm drastically reduced nucleation, and the same mutations disrupted the ability of the purified p40/ARPC1 arm domain to bind the VCA domain of WASp. Together, these data indicate that p40/ARPC1 performs at least three distinct, essential functions in regulating Arp2/3 complex-mediated actin assembly: 1) suppression of spontaneous nucleation by the Arp2/3 complex, which requires proper contacts with p15/ARPC5; 2) propagation of WASp activation signals via contacts with p19/ARPC2; and 3) direct facilitation of actin nucleation through interactions of the extended arm with the VCA domain of WASp.     

Activation of nucleation promoting factors for directional actin filament elongation: Allosteric regulation and multimerization on the membrane

Nucleation promoting factors (NPFs) activate the Arp2/3 complex to produce branched actin filaments. Branched actin filaments are observed in most organelles, and specific NPFs, such as WASP, N-WASP, WAVEs, WASH, and WHAMM, exist for each organelle. Interestingly, Arp2/3 and NPFs are both inactive by themselves, and thus require activation. The exposure of the Arp2/3 activating region, the VCA fragment, is recognized to be a key event in the activation of the NPFs. Together, small GTPase binding, phosphorylation, SH3 binding, and membrane binding promote VCA exposure synergistically. The increase in the local concentration of NPF by multimerization is thought to occur with the combination of such activators, to maximally activate the NPF and confine the region of actin polymerization. The mechanism of uni-directional filament extension beneath the membrane also is discussed.     

N-wasp and the arp2/3 complex are critical regulators of actin in the development of dendritic spines and synapses

Changes in the number, size, and shape of dendritic spines are associated with synaptic plasticity, which underlies cognitive functions such as learning and memory. This plasticity is attributed to reorganization of actin, but the molecular signals that regulate this process are poorly understood. In this study, we show neural Wiskott-Aldrich syndrome protein (N-WASP) regulates the formation of dendritic spines and synapses in hippocampal neurons. N-WASP localized to spines and active, functional synapses as shown by loading with FM4-64 dye. Knock down of endogenous N-WASP expression by RNA interference or inhibition of its activity by treatment with a specific inhibitor, wiskostatin, caused a significant decrease in the number of spines and excitatory synapses. Deletion of the C-terminal VCA region of N-WASP, which binds and activates the actin-related protein 2/3 (Arp2/3) complex, dramatically decreased the number of spines and synapses, suggesting activation of the Arp2/3 complex is critical for spine and synapse formation. Consistent with this, Arp3, like N-WASP, was enriched in spines and excitatory synapses and knock down of Arp3 expression impaired spine and synapse formation. A similar defect in spine and synapse formation was observed when expression of an N-WASP activator, Cdc42, was knocked down. Thus, activation of N-WASP and, subsequently, the Arp2/3 complex appears to be an important molecular signal for regulating spines and synapses. Arp2/3-mediated branching of actin could be a mechanism by which dendritic spine heads enlarge and subsequently mature. Collectively, our results point to a critical role for N-WASP and the Arp2/3 complex in spine and synapse formation.     

Interactions of Isolated C-terminal Fragments of Neural Wiskott-Aldrich Syndrome Protein (N-WASP) with Actin and Arp2/3 Complex

Wiskott-Aldrich syndrome proteins (WASP) are a family of proteins that all catalyze actin filament branching with the Arp2/3 complex in a variety of actin-based motile processes. The constitutively active C-terminal domain, called VCA, harbors one or more WASP homology 2 (WH2) domains that bind G-actin, whereas the CA extension binds the Arp2/3 complex. The VCA·actin·Arp2/3 entity associates with a mother filament to form a branched junction from which a daughter filament is initiated. The number and function of WH2-bound actin(s) in the branching process are not known, and the stoichiometry of the VCA·actin·Arp2/3 complex is debated. We have expressed the tandem WH2 repeats of N-WASP, either alone (V) or associated with the C (VC) and CA (VCA) extensions. We analyzed the structure of actin in complex with V, VC, and VCA using protein crystallography and hydrodynamic and spectrofluorimetric methods. The partial crystal structure of the VC·actin 1:1 complex shows two actins in the asymmetric unit with extensive actin-actin contacts. In solution, each of the two WH2 domains in V, VC, and VCA binds G-actin in 1:2 complexes that participate in barbed end assembly. V, VC, and VCA enhance barbed end depolymerization like profilin but neither nucleate nor sever filaments, in contrast with other WH2 repeats. VCA binds the Arp2/3 complex in a 1:1 complex even in the presence of a large excess of VCA. VCA·Arp2/3 binds one actin in a latrunculin A-sensitive fashion, in a 1:1:1 complex, indicating that binding of the second actin to VCA is weakened in the ternary complex.     

Essential role of neural Wiskott-Aldrich syndrome protein in neurite extension in PC12 cells and rat hippocampal primary culture cells

Neural Wiskott-Aldrich syndrome protein (N-WASP) is an actin-regulating protein that induces filopodium formation downstream of Cdc42. It has been shown that filopodia actively extend from the growth cone, a guidance apparatus located at the tip of neurites, suggesting their role in neurite extension. Here we examined the possible involvement of N-WASP in the neurite extension process. Since verprolin, cofilin homology and acidic region (VCA) of N-WASP is known to be required for the activation of Arp2/3 complex that induces actin polymerization, we prepared a mutant (Deltacof) lacking four amino acid residues in the cofilin homology region. The corresponding residues in WASP had been reported to be mutated in some Wiskott-Aldrich syndrome patients. Expression of Deltacof N-WASP suppressed neurite extension of PC12 cells. In support of this, the VCA region of Deltacof cannot activate Arp2/3 complex enough compared with wild-type VCA. Furthermore, H208D mutant, which has been shown unable to bind to Cdc42, also works as a dominant negative mutant in neurite extension assay. Interestingly, the expression of H208D-Deltacof double mutant has no significant dominant negative effect. Finally, the expression of the Deltacof mutant also severely inhibited the neurite extension of primary neurons from rat hippocampus. Thus, N-WASP is thought to be a general regulator of the actin cytoskeleton indispensable for neurite extension, which is probably caused through Cdc42 signaling and Arp2/3 complex-induced actin polymerization.     

Nucleotide- and Activator-Dependent Structural and Dynamic Changes of Arp2/3 Complex Monitored by Hydrogen/Deuterium Exchange and Mass Spectrometry

Arp2/3 complex plays a central role in the de novo nucleation of filamentous actin as branches on existing filaments. The complex must bind ATP, protein activators [e.g., Wiskott-Aldrich syndrome protein (WASp)], and the side of an actin filament to form a new actin filament. Amide hydrogen/deuterium exchange coupled with mass spectrometry was used to examine the structural and dynamic properties of the mammalian Arp2/3 complex in the presence of both ATP and the activating peptide segment from WASp. Changes in the rate of hydrogen exchange indicate that ATP binding causes conformational rearrangements of Arp2 and Arp3 that are transmitted allosterically to the Arp complex (ARPC)1, ARPC2, ARPC4, and ARPC5 subunits. These data are consistent with the closure of nucleotide-binding cleft of Arp3 upon ATP binding, resulting in structural rearrangements that propagate throughout the complex. Binding of the VCA domain of WASp to ATP-Arp2/3 further modulates the rates of hydrogen exchange in these subunits, indicating that a global conformational reorganization is occurring. These effects may include the direct binding of activators to Arp3, Arp2, and ARPC1; alterations in the relative orientations of Arp2 and Arp3; and the long-range transmission of activator-dependent signals to segments proposed to be involved in binding the F-actin mother filament.     

A novel role for WAVE1 in controlling actin network growth rate and architecture

Branched actin filament networks in cells are assembled through the combined activities of Arp2/3 complex and different WASP/WAVE proteins. Here we used TIRF and electron microscopy to directly compare for the first time the assembly kinetics and architectures of actin filament networks produced by Arp2/3 complex and dimerized VCA regions of WAVE1, WAVE2, or N-WASP. WAVE1 produced strikingly different networks from WAVE2 or N-WASP, which comprised unexpectedly short filaments. Further analysis showed that the WAVE1-specific activity stemmed from an inhibitory effect on filament elongation both in the presence and absence of Arp2/3 complex, which was observed even at low stoichiometries of WAVE1 to actin monomers, precluding an effect from monomer sequestration. Using a series of VCA chimeras, we mapped the elongation inhibitory effects of WAVE1 to its WH2 (“V”) domain. Further, mutating a single conserved lysine residue potently disrupted WAVE1''s inhibitory effects. Taken together, our results show that WAVE1 has unique activities independent of Arp2/3 complex that can govern both the growth rates and architectures of actin filament networks. Such activities may underlie previously observed differences between the cellular functions of WAVE1 and WAVE2.     

ARPC1/Arc40 mediates the interaction of the actin-related protein 2 and 3 complex with Wiskott-Aldrich syndrome protein family activators

The actin-related protein 2 and 3 (Arp2/3) complex is a seven-subunit protein complex that nucleates actin filaments at the cell cortex. Despite extensive cross-linking, crystallography, genetic and biochemical studies, the contribution of each subunit to the activity of the complex remains largely unclear. In this study we characterized the function of the 40-kDa subunit, ARPC1/Arc40, of the yeast Arp2/3 complex. We showed that this subunit is indeed a stable component of the Arp2/3 complex, but its highly unusual electrophoretic mobility eluded detection in previous studies. Recombinant Arc40 bound the VCA domain of Wiskott-Aldrich syndrome protein family activators at a K(d) of 0.45 mum, close to that of the full complex with VCA (0.30 microm), and this interaction was dependent on the conserved tryptophan at the COOH terminus of VCA. Using a newly constructed Delta arc40 yeast strain, we showed that loss of Arc40 severely reduced the binding affinity of the Arp2/3 complex with VCA as well as the nucleation activity of the complex, suggesting that Arc40 contains an important contact site of the Arp2/3 complex with VCA. The Delta arc40 cells exhibited reduced growth rate, loss of actin patches, and accumulation of cables like actin aggregates, phenotypes typical of other subunit nulls, suggesting that Arc40 functions exclusively within the Arp2/3 complex.     

N-WASP involvement in dorsal ruffle formation in mouse embryonic fibroblasts

The Wiskott-Aldrich syndrome protein (WASP) family activates the Arp2/3 complex leading to the formation of new actin filaments. Here, we study the involvement of Scar1, Scar2, N-WASP, and Arp2/3 complex in dorsal ruffle formation in mouse embryonic fibroblasts (MEFs). Using platelet-derived growth factor to stimulate circular dorsal ruffle assembly in primary E13 and immortalized E9 Scar1(+/+) and Scar1 null MEFs, we establish that Scar1 loss does not impair the formation of dorsal ruffles. Reduction of Scar2 protein levels via small interfering RNA (siRNA) also did not affect dorsal ruffle production. In contrast, wiskostatin, a chemical inhibitor of N-WASP, potently suppressed dorsal ruffle formation in a dose-dependent manner. Furthermore, N-WASP and Arp2 siRNA treatment significantly decreased the formation of dorsal ruffles in MEFs. In addition, the expression of an N-WASP truncation mutant that cannot bind Arp2/3 complex blocked the formation of these structures. Finally, N-WASP(-/-) fibroblast-like cells generated aberrant dorsal ruffles. These ruffles were highly unstable, severely depleted of Arp2/3 complex, and diminished in size. We hypothesize that N-WASP and Arp2/3 complex are part of a multiprotein assembly important for the generation of dorsal ruffles and that Scar1 and Scar2 are dispensable for this process.     

Identification and characterisation of a novel human isoform of Arp2/3 complex subunit p16-ARC/ARPC5

The Arp2/3 complex is an actin filament nucleator that activates regulated actin assembly in response to extracellular signals. The mammalian complex is composed of seven subunits, the smallest of which is known as ARPC5 or p16-Arc. We have identified a human cDNA sequence with homology to ARPC5 and here provide evidence that this encodes a novel ARPC5 isoform. Specific antibodies were generated against the novel protein, which we have termed ARPC5B, as well as the previously characterised ARPC5 isoform, henceforth ARPC5A. The presence of both ARPC5 isoforms was detected in Arp2/3 complex affinity purified from human neutrophil extract. The tissue distribution of ARPC5A and B was analysed using the isoform-specific antibodies and it was found that the two isoforms exhibited significant differences; ARPC5A was found to be highly enriched in spleen and thymus, while ARPC5B exhibits a more regular expression, with levels in the brain being highest. Myc-tagged ARPC5A and B co-localised with the Arp2/3 complex when expressed in C2C12 cells and the cellular distribution of the two isoforms could not be distinguished. Our data show for the first time that mammalian cells contain multiple forms of the Arp2/3 complex.     

Phosphorylation of the Arp2 subunit relieves auto-inhibitory interactions for Arp2/3 complex activation

Actin filament assembly by the actin-related protein (Arp) 2/3 complex is necessary to build many cellular structures, including lamellipodia at the leading edge of motile cells and phagocytic cups, and to move endosomes and intracellular pathogens. The crucial role of the Arp2/3 complex in cellular processes requires precise spatiotemporal regulation of its activity. While binding of nucleation-promoting factors (NPFs) has long been considered essential to Arp2/3 complex activity, we recently showed that phosphorylation of the Arp2 subunit is also necessary for Arp2/3 complex activation. Using molecular dynamics simulations and biochemical assays with recombinant Arp2/3 complex, we now show how phosphorylation of Arp2 induces conformational changes permitting activation. The simulations suggest that phosphorylation causes reorientation of Arp2 relative to Arp3 by destabilizing a network of salt-bridge interactions at the interface of the Arp2, Arp3, and ARPC4 subunits. Simulations also suggest a gain-of-function ARPC4 mutant that we show experimentally to have substantial activity in the absence of NPFs. We propose a model in which a network of auto-inhibitory salt-bridge interactions holds the Arp2 subunit in an inactive orientation. These auto-inhibitory interactions are destabilized upon phosphorylation of Arp2, allowing Arp2 to reorient to an activation-competent state.     

Activation of the CDC42 effector N-WASP by the Shigella flexneri IcsA protein promotes actin nucleation by Arp2/3 complex and bacterial actin-based motility.

To propel itself in infected cells, the pathogen Shigella flexneri subverts the Cdc42-controlled machinery responsible for actin assembly during filopodia formation. Using a combination of bacterial motility assays in platelet extracts with Escherichia coli expressing the Shigella IcsA protein and in vitro analysis of reconstituted systems from purified proteins, we show here that the bacterial protein IcsA binds N-WASP and activates it in a Cdc42-like fashion. Dramatic stimulation of actin assembly is linked to the formation of a ternary IcsA-N-WASP-Arp2/3 complex, which nucleates actin polymerization. The Arp2/3 complex is essential in initiation of actin assembly and Shigella movement, as previously observed for Listeria monocytogenes. Activation of N-WASP by IcsA unmasks two domains acting together in insertional actin polymerization. The isolated COOH-terminal domain of N-WASP containing a verprolin-homology region, a cofilin-homology sequence, and an acidic terminal segment (VCA) interacts with G-actin in a unique profilin-like functional fashion. Hence, when N-WASP is activated, its COOH-terminal domain feeds barbed end growth of filaments and lowers the critical concentration at the bacterial surface. On the other hand, the NH(2)-terminal domain of N-WASP interacts with F-actin, mediating the attachment of the actin tail to the bacterium surface. VASP is not involved in Shigella movement, and the function of profilin does not require its binding to proline-rich regions.