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.     

Toca-1 mediates Cdc42-dependent actin nucleation by activating the N-WASP-WIP complex

An important signaling pathway to the actin cytoskeleton links the Rho family GTPase Cdc42 to the actin-nucleating Arp2/3 complex through N-WASP. Nevertheless, these previously identified components are not sufficient to mediate Cdc42-induced actin polymerization in a physiological context. In this paper, we describe the biochemical purification of Toca-1 (transducer of Cdc42-dependent actin assembly) as an essential component of the Cdc42 pathway. Toca-1 binds both N-WASP and Cdc42 and is a member of the evolutionarily conserved PCH protein family. Toca-1 promotes actin nucleation by activating the N-WASP-WIP/CR16 complex, the predominant form of N-WASP in cells. Thus, the cooperative actions of two distinct Cdc42 effectors, the N-WASP-WIP complex and Toca-1, are required for Cdc42-induced actin assembly. These findings represent a significantly revised view of Cdc42-signaling and shed light on the pathogenesis of Wiskott-Aldrich syndrome.     

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.     

Association of Cdc42/N-WASP/Arp2/3 signaling pathway with Golgi membranes

Recent findings indicate that Cdc42 regulates Golgi-to-ER (endoplasmic reticulum) protein transport through N-WASP and Arp2/3 (Luna et al. 2002, Mol. Biol. Cell, 13:866-879). To analyse the components of the Cdc42-governed signaling pathway in the secretory pathway, we localized Cdc42, N-WASP and Arp2/3 in the Golgi complex by cryoimmunoelectron microscopy. Cdc42 is found throughout the Golgi stack, particularly in cis/middle cisternae, whereas N-WASP and Arp3 (a component of the Arp2/3 complex) are restricted to cis cisternae. Arp3 also colocalized in peri-Golgi tubulovesicular structures with either KDEL receptor or GM130. Even though Arp3 is not found in TGN46-positive cisternal elements, a small fraction of Arp3-labeled tubulo-vesicular elements showed TGN46 labeling. Active Cdc42 (GTP-bound form) induced relocation of N-WASP and Arp3 to the lateral rims of Golgi cisternae. These results show that the actin nucleation and polymerization signaling pathway governed by Cdc42/N-WASP/Arp operates in the Golgi complex of mammalian cells, further implicating actin dynamics in Golgi-associated membrane trafficking.     

Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4, 5-bisphosphate

Neuronal Wiskott-Aldrich Syndrome protein (N-WASP) transmits signals from Cdc42 to the nucleation of actin filaments by Arp2/3 complex. Although full-length N-WASP is a weak activator of Arp2/3 complex, its activity can be enhanced by upstream regulators such as Cdc42 and PI(4,5)P(2). We dissected this activation reaction and found that the previously described physical interaction between the NH(2)-terminal domain and the COOH-terminal effector domain of N-WASP is a regulatory interaction because it can inhibit the actin nucleation activity of the effector domain by occluding the Arp2/3 binding site. This interaction between the NH(2)- and COOH termini must be intramolecular because in solution N-WASP is a monomer. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) influences the activity of N-WASP through a conserved basic sequence element located near the Cdc42 binding site rather than through the WASp homology domain 1. Like Cdc42, PI(4,5)P(2) reduces the affinity between the NH(2)- and COOH termini of the molecule. The use of a mutant N-WASP molecule lacking this basic stretch allowed us to delineate a signaling pathway in Xenopus extracts leading from PI(4, 5)P(2) to actin nucleation through Cdc42, N-WASP, and Arp2/3 complex. In this pathway, PI(4,5)P(2) serves two functions: first, as an activator of N-WASP; and second, as an indirect activator of Cdc42.     

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.     

Profilin is required for sustaining efficient intra- and intercellular spreading of Shigella flexneri

The ability of Shigella to mediate actin-based motility within the host cell is a prominent pathogenic feature of bacillary dysentery. The ability is dependent on the interaction of VirG with neural Wiskott-Aldrich syndrome protein (N-WASP), which in turn mediates recruitment of Arp2/3 complex and several actin-related proteins. In the present study, we show that profilin I is essential to the rapid movement of Shigella in epithelial cells, for which the capacity of profilin to interact with G-actin and N-WASP is critical. In COS-7 cells overexpressing either mutated profilin H119E, which failed to bind G-actin, or H133S, which is unable to interact with poly-l-proline, Shigella motility was significantly inhibited. Similarly, depletion of profilin from Xenopus egg extracts resulted in a decrease in bacterial motility that was completely rescued by adding back profilin I but not H119E or H133S. In COS-7 cells overexpressing a N-WASP mutant lacking the proline-rich domain (Deltap) unable to interact with profilin, the actin tail formation of intracellular Shigella was inhibited. In N-WASP-depleted extracts, addition of Deltap but not full-length N-WASP was unable to restore the bacterial motility. Furthermore, in a plaque formation assay with Madin-Darby canine kidney cell monolayers infected by Shigella, Madin-Darby canine kidney cells stably expressing H119E, H133S, or Deltap reduced the bacterial cell-to-cell spreading. These results indicate that profilin I associated with N-WASP is an essential host factor for sustaining efficient intra- and intercellular spreading of Shigella.     

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.     

Differential regulation of WASP and N-WASP by Cdc42, Rac1, Nck, and PI(4,5)P2

The Wiskott-Aldrich syndrome protein (WASP) and neural WASP (N-WASP) are key players in regulating actin cytoskeleton via the Arp2/3 complex. It has been widely reported that the WASP proteins are activated by Rho family small GTPase Cdc42 and that Rac1 acts through SCAR/WAVE proteins. However, a systematic study of the specificity of different GTPases for different Arp2/3 activators has not been conducted. In this study, we have expressed, purified, and characterized completely soluble, highly active, and autoinhibited full-length human WASP and N-WASP from mammalian cells. We show a novel N-WASP activation by Rho family small GTPase Rac1. This GTPase exclusively stimulates N-WASP and has no effects on WASP. Rac1 is a significantly more potent N-WASP activator than Cdc42. In contrast, Cdc42 is a more effective activator of WASP than N-WASP. Lipid vesicles containing PIP2 significantly improve actin nucleation by the Arp2/3 complex and N-WASP in the presence of Rac1 or Cdc42. PIP2 vesicles have no effect on WASP activity alone. Moreover, the inhibition of WASP-stimulated actin nucleation in the presence of Cdc42 and PIP2 vesicles has been observed. We found that adaptor proteins Nck1 or Nck2 are the most potent WASP and N-WASP activators with distinct effects on the WASP family members. Our in vitro data demonstrates differential regulation of full-length WASP and N-WASP by cellular activators that highlights fundamental differences of response at the protein-protein level.     

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.     

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.     

WIP regulates N-WASP-mediated actin polymerization and filopodium formation

Induction of filopodia is dependent on activation of the small GTPase Cdc42 and on neural Wiskott-Aldrich-syndrome protein (N-WASP). Here we show that WASP-interacting protein (WIP) interacts directly with N-WASP and actin. WIP retards N-WASP/Cdc42-activated actin polymerization mediated by the Arp2/3 complex, and stabilizes actin filaments. Microinjection of WIP into NIH 3T3 fibroblasts induces filopodia; this is inhibited by microinjection of anti-N-WASP antibody. Microinjection of anti-WIP antibody inhibits induction of filopodia by bradykinin, by an active Cdc42 mutant (Cdc42(V12)) and by N-WASP. Our results indicate that WIP and N-WASP may act as a functional unit in filopodium formation, which is consistent with their role in actin-tail formation in cells infected with vaccinia virus or Shigella.     

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.     

The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly

Although small GTP-binding proteins of the Rho family have been implicated in signaling to the actin cytoskeleton, the exact nature of the linkage has remained obscure. We describe a novel mechanism that links one Rho family member, Cdc42, to actin polymerization. N-WASP, a ubiquitously expressed Cdc42-interacting protein, is required for Cdc42-stimulated actin polymerization in Xenopus egg extracts. The C terminus of N-WASP binds to the Arp2/3 complex and dramatically stimulates its ability to nucleate actin polymerization. Although full-length N-WASP is less effective, its activity can be greatly enhanced by Cdc42 and phosphatidylinositol (4,5) bisphosphate. Therefore, N-WASP and the Arp2/3 complex comprise a core mechanism that directly connects signal transduction pathways to the stimulation of actin polymerization.     

Actin Dynamics Regulated by the Balance of Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) and Cofilin Activities Determines the Biphasic Response of Glucose-induced Insulin Secretion

Actin dynamics in pancreatic β-cells is involved in insulin secretion. However, the molecular mechanisms of the regulation of actin dynamics by intracellular signals in pancreatic β-cells and its role in phasic insulin secretion are largely unknown. In this study, we elucidate the regulation of actin dynamics by neuronal Wiskott-Aldrich syndrome protein (N-WASP) and cofilin in pancreatic β-cells and demonstrate its role in glucose-induced insulin secretion (GIIS). N-WASP, which promotes actin polymerization through activation of the actin nucleation factor Arp2/3 complex, was found to be activated by glucose stimulation in insulin-secreting clonal pancreatic β-cells (MIN6-K8 β-cells). Introduction of a dominant-negative mutant of N-WASP, which lacks G-actin and Arp2/3 complex-binding region VCA, into MIN6-K8 β-cells or knockdown of N-WASP suppressed GIIS, especially the second phase. We also found that cofilin, which severs F-actin in its dephosphorylated (active) form, is converted to the phosphorylated (inactive) form by glucose stimulation in MIN6-K8 β-cells, thereby promoting F-actin remodeling. In addition, the dominant-negative mutant of cofilin, which inhibits activation of endogenous cofilin, or knockdown of cofilin reduced the second phase of GIIS. However, the first phase of GIIS occurs in the G-actin predominant state, in which cofilin activity predominates over N-WASP activity. Thus, actin dynamics regulated by the balance of N-WASP and cofilin activities determines the biphasic response of GIIS.     

Regulation of N-WASP and the Arp2/3 complex by Abp1 controls neuronal morphology

Polymerization and organization of actin filaments into complex superstructures is indispensable for structure and function of neuronal networks. We here report that knock down of the F-actin-binding protein Abp1, which is important for endocytosis and synaptic organization, results in changes in axon development virtually identical to Arp2/3 complex inhibition, i.e., a selective increase of axon length. Our in vitro and in vivo experiments demonstrate that Abp1 interacts directly with N-WASP, an activator of the Arp2/3 complex, and releases the autoinhibition of N-WASP in cooperation with Cdc42 and thereby promotes N-WASP-triggered Arp2/3 complex-mediated actin polymerization. In line with our mechanistical studies and the colocalization of Abp1, N-WASP and Arp2/3 at sites of actin polymerization in neurons, we reveal an essential role of Abp1 and its cooperativity with Cdc42 in N-WASP-induced rearrangements of the neuronal cytoskeleton. We furthermore show that introduction of N-WASP mutants lacking the ability to bind Abp1 or Cdc42, Arp2/3 complex inhibition, Abp1 knock down, N-WASP knock down and Arp3 knock down, all cause identical neuromorphological phenotypes. Our data thus strongly suggest that these proteins and their complex formation are important for cytoskeletal processes underlying neuronal network formation.     

Activation by Cdc42 and PIP(2) of Wiskott-Aldrich syndrome protein (WASp) stimulates actin nucleation by Arp2/3 complex

We purified native WASp (Wiskott-Aldrich Syndrome protein) from bovine thymus and studied its ability to stimulate actin nucleation by Arp2/3 complex. WASp alone is inactive in the presence or absence of 0.5 microM GTP-Cdc42. Phosphatidylinositol 4,5 bisphosphate (PIP(2)) micelles allowed WASp to activate actin nucleation by Arp2/3 complex, and this was further enhanced twofold by GTP-Cdc42. Filaments nucleated by Arp2/3 complex and WASp in the presence of PIP(2) and Cdc42 concentrated around lipid micelles and vesicles, providing that Cdc42 was GTP-bound and prenylated. Thus, the high concentration of WASp in neutrophils (9 microM) is dependent on interactions with both acidic lipids and GTP-Cdc42 to activate actin nucleation by Arp2/3 complex. The results also suggest that membrane binding increases the local concentrations of Cdc42 and WASp, favoring their interaction.     

Two pathways through Cdc42 couple the N-formyl receptor to actin nucleation in permeabilized human neutrophils

We developed a permeabilization method that retains coupling between N-formyl-methionyl-leucyl-phenylalanine tripeptide (FMLP) receptor stimulation, shape changes, and barbed-end actin nucleation in human neutrophils. Using GTP analogues, phosphoinositides, a phosphoinositide-binding peptide, constitutively active or inactive Rho GTPase mutants, and activating or inhibitory peptides derived from neural Wiskott-Aldrich syndrome family proteins (N-WASP), we identified signaling pathways leading from the FMLP receptor to actin nucleation that require Cdc42, but then diverge. One branch traverses the actin nucleation pathway involving N-WASP and the Arp2/3 complex, whereas the other operates through active Rac to promote actin nucleation. Both pathways depend on phosphoinositide expression. Since maximal inhibition of the Arp2/3 pathway leaves an N17Rac inhibitable alternate pathway intact, we conclude that this alternate involves phosphoinositide-mediated uncapping of actin filament barbed ends.     

FMNL2 drives actin-based protrusion and migration downstream of Cdc42

Cell migration entails protrusion of lamellipodia, densely packed networks of actin filaments at the cell front. Filaments are generated by nucleation, likely mediated by Arp2/3 complex and its activator Scar/WAVE. It is unclear whether formins contribute to lamellipodial actin filament nucleation or serve as elongators of filaments nucleated by Arp2/3 complex. Here we show that the Diaphanous-related formin FMNL2, also known as FRL3 or FHOD2, accumulates at lamellipodia and filopodia tips. FMNL2 is cotranslationally modified by myristoylation and regulated by interaction with the Rho-guanosine triphosphatase Cdc42. Abolition of myristoylation or Cdc42 binding interferes with proper FMNL2 activation, constituting an essential prerequisite for subcellular targeting. In vitro, C-terminal FMNL2 drives elongation rather than nucleation of actin filaments in the presence of profilin. In addition, filament ends generated by Arp2/3-mediated branching are captured and efficiently elongated by the formin. Consistent with these biochemical properties, RNAi-mediated silencing of FMNL2 expression decreases the rate of lamellipodia protrusion and, accordingly, the efficiency of cell migration. Our data establish that the FMNL subfamily member FMNL2 is a novel elongation factor of actin filaments that constitutes the first Cdc42 effector promoting cell migration and actin polymerization at the tips of lamellipodia.