A novel role for 14-kDa phosphohistidine phosphatase in lamellipodia formation

Cell migration involves dynamic regulation of the actin cytoskeleton, which exhibits rapid actin polymerization at the leading edge of migrating cells. This process relies on regulated recruitment of actin nucleators and actin-binding proteins to the leading edge to polymerize new actin filaments. Many of these proteins have been identified, including the actin-related protein (Arp) 2/3 complex, which has emerged as the core player in the initiation of actin polymerization. However, the functional coordination of these proteins is unclear. Previously, we have demonstrated that the 14-kDa phosphohistidine phosphatase (PHP14) is involved in cell migration regulation and affects actin cytoskeleton reorganization. Here, we show that PHP14 may regulate actin remodeling directly and play an important role in dynamic regulation of the actin cytoskeleton. We observed a colocalization of PHP14 with Arp3 and F-actin at the leading edge of migrating cells. Moreover, PHP14 was recruited to the actin remodeling sites in parallel with Arp3 during lamellipodia formation. Furthermore, PHP14 knockdown impaired Arp3 localization at the leading edge of lamellipodia, as well as lamellipodia formation. Most importantly, we found that PHP14 was a novel F-actin-binding protein, displaying an Arp2/3-dependent localization to the leading edge. Collectively, our results indicated a crucial role for PHP14 in the dynamic regulation of the actin cytoskeleton and cell migration.     

Novel roles of formin mDia2 in lamellipodia and filopodia formation in motile cells

Actin polymerization-driven protrusion of the leading edge is a key element of cell motility. The important actin nucleators formins and the Arp2/3 complex are believed to have nonoverlapping functions in inducing actin filament bundles in filopodia and dendritic networks in lamellipodia, respectively. We tested this idea by investigating the role of mDia2 formin in leading-edge protrusion by loss-of-function and gain-of-function approaches. Unexpectedly, mDia2 depletion by short interfering RNA (siRNA) severely inhibited lamellipodia. Structural analysis of the actin network in the few remaining lamellipodia suggested an mDia2 role in generation of long filaments. Consistently, constitutively active mDia2 (ΔGBD-mDia2) induced accumulation of long actin filaments in lamellipodia and increased persistence of lamellipodial protrusion. Depletion of mDia2 also inhibited filopodia, whereas expression of ΔGBD-mDia2 promoted their formation. Correlative light and electron microscopy showed that ΔGBD-mDia2–induced filopodia were formed from lamellipodial network through gradual convergence of long lamellipodial filaments into bundles. Efficient filopodia induction required mDia2 targeting to the membrane, likely through a scaffolding protein Abi1. Furthermore, mDia2 and Abi1 interacted through the N-terminal regulatory sequences of mDia2 and the SH3-containing Abi1 sequences. We propose that mDia2 plays an important role in formation of lamellipodia by nucleating and/or protecting from capping lamellipodial actin filaments, which subsequently exhibit high tendency to converge into filopodia.     

WAVE and Arp2/3 jointly inhibit filopodium formation by entering into a complex with mDia2

Lamellipodia/ruffles and filopodia are protruding organelles containing short and highly branched or long and unbranched actin filaments, respectively. The microscopic morphology, dynamic development and protein signature of both lamellipodia/ruffles and filopodia have been investigated; however, little is known about the mechanisms by which cells coordinate the formation of these actin-based extensions. Here, we show that WAVE holds mDia2 and the Arp2/3 complex in a multimolecular complex. WAVE- and Arp2/3-dependent ruffling induced by EGF does not require mDia2. Conversely, the emission of mDia2-dependent filopodia correlates with its disengagement from WAVE. Consistently, the ability of EGF, Cdc42 and serum to induce mDia2-dependent formation of filopodia is increased in the absence of either the WAVE/Abi1/Nap1/PIR121 (WANP) or the Arp2/3 complex. Reintroduction of WAVE2 into WANP-complex knockdown cells markedly reduces filopodia formation independently of actin polymerization. Thus, WAVE and the Arp2/3 complex jointly orchestrate different types of actin-based plasma membrane protrusions by promoting ruffling and inhibiting mDia2-induced filopodia.     

Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation

The Rho-GTPase Rac1 stimulates actin remodelling at the cell periphery by relaying signals to Scar/WAVE proteins leading to activation of Arp2/3-mediated actin polymerization. Scar/WAVE proteins do not interact with Rac1 directly, but instead assemble into multiprotein complexes, which was shown to regulate their activity in vitro. However, little information is available on how these complexes function in vivo. Here we show that the specifically Rac1-associated protein-1 (Sra-1) and Nck-associated protein 1 (Nap1) interact with WAVE2 and Abi-1 (e3B1) in resting cells or upon Rac activation. Consistently, Sra-1, Nap1, WAVE2 and Abi-1 translocated to the tips of membrane protrusions after microinjection of constitutively active Rac. Moreover, removal of Sra-1 or Nap1 by RNA interference abrogated the formation of Rac-dependent lamellipodia induced by growth factor stimulation or aluminium fluoride treatment. Finally, microinjection of an activated Rac failed to restore lamellipodia protrusion in cells lacking either protein. Thus, Sra-1 and Nap1 are constitutive and essential components of a WAVE2- and Abi-1-containing complex linking Rac to site-directed actin assembly.     

Localized zones of Rho and Rac activities drive initiation and expansion of epithelial cell-cell adhesion

Spatiotemporal coordination of cell-cell adhesion involving lamellipodial interactions, cadherin engagement, and the lateral expansion of the contact is poorly understood. Using high-resolution live-cell imaging, biosensors, and small molecule inhibitors, we investigate how Rac1 and RhoA regulate actin dynamics during de novo contact formation between pairs of epithelial cells. Active Rac1, the Arp2/3 complex, and lamellipodia are initially localized to de novo contacts but rapidly diminish as E-cadherin accumulates; further rounds of activation and down-regulation of Rac1 and Arp2/3 occur at the contacting membrane periphery, and this cycle repeats as a restricted membrane zone that moves outward with the expanding contact. The cortical bundle of actin filaments dissolves beneath the expanding contacts, leaving actin bundles at the contact edges. RhoA and actomyosin contractility are activated at the contact edges and are required to drive expansion and completion of cell-cell adhesion. We show that zones of Rac1 and lamellipodia activity and of RhoA and actomyosin contractility are restricted to the periphery of contacting membranes and together drive initiation, expansion, and completion of cell-cell adhesion.     

Sphingosine-1-phosphate signaling regulates lamellipodia localization of cortactin complexes in endothelial cells

Sphingosine-1-phosphate (S1P), a serum-borne lipid mediator, was demonstrated to be a potent chemoattractant of endothelial cells. It was recently shown that the colocalization of cortactin and actin related protein 2/3 (Arp2/3) in the lamellipodia is critical to S1P-induced endothelial chemotaxis. In this report, we describe that S1P-stimulated cortactin translocation to the cell periphery to form lamellipodia is specifically mediated by the endothelial S1P1 G-protein coupled receptor, and is regulated by G_i-mediated Akt-dependent S1P1 receptor phosphorylation and Cdc42/Rac activation pathways. In contrast to Src-dependent fibroblast growth factor-induced cortactin translocation, tyrosine phosphorylation cascades are not required for S1P-mediated lamellipodia formation and chemotaxis. Furthermore, we also demonstrate that S1P signaling, via the G_i/Akt/S1P1 phosphorylation/Rac pathway, regulates the cortactin–Arp2/3 complex formation, which ultimately results in membrane ruffling, formation of the lamellipodia and endothelial migration.J.F. Lee and H. Ozaki contributed equally to this work     

Arp2/3 branched actin network mediates filopodia-like bundles formation in vitro

During cellular migration, regulated actin assembly takes place at the cell leading edge, with continuous disassembly deeper in the cell interior. Actin polymerization at the plasma membrane results in the extension of cellular protrusions in the form of lamellipodia and filopodia. To understand how cells regulate the transformation of lamellipodia into filopodia, and to determine the major factors that control their transition, we studied actin self-assembly in the presence of Arp2/3 complex, WASp-VCA and fascin, the major proteins participating in the assembly of lamellipodia and filopodia. We show that in the early stages of actin polymerization fascin is passive while Arp2/3 mediates the formation of dense and highly branched aster-like networks of actin. Once filaments in the periphery of an aster get long enough, fascin becomes active, linking the filaments into bundles which emanate radially from the aster's surface, resulting in the formation of star-like structures. We show that the number of bundles nucleated per star, as well as their thickness and length, is controlled by the initial concentration of Arp2/3 complex ([Arp2/3]). Specifically, we tested several values of [Arp2/3] and found that for given initial concentrations of actin and fascin, the number of bundles per star, as well as their length and thickness are larger when [Arp2/3] is lower. Our experimental findings can be interpreted and explained using a theoretical scheme which combines Kinetic Monte Carlo simulations for aster growth, with a simple mechanistic model for bundles' formation and growth. According to this model, bundles emerge from the aster's (sparsely branched) surface layer. Bundles begin to form when the bending energy associated with bringing two filaments into contact is compensated by the energetic gain resulting from their fascin linking energy. As time evolves the initially thin and short bundles elongate, thus reducing their bending energy and allowing them to further associate and create thicker bundles, until all actin monomers are consumed. This process is essentially irreversible on the time scale of actin polymerization. Two structural parameters, L, which is proportional to the length of filament tips at the aster periphery and b, the spacing between their origins, dictate the onset of bundling; both depending on [Arp2/3]. Cells may use a similar mechanism to regulate filopodia formation along the cell leading edge. Such a mechanism may allow cells to have control over the localization of filopodia by recruiting specific proteins that regulate filaments length (e.g., Dia2) to specific sites along lamellipodia.     

Filopodia formation in the absence of functional WAVE- and Arp2/3-complexes

Cell migration is initiated by plasma membrane protrusions, in the form of lamellipodia and filopodia. The latter rod-like projections may exert sensory functions and are found in organisms as distant in evolution as mammals and amoeba such as Dictyostelium discoideum. In mammals, lamellipodia protrusion downstream of the small GTPase Rac1 requires a multimeric protein assembly, the WAVE-complex, which activates Arp2/3-mediated actin filament nucleation and actin network assembly. A current model of filopodia formation postulates that these structures arise from a dendritic network of lamellipodial actin filaments by selective elongation and bundling. Here, we have analyzed filopodia formation in mammalian cells abrogated in expression of essential components of the lamellipodial actin polymerization machinery. Cells depleted of the WAVE-complex component Nck-associated protein 1 (Nap1), and, in consequence, of lamellipodia, exhibited normal filopodia protrusion. Likewise, the Arp2/3-complex, which is essential for lamellipodia protrusion, is dispensable for filopodia formation. Moreover, genetic disruption of nap1 or the WAVE-orthologue suppressor of cAMP receptor (scar) in Dictyostelium was also ineffective in preventing filopodia protrusion. These data suggest that the molecular mechanism of filopodia formation is conserved throughout evolution from Dictyostelium to mammals and show that lamellipodia and filopodia formation are functionally separable.     

Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia.

The leading edge (approximately 1 microgram) of lamellipodia in Xenopus laevis keratocytes and fibroblasts was shown to have an extensively branched organization of actin filaments, which we term the dendritic brush. Pointed ends of individual filaments were located at Y-junctions, where the Arp2/3 complex was also localized, suggesting a role of the Arp2/3 complex in branch formation. Differential depolymerization experiments suggested that the Arp2/3 complex also provided protection of pointed ends from depolymerization. Actin depolymerizing factor (ADF)/cofilin was excluded from the distal 0.4 micrometer++ of the lamellipodial network of keratocytes and in fibroblasts it was located within the depolymerization-resistant zone. These results suggest that ADF/cofilin, per se, is not sufficient for actin brush depolymerization and a regulatory step is required. Our evidence supports a dendritic nucleation model (Mullins, R.D., J.A. Heuser, and T.D. Pollard. 1998. Proc. Natl. Acad. Sci. USA. 95:6181-6186) for lamellipodial protrusion, which involves treadmilling of a branched actin array instead of treadmilling of individual filaments. In this model, Arp2/3 complex and ADF/cofilin have antagonistic activities. Arp2/3 complex is responsible for integration of nascent actin filaments into the actin network at the cell front and stabilizing pointed ends from depolymerization, while ADF/cofilin promotes filament disassembly at the rear of the brush, presumably by pointed end depolymerization after dissociation of the Arp2/3 complex.     

Abi1 is essential for the formation and activation of a WAVE2 signalling complex

WAVE2 belongs to a family of proteins that mediates actin reorganization by relaying signals from Rac to the Arp2/3 complex, resulting in lamellipodia protrusion. WAVE2 displays Arp2/3-dependent actin nucleation activity in vitro, and does not bind directly to Rac. Instead, it forms macromolecular complexes that have been reported to exert both positive and negative modes of regulation. How these complexes are assembled, localized and activated in vivo remains to be established. Here we use tandem mass spectrometry to identify an Abi1-based complex containing WAVE2, Nap1 (Nck-associated protein) and PIR121. Abi1 interacts directly with the WHD domain of WAVE2, increases WAVE2 actin polymerization activity and mediates the assembly of a WAVE2-Abi1-Nap1-PIR121 complex. The WAVE2-Abi1-Nap1-PIR121 complex is as active as the WAVE2-Abi1 sub-complex in stimulating Arp2/3, and after Rac activation it is re-localized to the leading edge of ruffles in vivo. Consistently, inhibition of Abi1 by RNA interference (RNAi) abrogates Rac-dependent lamellipodia protrusion. Thus, Abi1 orchestrates the proper assembly of the WAVE2 complex and mediates its activation at the leading edge in vivo.     

Arp2/3 is a negative regulator of growth cone translocation

Arp2/3 is an actin binding complex that is enriched in the peripheral lamellipodia of fibroblasts, where it forms a network of short, branched actin filaments, generating the protrusive force that extends lamellipodia and drives fibroblast motility. Although it has been assumed that Arp2/3 would play a similar role in growth cones, our studies indicate that Arp2/3 is enriched in the central, not the peripheral, region of growth cones and that the growth cone periphery contains few branched actin filaments. Arp2/3 inhibition in fibroblasts severely disrupts actin organization and membrane protrusion. In contrast, Arp2/3 inhibition in growth cones minimally affects actin organization and does not inhibit lamellipodia protrusion or de novo filopodia formation. Surprisingly, Arp2/3 inhibition significantly enhances axon elongation and causes defects in growth cone guidance. These results indicate that Arp2/3 is a negative regulator of growth cone translocation.     

Arp2/3 complex is important for filopodia formation, growth cone motility, and neuritogenesis in neuronal cells

A role of Arp2/3 complex in lamellipodia is well established, whereas its roles in filopodia formation remain obscure. We addressed this question in neuronal cells, in which motility is heavily based on filopodia, and we found that Arp2/3 complex is involved in generation of both lamellipodia and filopodia in growth cones, and in neuritogenesis, the processes thought to occur largely in Arp2/3 complex-independent manner. Depletion of Arp2/3 complex in primary neurons and neuroblastoma cells by small interfering RNA significantly decreased the F-actin contents and inhibited lamellipodial protrusion and retrograde flow in growth cones, but also initiation and dynamics of filopodia. Using electron microscopy, immunochemistry, and gene expression, we demonstrated the presence of the Arp2/3 complex-dependent dendritic network of actin filaments in growth cones, and we showed that individual actin filaments in filopodia originated at Arp2/3 complex-dependent branch points in lamellipodia, thus providing a mechanistic explanation of Arp2/3 complex functions during filopodia formation. Additionally, Arp2/3 complex depletion led to formation of multiple neurites, erratic pattern of neurite extension, and excessive formation of stress fibers and focal adhesions. Consistent with this phenotype, RhoA activity was increased in Arp2/3 complex-depleted cells, indicating that besides nucleating actin filaments, Arp2/3 complex may influence cell motility by altering Rho GTPase signaling.     

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.     

Focal adhesion kinase controls actin assembly via a FERM-mediated interaction with the Arp2/3 complex

Networks of actin filaments, controlled by the Arp2/3 complex, drive membrane protrusion during cell migration. How integrins signal to the Arp2/3 complex is not well understood. Here, we show that focal adhesion kinase (FAK) and the Arp2/3 complex associate and colocalize at transient structures formed early after adhesion. Nascent lamellipodia, which originate at these structures, do not form in FAK-deficient cells, or in cells in which FAK mutants cannot be autophosphorylated after integrin engagement. The FERM domain of FAK binds directly to Arp3 and can enhance Arp2/3-dependent actin polymerization. Critically, Arp2/3 is not bound when FAK is phosphorylated on Tyr 397. Interfering peptides and FERM-domain point mutants show that FAK binding to Arp2/3 controls protrusive lamellipodia formation and cell spreading. This establishes a new function for the FAK FERM domain in forming a phosphorylation-regulated complex with Arp2/3, linking integrin signalling directly with the actin polymerization machinery.     

PtdIns(3,4,5)P3 binding is necessary for WAVE2-induced formation of lamellipodia

Polarized cell movement is triggered by the development of a PtdIns(3,4,5)P(3) gradient at the membrane, which is followed by rearrangement of the actin cytoskeleton. The WASP family verprolin homologous protein (WAVE) is essential for lamellipodium formation at the leading edge by activating the Arp2/3 complex downstream of Rac GTPase. Here, we report that WAVE2 binds to PtdIns(3,4,5)P(3) through its basic domain. The amino-terminal portion of WAVE2, which includes the PtdIns(3,4,5)P(3)-binding sequence, was localized at the leading edge of lamellipodia induced by an active form of Rac (RacDA) or by treatment with platelet-derived growth factor (PDGF). Production of PtdIns(3,4,5)P(3) at the cell membrane by myristoylated phosphatidylinositol-3-OH kinase (PI(3)K) is sufficient to recruit WAVE2 in the presence of dominant-negative Rac and latrunculin, demonstrating that PtdIns(3,4,5)P(3) alone is able to recruit WAVE2. Expression of a full-length mutant of WAVE2 that lacks the lipid-binding activity inhibited proper formation of lamellipodia induced by RacDA. These results suggest that one of the products of PI(3)K, PtdIns(3,4,5)P(3), recruits WAVE2 to the polarized membrane and that this recruitment is essential for lamellipodium formation at the leading edge.     

F-actin binding region of SPIN90 C-terminus is essential for actin polymerization and lamellipodia formation

We recently reported that SPIN90 is able to bind with several proteins involved in regulating actin cytoskeleton networks, including dynamin, WASP, β PIX, and Nck. Based on these findings, we investigated how SPIN90 regulates the actin cytoskeleton and promotes actin assembly. This study demonstrated that aluminium fluoride-induced localization of SPIN90 to lamellipodia requires amino acids 582-722 at the SPIN90 C-terminus, which is also essential for F-actin binding and Arp2/3 complex mediated polymerization of actin into branched actin filaments. Furthermore, after deletion of the F-actin binding region (582-722 SPIN90) failed to localize at the membrane edge and was unable to promote lamellipodia formation, suggesting that the F-actin binding region in the SPIN90 C-terminus is essential for the formation of branched actin networks and regulation of the actin cytoskeleton at the leading edge of cells.     

Cofilin phosphorylation and actin cytoskeletal dynamics regulated by rho- and Cdc42-activated LIM-kinase 2

The rapid turnover of actin filaments and the tertiary meshwork formation are regulated by a variety of actin-binding proteins. Protein phosphorylation of cofilin, an actin-binding protein that depolymerizes actin filaments, suppresses its function. Thus, cofilin is a terminal effector of signaling cascades that evokes actin cytoskeletal rearrangement. When wild-type LIMK2 and kinase-dead LIMK2 (LIMK2/KD) were respectively expressed in cells, LIMK2, but not LIMK2/KD, phosphorylated cofilin and induced formation of stress fibers and focal complexes. LIMK2 activity toward cofilin phosphorylation was stimulated by coexpression of activated Rho and Cdc42, but not Rac. Importantly, expression of activated Rho and Cdc42, respectively, induced stress fibers and filopodia, whereas both Rho- induced stress fibers and Cdc42-induced filopodia were abrogated by the coexpression of LIMK2/KD. In contrast, the coexpression of LIMK2/KD with the activated Rac did not affect Rac-induced lamellipodia formation. These results indicate that LIMK2 plays a crucial role both in Rho- and Cdc42-induced actin cytoskeletal reorganization, at least in part by inhibiting the functions of cofilin. Together with recent findings that LIMK1 participates in Rac-induced lamellipodia formation, LIMK1 and LIMK2 function under control of distinct Rho subfamily GTPases and are essential regulators in the Rho subfamilies-induced actin cytoskeletal reorganization.     

Novel mechanism of regulation of Rac activity and lamellipodia formation by RET tyrosine kinase

Rac activation in neuronal cells plays an important role in lamellipodia formation that is a critical event for neuritogenesis. It is well known that the Rac activity is regulated via activation of phosphatidylinositol 3-kinase (PI3K) by a variety of receptor tyrosine kinases. Here we show that increased serine phosphorylation on RET receptor tyrosine kinase following cAMP elevation promotes lamellipodia formation of neuronal cells induced by glial cell line-derived neurotrophic factor (GDNF). We identified serine 696 in RET as a putative phosphorylation site by protein kinase A and found that mutation of this serine almost completely inhibited lamellipodia formation by GDNF without affecting activation of the PI3K/AKT signaling pathway. Mutation of tyrosine 1062 in RET, whose phosphorylation is crucial for activation of PI3K, also inhibited lamellipodia formation by GDNF. Inhibition of lamellipodia formation by mutation of either serine 696 or tyrosine 1062 was associated with decrease of the Rac1-guanine nucleotide exchange factor (GEF) activity, suggesting that this activity is regulated by two different signaling pathways via serine 696 and tyrosine 1062 in RET. Moreover, in the presence of serine 696 mutation, lamellipodia formation was rescued by replacing tyrosine 687 with phenylalanine. These findings propose a novel mechanism that receptor tyrosine kinase modulates actin dynamics in neuronal cells via its cAMP-dependent phosphorylation.     

Pointed-end capping by tropomodulin3 negatively regulates endothelial cell motility

Actin filament pointed-end dynamics are thought to play a critical role in cell motility, yet regulation of this process remains poorly understood. We describe here a previously uncharacterized tropomodulin (Tmod) isoform, Tmod3, which is widely expressed in human tissues and is present in human microvascular endothelial cells (HMEC-1). Tmod3 is present in sufficient quantity to cap pointed ends of actin filaments, localizes to actin filament structures in HMEC-1 cells, and appears enriched in leading edge ruffles and lamellipodia. Transient overexpression of GFP-Tmod3 leads to a depolarized cell morphology and decreased cell motility. A fivefold increase in Tmod3 results in an equivalent decrease in free pointed ends in the cells. Unexpectedly, a decrease in the relative amounts of F-actin, free barbed ends, and actin-related protein 2/3 (Arp2/3) complex in lamellipodia are also observed. Conversely, decreased expression of Tmod3 by RNA interference leads to faster average cell migration, along with increases in free pointed and barbed ends in lamellipodial actin filaments. These data collectively demonstrate that capping of actin filament pointed ends by Tmod3 inhibits cell migration and reveal a novel control mechanism for regulation of actin filaments in lamellipodia.     

Scar1 and the related Wiskott–Aldrich syndrome protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex

Background: The actin-related proteins Arp2 and Arp3 are part of a seven-protein complex which is localized in the lamellipodia of a variety of cell types, and in actin-rich spots of unknown function. The Arp2/3 complex enhances actin nucleation and causes branching and crosslinking of actin filaments in vitro; in vivo it is thought to drive the formation of lamellipodia and to be a control center for actin-based motility. The Wiskott–Aldrich syndrome protein, WASP, is an adaptor protein implicated in the transmission of signals from tyrosine kinase receptors and small GTPases to the actin cytoskeleton. Scar1 is a member of a new family of proteins related to WASP, and it may also have a role in regulating the actin cytoskeleton. Scar1 is the human homologue of Dictyostelium Scar1, which is thought to connect G-protein-coupled receptors to the actin cytoskeleton. The mammalian Scar family contains at least four members. We have examined the relationships between WASP, Scar1, and the Arp2/3 complex.Results: We have identified WASP and its relative Scar1 as proteins that interact with the Arp2/3 complex. We have used deletion analysis to show that both WASP and Scar1 interact with the p21 subunit of the Arp2/3 complex through their carboxyl termini. Overexpression of carboxy-terminal fragments of Scar1 or WASP in cells caused a disruption in the localization of the Arp2/3 complex and, concomitantly, induced a complete loss of lamellipodia and actin spots. The induction of lamellipodia by platelet-derived growth factor was also suppressed by overexpression of the fragment of Scar1 that binds to the Arp2/3 complex.Conclusions: We have identified a conserved sequence domain in proteins of the WASP family that binds to the Arp2/3 complex. Overexpression of this domain in cells disrupts the localization of the Arp2/3 complex and inhibits lamellipodia formation. Our data suggest that WASP-related proteins may regulate the actin cytoskeleton through the Arp2/3 complex.