Linked regulation of motility and integrin function in activated migrating neutrophils revealed by interference in remodelling of the cytoskeleton

Neutrophils migrate rapidly by co-ordinating regulation of their beta2-integrin adhesion with turnover of filamentous F-actin. The seven-protein Arp2/3 complex regulates actin polymerisation upon activation by proteins of the WASP-family. To investigate links between actin polymerisation, adhesion, and migration, we used a novel osmotic-shock method to load neutrophils with peptides: (1). WASP-WA and Scar-WA (which incorporate the actin- and Arp2/3-binding regions of WASP and Scar1), to compete with endogenous WASP-family members; (2). proline rich motifs (PRM) from the ActA protein of L. monocytogenes or from vinculin, which bind vasodilator-stimulated phosphoprotein (VASP), a regulator of cytoskeleton assembly. In a flow system, rolling-adherent neutrophils were stimulated with formyl tri-peptide. This caused rapid immobilisation, followed by migration with increasing velocity, supported by activated beta2-integrin CD11b/CD18. Loading ActA PRM (but not vinculin PRM) caused concentration-dependent reduction in migration velocity. At the highest concentration, unstimulated neutrophils had elevated F-actin and were rigid, but could not change their F-actin content or shape upon stimulation. Scar-WA also caused marked reduction in migration rate, but WASP-WA had a lesser effect. Scar-WA did not modify activation-dependent formation of F-actin or change in shape. However, a reduction in rate of downregulation of integrin adhesion appeared to contribute to impaired migration. These studies show that interference in cytoskeletal reorganisation that follows activation in neutrophils, can impair regulation of integrin function as well as motility. They also suggest a role of the Arp2/3 complex and WASP-family in co-ordinating actin polymerisation and integrin function in migrating neutrophils.     

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.     

Activation of Arp2/3 complex: addition of the first subunit of the new filament by a WASP protein triggers rapid ATP hydrolysis on Arp2

In response to activation by WASP-family proteins, the Arp2/3 complex nucleates new actin filaments from the sides of preexisting filaments. The Arp2/3-activating (VCA) region of WASP-family proteins binds both the Arp2/3 complex and an actin monomer and the Arp2 and Arp3 subunits of the Arp2/3 complex bind ATP. We show that Arp2 hydrolyzes ATP rapidly—with no detectable lag—upon nucleation of a new actin filament. Filamentous actin and VCA together do not stimulate ATP hydrolysis on the Arp2/3 complex, nor do monomeric and filamentous actin in the absence of VCA. Actin monomers bound to the marine macrolide Latrunculin B do not polymerize, but in the presence of phalloidin-stabilized actin filaments and VCA, they stimulate rapid ATP hydrolysis on Arp2. These data suggest that ATP hydrolysis on the Arp2/3 complex is stimulated by interaction with a single actin monomer and that the interaction is coordinated by VCA. We show that capping of filament pointed ends by the Arp2/3 complex (which occurs even in the absence of VCA) also stimulates rapid ATP hydrolysis on Arp2, identifying the actin monomer that stimulates ATP hydrolysis as the first monomer at the pointed end of the daughter filament. We conclude that WASP-family VCA domains activate the Arp2/3 complex by driving its interaction with a single conventional actin monomer to form an Arp2–Arp3–actin nucleus. This actin monomer becomes the first monomer of the new daughter filament.     

Arp2/3 complex-independent actin regulatory function of WAVE

We report that WAVE1/Scar1, a WASP-family protein that functions downstream of Rac in membrane ruffling, can induce part of the reorganization of the actin cytoskeleton without Arp2/3 complex. WAVE1 has been reported to associate and activate Arp2/3 complex at its C-terminal region that is rich in acidic residues. The deletion of the acidic residues abolished the interaction with and the activation ability of Arp2/3 complex. The expression of the mutant WAVE1 lacking the acidic residues (DeltaA), however, induced actin-clustering in cells as the wild-type WAVE1 did. In addition, this actin-clustering could not be suppressed by the coexpression of the Arp2/3 complex-sequestering fragment (CA-region) derived from N-WASP, which clearly inhibits Rac-induced membrane ruffling. This study therefore demonstrates that WAVE1 reorganizes the actin cytoskeleton not only through Arp2/3 complex but also through another unidentified mechanism that may be important but has been neglected thus far.     

Src-dependent phosphorylation of Scar1 promotes its association with the Arp2/3 complex

The WAVE/Scar proteins regulate actin polymerisation at the leading edge of motile cells via activation of the Arp2/3 complex in response to extracellular cues. Within cells they form part of a pentameric complex that is thought to regulate their ability to interact and activate the Arp2/3 complex. However, the exact mechanism for this is not known. We set out to assess whether phosphorylation of Scar1 by the non-receptor tyrosine kinase Src may influence the function of Scar1 and its ability to regulate Arp2/3-mediated actin polymerisation. We show that Scar1 is phosphorylated by Src in vitro and in vivo and identify tyrosine 125 as the major site in Scar1 to be phosphorylated in cells. Src-dependent phosphorylation of Scar1 on tyrosine 125 enhances its ability to bind to the Arp2/3 complex and regulates its ability to control actin polymerisation in cells. Thus, Src may act as an intermediary to regulate the activity of the Arp2/3 complex in response to external stimuli, via modulation of its interaction with WAVE/Scar proteins.     

The WASP-Arp2/3 pathway: genetic insights

Arp2/3 complex nucleates the formation of dendritic actin filament arrays, which are especially prominent at the leading edges of motile cells. Recent genetic and other loss-of-function studies have highlighted the importance of the Arp2/3 complex for normal cell functions, and especially for cell motility. WASP/Scar family proteins regulate the activity of the Arp2/3 complex, and also link it to several signaling pathways. Recent studies suggest that Scar is a more important regulator of Arp2/3 activity in actin-dependent morphological processes than WASP, which may have a more restricted role in specialized cellular events. It has also become clear that precise regulation of both Scar and WASP activity is of the utmost importance for their physiological functions.     

Synthetic Triterpenoids Target the Arp2/3 Complex and Inhibit Branched Actin Polymerization

Synthetic triterpenoids are anti-tumor agents that affect numerous cellular functions including apoptosis and growth inhibition. Here, we used mass spectrometric and protein array approaches and uncovered that triterpenoids associate with proteins of the actin cytoskeleton, including actin-related protein 3 (Arp3). Arp3, a subunit of the Arp2/3 complex, is involved in branched actin polymerization and the formation of lamellipodia. 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)-Im and CDDO-Me were observed to 1) inhibit the localization of Arp3 and actin at the leading edge of cells, 2) abrogate cell polarity, and 3) inhibit Arp2/3-dependent branched actin polymerization. We confirmed our drug effects with siRNA targeting of Arp3 and observed a decrease in Rat2 cell migration. Taken together, our data suggest that synthetic triterpenoids target Arp3 and branched actin polymerization to inhibit cell migration.     

Phosphorylation of the Arp2/3 complex is necessary to nucleate actin filaments

The actin-related protein 2/3 (Arp2/3) complex is the primary nucleator of new actin filaments in most crawling cells. Nucleation-promoting factors (NPFs) of the Wiskott-Aldrich syndrome protein (WASP)/Scar family are the currently recognized activators of the Arp2/3 complex. We now report that the Arp2/3 complex must be phosphorylated on either threonine or tyrosine residues to be activated by NPFs. Phosphorylation of the Arp2/3 complex is not necessary to bind NPFs or the sides of actin filaments but is critical for binding the pointed end of actin filaments and nucleating actin filaments. Mass spectrometry revealed phosphorylated Thr237 and Thr238 in Arp2, which are evolutionarily conserved residues. In cells, phosphorylation of only the Arp2 subunit increases in response to growth factors, and alanine substitutions of Arp2 T237 and T238 or Y202 inhibits membrane protrusion. These findings reveal an additional level of regulation of actin filament assembly independent of WASP proteins, and show that phosphorylation of the Arp2/3 complex provides a logical “or gate” capable integrating diverse upstream signals.     

Different WASP family proteins stimulate different Arp2/3 complex-dependent actin-nucleating activities

Background: Assembly and organization of actin filaments are required for many cellular processes, including locomotion and division. In many cases, actin assembly is initiated when proteins of the WASP/Scar family respond to signals from Rho family G proteins and stimulate the actin-nucleating activity of the Arp2/3 complex. Two questions of fundamental importance raised in the study of actin dynamics concern the molecular mechanism of Arp2/3-dependent actin nucleation and how different signaling pathways that activate the same Arp2/3 complex produce actin networks with different three-dimensional architectures?Results: We directly compared the activity of the Arp2/3 complex in the presence of saturating concentrations of the minimal Arp2/3-activating domains of WASP, N-WASP, and Scar1 and found that each induces unique kinetics of actin assembly. In cell extracts, N-WASP induces rapid actin polymerization, while Scar1 fails to induce detectable polymerization. Using purified proteins, Scar1 induces the slowest rate of nucleation. WASP activity is 16-fold higher, and N-WASP activity is 70-fold higher. The data for all activators fit a mathematical model in which one activated Arp2/3 complex, one actin monomer, and an actin filament combine into a preactivation complex which then undergoes a first-order activation step to become a nucleus. The differences between Scar and N-WASP activity are explained by differences in the rate constants for the activation step. Changing the number of actin binding sites on a WASP family protein, either by removing a WH2 domain from N-WASP or by adding WH2 domains to Scar1, has no significant effect on nucleation activity. The addition of a three amino acid insertion found in the C-terminal acidic domains of WASP and N-WASP, however, increases the activity of Scar1 by more than 20-fold. Using chemical crosslinking assays, we determined that both N-WASP and Scar1 induce a conformational change in the Arp2/3 complex but crosslink with different efficiencies to the small molecular weight subunits p18 and p14.Conclusion: The WA domains of N-WASP, WASP, and Scar1 bind actin and Arp2/3 with nearly identical affinities but stimulate rates of actin nucleation that vary by almost 100-fold. The differences in nucleation rate are caused by differences in the number of acidic amino acids at the C terminus, so each protein is tuned to produce a different rate of actin filament formation. Arp2/3, therefore, is not regulated by a simple on-off switch. Precise tuning of the filament formation rate may help determine the architecture of actin networks produced by different nucleation-promoting factors.     

Arp2/3 complex regulates asymmetric division and cytokinesis in mouse oocytes

Mammalian oocyte meiotic maturation involves oocyte polarization and a unique asymmetric division, but until now, the underlying mechanisms have been poorly understood. Arp2/3 complex has been shown to regulate actin nucleation and is widely involved in a diverse range of processes such as cell locomotion, phagocytosis and the establishment of cell polarity. Whether Arp2/3 complex participates in oocyte polarization and asymmetric division is unknown. The present study investigated the expression and functions of Arp2/3 complex during mouse oocyte meiotic maturation. Immunofluorescent staining showed that the Arp2/3 complex was restricted to the cortex, with a thickened cap above the meiotic apparatus, and that this localization pattern was depended on actin. Disruption of Arp2/3 complex by a newly-found specific inhibitor CK666, as well as by Arpc2 and Arpc3 RNAi, resulted in a range of effects. These included the failure of asymmetric division, spindle migration, and the formation and completion of oocyte cytokinesis. The formation of the actin cap and cortical granule-free domain (CGFD) was also disrupted, which further confirmed the disruption of spindle migration. Our data suggest that the Arp2/3 complex probably regulates oocyte polarization through its effect on spindle migration, asymmetric division and cytokinesis during mouse oocyte meiotic maturation.     

The Arp2/3 complex is essential for the actin-based motility of Listeria monocytogenes.

Actin polymerisation is thought to drive the movement of eukaryotic cells and some intracellular pathogens such as Listeria monocytogenes. The Listeria surface protein ActA synergises with recruited host proteins to induce actin polymerisation, propelling the bacterium through the host cytoplasm [1]. The Arp2/3 complex is one recruited host factor [2] [3]; it is also believed to regulate actin dynamics in lamellipodia [4] [5]. The Arp2/3 complex promotes actin filament nucleation in vitro, which is further enhanced by ActA [6] [7]. The Arp2/3 complex also interacts with members of the Wiskott-Aldrich syndrome protein (WASP) [8] family - Scar1 [9] [10] and WASP itself [11]. We interfered with the targeting of the Arp2/3 complex to Listeria by using carboxy-terminal fragments of Scar1 that bind the Arp2/3 complex [11]. These fragments completely blocked actin tail formation and motility of Listeria, both in mouse brain extract and in Ptk2 cells overexpressing Scar1 constructs. In both systems, Listeria could initiate actin cloud formation, but tail formation was blocked. Full motility in vitro was restored by adding purified Arp2/3 complex. We conclude that the Arp2/3 complex is a host-cell factor essential for the actin-based motility of L. monocytogenes, suggesting that it plays a pivotal role in regulating the actin cytoskeleton.     

The branched actin nucleator Arp2/3 promotes nuclear migrations and cell polarity in the C. elegans zygote

Regulated movements of the nucleus are essential during zygote formation, cell migrations, and differentiation of neurons. The nucleus moves along microtubules (MTs) and is repositioned on F-actin at the cellular cortex. Two families of nuclear envelope proteins, SUN and KASH, link the nucleus to the actin and MT cytoskeletons during nuclear movements. However, the role of actin nucleators in nuclear migration and positioning is poorly understood. We show that the branched actin nucleator, Arp2/3, affects nuclear movements throughout embryonic and larval development in C. elegans, including nuclear migrations in epidermal cells and neuronal precursors. In one-cell embryos the migration of the male pronucleus to meet the female pronucleus after fertilization requires Arp2/3. Loss of Arp2/3 or its activators changes the dynamics of non-muscle myosin, NMY-2, and alters the cortical accumulation of posterior PAR proteins. Reduced establishment of the posterior microtubule cytoskeleton in Arp2/3 mutants correlates with reduced male pronuclear migration. The UNC-84/SUN nuclear envelope protein that links the nucleus to the MT and actin cytoskeleton is known to regulate later nuclear migrations. We show here it also positions the male pronucleus. These studies demonstrate a global role for Arp2/3 in nuclear migrations. In the C. elegans one-cell embryo Arp2/3 promotes the establishment of anterior/posterior polarity and promotes MT growth that propels the anterior migration of the male pronucleus. In contrast with previous studies emphasizing pulling forces on the male pronucleus, we propose that robust MT nucleation pushes the male pronucleus anteriorly to join the female pronucleus.     

Nucleation Promoting Factors Regulate the Expression and Localization of Arp2/3 Complex during Meiosis of Mouse Oocytes

The actin nucleation factor Arp2/3 complex is a main regulator of actin assembly and is involved in multiple processes like cell migration and adhesion, endocytosis, and the establishment of cell polarity in mitosis. Our previous work showed that the Arp2/3 complex was involved in the actin-mediated mammalian oocyte asymmetric division. However, the regulatory mechanisms and signaling pathway of Arp2/3 complex in meiosis is still unclear. In the present work, we identified that the nucleation promoting factors (NPFs) JMY and WAVE2 were necessary for the expression and localization of Arp2/3 complex in mouse oocytes. RNAi of both caused the degradation of actin cap intensity, indicating the roles of NPFs in the formation of actin cap. Moreover, JMY and WAVE2 RNAi decreased the expression of ARP2, a key component of Arp2/3 complex. However, knock down of Arp2/3 complex by Arpc2 and Arpc3 siRNA microinjection did not affect the expression and localization of JMY and WAVE2. Our results indicate that the NPFs, JMY and WAVE2, are upstream regulators of Arp2/3 complex in mammalian oocyte asymmetric division.     

Arabidopsis BRICK1/HSPC300 is an essential WAVE-complex subunit that selectively stabilizes the Arp2/3 activator SCAR2

The actin cytoskeleton dynamically reorganizes the cytoplasm during cell morphogenesis. The actin-related protein (Arp)2/3 complex is a potent nucleator of actin filaments that controls a variety of endomembrane functions including the endocytic internalization of plasma membrane , vacuole biogenesis , plasma-membrane protrusion in crawling cells , and membrane trafficking from the Golgi . Therefore, Arp2/3 is an important signaling target during morphogenesis. The evolutionarily conserved Rac-WAVE-Arp2/3 pathway links actin filament nucleation to cell morphogenesis . WAVE translates Rac-GTP signals into Arp2/3 activation by regulating the stability and/or localization of the activator subunit Scar/WAVE . The WAVE complex includes Sra1/PIR121/CYFIP1, Nap1/NAP125, Abi-1/Abi-2, Brick1(Brk1)/HSPC300, and Scar/WAVE : Defining the in vivo function of each subunit is an important step toward understanding this complicated signaling pathway. Brk1/HSPC300 has been the most recalcitrant WAVE-complex protein and has no known function. In this paper, we report that Arabidopsis brick1 (brk1) is a member of the "distorted group" of trichome morphology mutants, a group that defines a WAVE-ARP2/3 morphogenesis pathway . In this paper we provide the first strong genetic and biochemical evidence that BRK1 is a critical WAVE-complex subunit that selectively stabilizes the Arp2/3 activator SCAR2.     

The Rac1 inhibitor,NSC23766, depolarizes adhesive secretion,endomembrane cycling,and tip growth in the fucoid alga, <Emphasis Type="Italic">Silvetia compressa</Emphasis>

Proper cell morphogenesis is dependent on the establishment and expression of cellular polarity. In the fucoid zygote, cell shape is critical for establishing the developmental pattern of the adult, and is achieved by guiding insertion of new membrane and wall to the rhizoid tip. Selection and growth of the appropriate tip site are accompanied by formation of dynamic actin arrays associated with the actin-nucleating Arp2/3 complex. In eukaryotes, a major pathway for activation of the Arp2/3 complex is via the Rho family GTPase, Rac1, which stimulates the Scar/WAVE complex. To determine whether Rac1 controls actin nucleation in Silvetia compressa (J. Agardh) E. Serrao, T. O. Cho, S. M. Boo et Brawley, we tested the effects of the Rac1-specific inhibitory compound, NSC23766, on actin dependent processes and on actin arrays. We found that NSC23766 disrupted polar secretion of adhesive, polarization of endomembranes, and tip-focused growth in the rhizoid. Similarly, NSC23766 altered actin and Arp2 localization in the growing rhizoid. In contrast, NSC23766 had no effect on selection of the growth site or on cytokinesis. These data suggest that Rac1 participates in nucleation of specific actin arrays in the developing zygote.     

Fascin-induced actin protrusions are suppressed by dendritic networks in giant unilamellar vesicles

The interactions between actin networks and cell membrane are immensely important for eukaryotic cell functions including cell shape changes, motility, polarity establishment, and adhesion. Actin-binding proteins are known to compete and cooperate using a finite amount of actin monomers to form distinct actin networks. How actin-bundling protein fascin and actin-branching protein Arp2/3 complex compete to remodel membranes is not entirely clear. To investigate fascin- and Arp2/3-mediated actin network remodeling, we applied a reconstitution approach encapsulating bundled and dendritic actin networks inside giant unilamellar vesicles (GUVs). Independently reconstituted, membrane-bound Arp2/3 nucleation forms an actin cortex in GUVs, whereas fascin mediates formation of actin bundles that protrude out of GUVs. Coencapsulating both fascin and Arp2/3 complex leads to polarized dendritic aggregates and significantly reduces membrane protrusions, irrespective of whether the dendritic network is membrane bound or not. However, reducing Arp2/3 complex while increasing fascin restores membrane protrusion. Such changes in network assembly and the subsequent interplay with membrane can be attributed to competition between fascin and Arp2/3 complex to utilize a finite pool of actin.     

Activation of the Arp2/3 complex by the Listeria acta protein. Acta binds two actin monomers and three subunits of the Arp2/3 complex

ActA is a bacterially encoded protein that enables Listeria monocytogenes to hijack the host cell actin cytoskeleton. It promotes Arp2/3-dependent actin nucleation, but its interactions with cellular components of the nucleation machinery are not well understood. Here we show that two domains of ActA (residues 85-104 and 121-138) with sequence similarity to WASP homology 2 domains bind two actin monomers with submicromolar affinity. ActA binds Arp2/3 with a K(d) of 0.6 microm and competes for binding with the WASP family proteins N-WASP and Scar1. By chemical cross-linking, ActA, N-WASP, and Scar1 contact the same three subunits of the Arp2/3 complex, p40, Arp2, and Arp3. Interestingly, profilin competes with ActA for binding of Arp2/3, but actophorin (cofilin) does not. The minimal Arp2/3-binding site of ActA (residues 144-170) is C-terminal to both actin-binding sites and shares sequence homology with Arp2/3-binding regions of WASP family proteins. The maximal activity at saturating concentrations of ActA is identical to the most active domains of the WASP family proteins. We propose that ActA and endogenous WASP family proteins promote Arp2/3-dependent nucleation by similar mechanisms and require simultaneous binding of Arp2 and Arp3.     

Mechanism of a concentration-dependent switch between activation and inhibition of Arp2/3 complex by coronin

Arp2/3 complex is a key actin filament nucleator that assembles branched actin networks in response to cellular signals. The activity of Arp2/3 complex is regulated by both activating and inhibitory proteins. Coronins make up a large class of actin-binding proteins previously shown to inhibit Arp2/3 complex. Although coronins are known to play a role in controlling actin dynamics in diverse processes, including endocytosis and cell motility, the precise mechanism by which they regulate Arp2/3 complex is unclear. We conducted a detailed biochemical analysis of budding yeast coronin, Crn1, and found that it not only inhibits Arp2/3 complex but also activates it. We mapped regions required for activation and found that Crn1 contains a sequence called CA, which is conserved in WASp/Scar proteins, the prototypical activators of Arp2/3 complex. Point mutations in CA abolished activation of Arp2/3 complex by Crn1 in vitro. Confocal microscopy and quantitative actin patch tracking showed that these mutants had defective endocytic actin patch dynamics in Saccharomyces cerevisiae, indicating that activation of Arp2/3 complex by coronin is required for normal actin dynamics in vivo. The switch between the dual modes of regulation by Crn1 is controlled by concentration, and low concentrations of Crn1 enhance filament binding by Arp2/3 complex, whereas high concentrations block binding. Our data support a direct tethering recruitment model for activation of Arp2/3 complex by Crn1 and suggest that Crn1 indirectly inhibits Arp2/3 complex by blocking it from binding actin filaments.     

Extending the Arp2/3 complex and its regulation beyond the leading edge

Two studies characterizing Drosophila Arp2/3 complex and Scar mutants demonstrate that assembly of some actin structures in nonmotile cells of multicellular organisms utilizes the same proteins as are important for actin assembly in motile cells. These studies also show that assembly of other actin structures is independent of these proteins, suggesting that alternative mechanisms also exist.     

SCAR is a primary regulator of Arp2/3-dependent morphological events in Drosophila

The Arp2/3 complex and its activators, Scar/WAVE and Wiskott-Aldrich Syndrome protein (WASp), promote actin polymerization in vitro and have been proposed to influence cell shape and motility in vivo. We demonstrate that the Drosophila Scar homologue, SCAR, localizes to actin-rich structures and is required for normal cell morphology in multiple cell types throughout development. In particular, SCAR function is essential for cytoplasmic organization in the blastoderm, axon development in the central nervous system, egg chamber structure during oogenesis, and adult eye morphology. Highly similar developmental requirements are found for subunits of the Arp2/3 complex. In the blastoderm, SCAR and Arp2/3 mutations result in a reduction in the amount of cortical filamentous actin and the disruption of dynamically regulated actin structures. Remarkably, the single Drosophila WASp homologue, Wasp, is largely dispensable for these numerous Arp2/3-dependent functions, whereas SCAR does not contribute to cell fate decisions in which Wasp and Arp2/3 play an essential role. These results identify SCAR as a major component of Arp2/3-dependent cell morphology during Drosophila development and demonstrate that the Arp2/3 complex can govern distinct cell biological events in response to SCAR and Wasp regulation.