Abi Is Required for Modulation and Stability but Not Localization or Activation of the SCAR/WAVE Complex

The SCAR/WAVE complex drives actin-based protrusion, cell migration, and cell separation during cytokinesis. However, the contribution of the individual complex members to the activity of the whole remains a mystery. This is primarily because complex members depend on one another for stability, which limits the scope for experimental manipulation. Several studies suggest that Abi, a relatively small complex member, connects signaling to SCAR/WAVE complex localization and activation through its polyproline C-terminal tail. We generated a deletion series of the Dictyostelium discoideum Abi to investigate its exact role in regulation of the SCAR complex and identified a minimal fragment that would stabilize the complex. Surprisingly, loss of either the N terminus of Abi or the C-terminal polyproline tail conferred no detectable defect in complex recruitment to the leading edge or the formation of pseudopods. A fragment containing approximately 20% Abi—and none of the sites that couple to known signaling pathways—allowed the SCAR complex to function with normal localization and kinetics. However, expression of N-terminal Abi deletions exacerbated the cytokinesis defect of the Dictyostelium abi mutant, which was earlier shown to be caused by the inappropriate activation of SCAR. This demonstrates, unexpectedly, that Abi does not mediate the SCAR complex''s ability to make pseudopods, beyond its role in complex stability. Instead, we propose that Abi has a modulatory role when the SCAR complex is activated through other mechanisms.     

Arabidopsis NAP and PIR regulate actin-based cell morphogenesis and multiple developmental processes

The actin cytoskeleton mediates cellular processes through the dynamic regulation of the time, location, and extent of actin polymerization. Actin polymerization is controlled by several types of evolutionarily conserved proteins, including those comprising the ARP2/3 complex. In animal cells ARP2/3 activity is regulated by WAVE complexes that contain WAVE/SCAR proteins, PIR121, Nap125, and other proteins. The activity of the WAVE complex is regulated by Rho-GTPase-mediated signaling that leads to ARP2/3 activation by WAVE/SCAR proteins. We describe in this report Arabidopsis (Arabidopsis thaliana) genes encoding Nap and PIR proteins. Light-grown Atnap-1 and Atpir-1 mutant plants displayed altered leaf, inflorescence, silique, and seed set phenotypes. Dark-grown Atnap-1 and Atpir-1 seedlings also exhibited longer roots, enhanced skotomorphogenesis and Glc responses, and shorter thicker hypocotyls than those of wild type, showing that AtNAP and AtPIR participate in a variety of growth and developmental processes. Mutations in AtNAP and AtPIR caused cell morphology defects in cotyledon pavement cells and trichomes seen in mutants in ARP2/3 subunits and in plants expressing constitutively active Rop2 GTPase. The patterns and levels of actin polymerization observed in Atnap-1 and Atpir-1 mutant trichome cells and epidermal pavement cell morphology is consistent with Arabidopsis NAP and PIR proteins forming a WAVE complex that activates ARP2/3 activity. The multiple growth and developmental phenotypes of Atnap and Atpir mutants reveals these proteins are also required for a wider variety of cellular functions in addition to regulating trichome cell growth.     

Abi, Sra1, and Kette control the stability and localization of SCAR/WAVE to regulate the formation of actin-based protrusions

BACKGROUND: In animal cells, GTPase signaling pathways are thought to generate cellular protrusions by modulating the activity of downstream actin-regulatory proteins. Although the molecular events linking activation of a GTPase to the formation of an actin-based process with a characteristic morphology are incompletely understood, Rac-GTP is thought to promote the activation of SCAR/WAVE, whereas Cdc42 is thought to initiate the formation of filopodia through WASP. SCAR and WASP then activate the Arp2/3 complex to nucleate the formation of new actin filaments, which through polymerization exert a protrusive force on the membrane. RESULTS: Using RNAi to screen for genes regulating cell form in an adherent Drosophila cell line, we identified a set of genes, including Abi/E3B1, that are absolutely required for the formation of dynamic protrusions. These genes delineate a pathway from Cdc42 and Rac to SCAR and the Arp2/3 complex. Efforts to place Abi in this signaling hierarchy revealed that Abi and two components of a recently identified SCAR complex, Sra1 (p140/PIR121/CYFIP) and Kette (Nap1/Hem), protect SCAR from proteasome-mediated degradation and are critical for SCAR localization and for the generation of Arp2/3-dependent protrusions. CONCLUSIONS: In Drosophila cells, SCAR is regulated by Abi, Kette, and Sra1, components of a conserved regulatory SCAR complex. By controlling the stability, localization, and function of SCAR, these proteins may help to ensure that Arp2/3 activation and the generation of actin-based protrusions remain strictly dependant on local GTPase signaling.     

Membrane-targeted WAVE mediates photoreceptor axon targeting in the absence of the WAVE complex in Drosophila

A tight spatial-temporal coordination of F-actin dynamics is crucial for a large variety of cellular processes that shape cells. The Abelson interactor (Abi) has a conserved role in Arp2/3-dependent actin polymerization, regulating Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous protein (WAVE). In this paper, we report that Abi exerts nonautonomous control of photoreceptor axon targeting in the Drosophila visual system through WAVE. In abi mutants, WAVE is unstable but restored by reexpression of Abi, confirming that Abi controls the integrity of the WAVE complex in vivo. Remarkably, expression of a membrane-tethered WAVE protein rescues the axonal projection defects of abi mutants in the absence of the other subunits of the WAVE complex, whereas cytoplasmic WAVE only slightly affects the abi mutant phenotype. Thus complex formation not only stabilizes WAVE, but also provides further membrane-recruiting signals, resulting in an activation of WAVE.     

Abi activates WASP to promote sensory organ development

Actin polymerization is a key process for many cellular events during development. To a large extent, the formation of filamentous actin is controlled by the WASP and WAVE proteins that activate the Arp2/3 complex in different developmental processes. WAVE function is regulated through a protein complex containing Sra1, Kette and Abi. Using biochemical, cell biological and genetic tools, we show here that the Abi protein also has a central role in activating WASP-mediated processes. Abi binds WASP through its carboxy-terminal domain and acts as a potent stimulator of WASP-dependent F-actin formation. To elucidate the biological function of abi in Drosophila melanogaster, we studied bristle development, a process known to require wasp function. Reduction of abi function leads to a loss of bristles similar to that observed in wasp mutants. Activation of Abi results in the formation of ectopic bristles, a phenotype that is suppressed by a reduction of wasp activity but is not affected by the reduction of wave function. Thus, in vivo Abi may set the balance between WASP and WAVE in different actin-based developmental processes.     

IRREGULAR TRICHOME BRANCH1 in Arabidopsis encodes a plant homolog of the actin-related protein2/3 complex activator Scar/WAVE that regulates actin and microtubule organization

The dynamic actin cytoskeleton is important for a myriad of cellular functions, including intracellular transport, cell division, and cell shape. An important regulator of actin polymerization is the actin-related protein2/3 (Arp2/3) complex, which nucleates the polymerization of new actin filaments. In animals, Scar/WAVE family members activate Arp2/3 complex-dependent actin nucleation through interactions with Abi1, Nap1, PIR121, and HSCP300. Mutations in the Arabidopsis thaliana genes encoding homologs of Arp2/3 complex subunits PIR121 and NAP1 all show distorted trichomes as well as additional epidermal cell expansion defects, suggesting that a Scar/WAVE homolog functions in association with PIR121 and NAP1 to activate the Arp2/3 complex in Arabidopsis. In a screen for trichome branching defects, we isolated a mutant that showed irregularities in trichome branch positioning and expansion. We named this gene IRREGULAR TRICHOME BRANCH1 (ITB1). Positional cloning of the ITB1 gene showed that it encodes SCAR2, an Arabidopsis protein related to Scar/WAVE. Here, we show that itb1 mutants display cell expansion defects similar to those reported for the distorted class of trichome mutants, including disruption of actin and microtubule organization. In addition, we show that the scar homology domain (SHD) of ITB1/SCAR2 is necessary and sufficient for in vitro binding to Arabidopsis BRK1, the plant homolog of HSPC300. Overexpression of the SHD in transgenic plants causes a dominant negative phenotype. Our results extend the evidence that the Scar/WAVE pathway of Arp2/3 complex regulation exists in plants and plays an important role in regulating cell expansion.     

The N-terminus of Dictyostelium Scar interacts with Abi and HSPC300 and is essential for proper regulation and function

Scar/WAVE proteins, members of the conserved Wiskott-Aldrich syndrome (WAS) family, promote actin polymerization by activating the Arp2/3 complex. A number of proteins, including a complex containing Nap1, PIR121, Abi1/2, and HSPC300, interact with Scar/WAVE, though the role of this complex in regulating Scar function remains unclear. Here we identify a short N-terminal region of Dictyostelium Scar that is necessary and sufficient for interaction with HSPC300 and Abi in vitro. Cells expressing Scar lacking this N-terminal region show abnormalities in F-actin distribution, cell morphology, movement, and cytokinesis. This is true even in the presence of wild-type Scar. The data suggest that the first 96 amino acids of Scar are necessary for participation in a large-molecular-weight protein complex, and that this Scar-containing complex is responsible for the proper localization and regulation of Scar. The presence of mis-regulated or unregulated Scar has significant deleterious effects on cells and may explain the need to keep Scar activity tightly controlled in vivo either by assembly in a complex or by rapid degradation.     

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.     

NESH (Abi-3) is present in the Abi/WAVE complex but does not promote c-Abl-mediated phosphorylation

Abl interactor (Abi) was identified as an Abl tyrosine kinase-binding protein and subsequently shown to be a component of the macromolecular Abi/WAVE complex, which is a key regulator of Rac-dependent actin polymerization. Previous studies showed that Abi-1 promotes c-Abl-mediated phosphorylation of Mammalian Enabled (Mena) and WAVE2. In addition to Abi-1, mammals possess Abi-2 and NESH (Abi-3). In this study, we compared the three Abi proteins in terms of the promotion of c-Abl-mediated phosphorylation and the formation of Abi/WAVE complex. Although Abi-2, like Abi-1, promoted the c-Abl-mediated phosphorylation of Mena and WAVE2, NESH (Abi-3) had no such effect. This difference was likely due to their binding abilities as to c-Abl. Immunoprecipitation revealed that NESH (Abi-3) is present in the Abi/WAVE complex. Our results suggest that NESH (Abi-3), like Abi-1 and Abi-2, is a component of the Abi/WAVE complex, but likely plays a different role in the regulation of c-Abl.     

WAVE2 signaling mediates invasion of polarized epithelial cells by Salmonella typhimurium

The bacterial pathogen Salmonella penetrates the intestinal epithelium by inducing its own phagocytosis into epithelial cells. The dramatic reorganization of the actin cytoskeleton required for internalization is driven by bacterial manipulation of host signaling pathways, including activation of the Rho family GTPase Rac1 and subsequent activation of the Arp2/3 complex. However, the mechanisms linking these two events remain poorly understood. Rac1 is thought to promote activation of the Arp2/3 complex through its interaction with suppressor of cAMP receptor/WASP family verprolin-homologous (SCAR/WAVE) family proteins, but this interaction is apparently indirect. Two different Rac1 effectors have been shown to bind WAVE2: IRSp53, the SH3 domain of which binds the WAVE2 proline-rich domain, and PIR121/Sra-1, which forms a pentameric complex containing WAVE, Abi1, Nap1, and HSPC300. However, the extent to which each of these complexes contributes to Arp2/3 complex activation in the context of Salmonella infection is unclear. Here, we show that WAVE2 is necessary for efficient invasion of epithelial cells by Salmonella typhimurium. We found that although Salmonella infection strongly promotes the formation of an IRSp53/WAVE2 complex, IRSp53 is not necessary for bacterial internalization. In contrast, disruption of the PIR121/Nap1/Abi1/WAVE2/HSPC300 complex potently inhibits bacterial uptake. These results indicate that WAVE2 is an important component in signaling pathways leading to Salmonella invasion. Although infection leads to the formation of an IRSp53/WAVE2 complex, it is the association of WAVE2 with the Abi1/Nap1/PIR121/HSPC300 complex that regulates bacterial internalization.     

Abi1 regulates the activity of N-WASP and WAVE in distinct actin-based processes

Neural Wiskott-Aldrich syndrome protein (N-WASP) and WAVE are members of a family of proteins that use the Arp2/3 complex to stimulate actin assembly in actin-based motile processes. By entering into distinct macromolecular complexes, they act as convergent nodes of different signalling pathways. The role of WAVE in generating lamellipodial protrusion during cell migration is well established. Conversely, the precise cellular functions of N-WASP have remained elusive. Here, we report that Abi1, an essential component of the WAVE protein complex, also has a critical role in regulating N-WASP-dependent function. Consistently, Abi1 binds to N-WASP with nanomolar affinity and, cooperating with Cdc42, potently induces N-WASP activity in vitro. Molecular genetic approaches demonstrate that Abi1 and WAVE, but not N-WASP, are essential for Rac-dependent membrane protrusion and macropinocytosis. Conversely, Abi1 and N-WASP, but not WAVE, regulate actin-based vesicular transport, epidermal growth factor receptor (EGFR) endocytosis, and EGFR and transferrin receptor (TfR) cell-surface distribution. Thus, Abi1 is a dual regulator of WAVE and N-WASP activities in specific processes that are dependent on actin dynamics.     

SCAR knockouts in Dictyostelium: WASP assumes SCAR's position and upstream regulators in pseudopods

Under normal conditions, the Arp2/3 complex activator SCAR/WAVE controls actin polymerization in pseudopods, whereas Wiskott-Aldrich syndrome protein (WASP) assembles actin at clathrin-coated pits. We show that, unexpectedly, Dictyostelium discoideum SCAR knockouts could still spread, migrate, and chemotax using pseudopods driven by the Arp2/3 complex. In the absence of SCAR, some WASP relocated from the coated pits to the leading edge, where it behaved with similar dynamics to normal SCAR, forming split pseudopods and traveling waves. Pseudopods colocalized with active Rac, whether driven by WASP or SCAR, though Rac was activated to a higher level in SCAR mutants. Members of the SCAR regulatory complex, in particular PIR121, were not required for WASP regulation. We thus show that WASP is able to respond to all core upstream signals and that regulators coupled through the other members of SCAR's regulatory complex are not essential for pseudopod formation. We conclude that WASP and SCAR can regulate pseudopod actin using similar mechanisms.     

The role of Arabidopsis SCAR genes in ARP2-ARP3-dependent cell morphogenesis

The actin-nucleating ARP2-ARP3 complex controls cell shape in plants in many different cell types. Its activity is controlled by a multimeric complex containing BRK1 (also known as HSPC300), NAP1, SRA1, ABI and SCAR/WAVE. In this study, we focus on the function of the five putative SCAR homologues in Arabidopsis and we provide biochemical evidence that AtSCAR2 can activate the ARP2-ARP3 complex in vitro. Among the single mutants, mutations in only AtSCAR2 result in a subtle or weak phenotype similar to ARP2, ARP3 and other ;distorted' mutants. Double-mutant analysis revealed a redundancy with AtSCAR4. Systematic application of the yeast two-hybrid system and Bimolecular Fluorescence Complementation (BiFC) revealed a complex protein-interaction network between the ARP2-ARP3 complex and its genetically defined regulators. In addition to protein interactions known in other systems, we identified several new interactions, suggesting that SPIKE1 may be an integral component of the SCAR/WAVE complex and that SCAR proteins in plants might act as direct effectors of ROP GTPases.     

WAVE/SCAR, a multifunctional complex coordinating different aspects of neuronal connectivity

Although it is well established that the WAVE/SCAR complex transduces Rac1 signaling to trigger Arp2/3-dependent actin nucleation, regulatory mechanisms of this complex and its versatile function in the nervous system are poorly understood. Here we show that the Drosophila proteins SCAR, CYFIP and Kette, orthologs of WAVE/SCAR complex components, all show strong accumulation in axons of the central nervous system and indeed form a complex in vivo. Neuronal defects of SCAR, CYFIP and Kette mutants are, despite the initially proposed function of CYFIP and Kette as SCAR silencers, indistinguishable and are as diverse as ectopic midline crossing and nerve branching as well as synapse undergrowth at the larval neuromuscular junction. The common phenotypes of the single mutants are readily explained by the finding that loss of any one of the three proteins leads to degradation of its partners. As a consequence, each mutant is unambiguously to be judged as defective in multiple components of the complex even though each component affects different signaling pathways. Indeed, SCAR-Arp2/3 signaling is known to control axonogenesis whereas CYFIP signaling to the Fragile X Mental Retardation Protein fly ortholog contributes to synapse morphology. Thus, our results identify the Drosophila WAVE/SCAR complex as a multifunctional unit orchestrating different pathways and aspects of neuronal connectivity.     

WAVE forms hetero- and homo-oligomeric complexes at integrin junctions in Drosophila visualized by bimolecular fluorescence complementation

Dynamic actin polymerization drives a variety of morphogenetic events during metazoan development. Members of the WASP/WAVE protein family are central nucleation-promoting factors. They are embedded within regulatory networks of macromolecular complexes controlling Arp2/3-mediated actin nucleation in time and space. WAVE (Wiskott-Aldrich syndrome protein family verprolin-homologous protein) proteins are found in a conserved pentameric heterocomplex that contains Abi, Kette/Nap1, Sra-1/CYFIP, and HSPC300. Formation of the WAVE complex contributes to the localization, activity, and stability of the various WAVE proteins. Here, we established the Bimolecular Fluorescence Complementation (BiFC) technique in Drosophila to determine the subcellular localization of the WAVE complex in living flies. Using different split-YFP combinations, we are able to visualize the formation of the WAVE-Abi complex in vivo. We found that WAVE also forms dimers that are capable of forming higher order clusters with endogenous WAVE complex components. The N-terminal WAVE homology domain (WHD) of the WAVE protein mediates both WAVE-Abi and WAVE-WAVE interactions. Detailed localization analyses show that formation of WAVE complexes specifically takes place at basal cell compartments promoting actin polymerization. In the wing epithelium, hetero- and homooligomeric WAVE complexes co-localize with Integrin and Talin suggesting a role in integrin-mediated cell adhesion. RNAi mediated suppression of single components of the WAVE and the Arp2/3 complex in the wing further suggests that WAVE-dependent Arp2/3-mediated actin nucleation is important for the maintenance of stable integrin junctions.     

Identification of two human WAVE/SCAR homologues as general actin regulatory molecules which associate with the Arp2/3 complex.

WAVE/SCAR protein was identified as a protein which has similarity to WASP and N-WASP, especially in its C terminal. Recently, WAVE/SCAR protein has been shown to cooperate with the Arp2/3 complex, a nucleation core for actin polymerization in vitro. However, in spite of its general function, WAVE/SCAR expression is mainly restricted to the brain, suggesting the existence of related molecule(s). We here identified two human WAVE/SCAR homologues, which cover other organs. We named the original WAVE1 and newly identified ones WAVE2 and WAVE3. WAVE2 had a very wide distribution with strong expression in peripheral blood leukocytes and mapped on chromosome Xp11.21, next to the WASP locus. WAVE3 and WAVE1 had similar distributions. WAVE3 was strongly expressed in brain and mapped on chromosome 13q12. WAVE1 was mapped on chromosome 6q21-22. Ectopically expressed WAVE2 and WAVE3 induced actin filament clusters in a similar manner with WAVE1. These actin cluster formations were suppressed by deletion of their C-terminal VPH (verproline homology)/WH2 (WASP homology 2) domain. Further, WAVE2 and WAVE3 associate with the Arp2/3 complex as does WAVE1. Our identification of WAVE homologues suggests that WAVE family proteins have general function for regulating the actin cytoskeleton in many tissues.     

Negative regulation of yeast WASp by two SH3 domain-containing proteins

BACKGROUND: WASp family proteins promote actin filament assembly by activating Arp2/3 complex and are regulated spatially and temporally to assemble specialized actin structures used in diverse cellular processes. Some WASp family members are autoinhibited until bound by activating ligands; however, regulation of the budding yeast WASp homolog (Las17/Bee1) has not yet been explored. RESULTS: We isolated full-length Las17 and characterized its biochemical activities on yeast Arp2/3 complex. Purified Las17 was not autoinhibited; in this respect, it is more similar to SCAR/WAVE than to WASp proteins. Las17 was a much stronger activator of Arp2/3 complex than its carboxyl-terminal (WA) fragment. In addition, actin polymerization stimulated by Las17-Arp2/3 was much less sensitive to the inhibitory effects of profilin compared to polymerization stimulated by WA-Arp2/3. Two SH3 domain-containing binding partners of Las17, Sla1 and Bbc1, were purified and were shown to cooperate in inhibiting Las17 activity. The two SLA1 SH3 domains required for this inhibitory activity in vitro were also required in vivo, in combination with BBC1, for cell viability and normal actin organization. CONCLUSIONS: Full-length Las17 is not autoinhibited and activates Arp2/3 complex more strongly than its WA domain alone, revealing an important role for the Las17 amino terminus in Arp2/3 complex activation. Two of the SH3 domain-containing ligands of Las17, Sla1 and Bbc1, cooperate to inhibit Las17 activity in vitro and are required for a shared function in actin organization in vivo. Our results show that, like SCAR/WAVE, WASp proteins can be controlled by negative regulation through the combined actions of multiple ligands.     

Arabidopsis SCARs function interchangeably to meet actin-related protein 2/3 activation thresholds during morphogenesis

During polarized growth and tissue morphogenesis, cells must reorganize their cytoplasm and change shape in response to growth signals. Dynamic polymerization of actin filaments is one cellular component of polarized growth, and the actin-related protein 2/3 (ARP2/3) complex is an important actin filament nucleator in plants. ARP2/3 alone is inactive, and the Arabidopsis thaliana WAVE complex translates Rho-family small GTPase signals into an ARP2/3 activation response. The SCAR subunit of the WAVE complex is the primary activator of ARP2/3, and plant and vertebrate SCARs are encoded by a small gene family. However, it is unclear if SCAR isoforms function interchangeably or if they have unique properties that customize WAVE complex functions. We used the Arabidopsis distorted group mutants and an integrated analysis of SCAR gene and protein functions to address this question directly. Genetic results indicate that each of the four SCARs functions in the context of the WAVE-ARP2/3 pathway and together they define the lone mechanism for ARP2/3 activation. Genetic interactions among the scar mutants and transgene complementation studies show that the activators function interchangeably to meet the threshold for ARP2/3 activation in the cell. Interestingly, double, triple, and quadruple mutant analyses indicate that individual SCAR genes vary in their relative importance depending on the cell type, tissue, or organ that is analyzed. Differences among SCARs in mRNA levels and the biochemical efficiency of ARP2/3 activation may explain the functional contributions of individual genes.     

A role for ABIL3 in plant cell morphogenesis

Actin nucleation facilitated by the ARP2/3 complex plays a central role in plant cell shape development. The molecular characterization of the distorted class of trichome mutants has recently revealed the SCAR/WAVE complex as an essential upstream activator of ARP2/3 function in plants. The SCAR/WAVE complex is conserved from animals to plants and, generally, is composed of the five subunits SCAR/WAVE, PIR121, NAP125, BRICK and ABI. In plants, four of the five subunits have been shown to participate in trichome and pavement morphogenesis. Plant ABI‐like proteins (ABIL), however, which constitute a small four‐member protein family in Arabidopsis thaliana, have not been characterized functionally, so far. Here we demonstrate that microRNA knock‐down of the ABIL3 gene leads to a distorted trichome phenotype reminiscent of ARP2/3 mutant phenotypes and consistent with a crucial role of the ABIL3 protein in an ARP2/3‐activating SCAR/WAVE complex. In contrast to ARP2/3 mutants, however, the ABIL3 knock‐down stimulated cell elongation in the root, indicating distinct functions of the ABIL3 protein in different tissues. Furthermore, we provide evidence that ABIL3 associates with microtubules in vivo, opening up the intriguing possibility that ABI‐like proteins have a function in linking SCAR/WAVE‐dependent actin nucleation with organization of the microtubule cytoskeleton.     

The WAVE/SCAR complex promotes polarized cell movements and actin enrichment in epithelia during C. elegans embryogenesis

The WAVE/SCAR complex promotes actin nucleation through the Arp2/3 complex, in response to Rac signaling. We show that loss of WVE-1/GEX-1, the only C. elegans WAVE/SCAR homolog, by genetic mutation or by RNAi, has the same phenotype as loss of GEX-2/Sra1/p140/PIR121, GEX-3/NAP1/HEM2/KETTE, or ABI-1/ABI, the three other components of the C. elegans WAVE/SCAR complex. We find that the entire WAVE/SCAR complex promotes actin-dependent events at different times and in different tissues during development. During C. elegans embryogenesis loss of CED-10/Rac1, WAVE/SCAR complex components, or Arp2/3 blocks epidermal cell migrations despite correct epidermal cell differentiation. 4D movies show that this failure occurs due to decreased membrane dynamics in specific epidermal cells. Unlike myoblasts in Drosophila, epidermal cell fusions in C. elegans can occur in the absence of WAVE/SCAR or Arp2/3. Instead we find that subcellular enrichment of F-actin in epithelial tissues requires the Rac-WAVE/SCAR-Arp2/3 pathway. Intriguingly, we find that at the same stage of development both F-actin and WAVE/SCAR proteins are enriched apically in one epithelial tissue and basolaterally in another. We propose that temporally and spatially regulated actin nucleation by the Rac-WAVE/SCAR-Arp2/3 pathway is required for epithelial cell organization and movements during morphogenesis.