The WAVE regulatory complex is inhibited

Howard Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, USA.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 05/2009; 16(5):561-3. DOI: 10.1038/nsmb.1587
Source: PubMed


The WAVE regulatory complex (WRC) transmits information from the Rac GTPase to the actin nucleator Arp2/3 complex. We have reconstituted recombinant human and Drosophila WRC in several forms and shown that they are inactive toward Arp2/3 complex but can be activated by Rac in a nucleotide-dependent fashion. Our observations identify core components needed for WAVE inhibition, reconcile contradictory existing mechanisms and reveal common regulatory principles for the WAVE/WASP family of proteins.

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    • "The analyses of single and double mutants that possess defects in Arp2/3-induced actin polymerization demonstrated that Scar-dependent Arp2/3 activation is essential for longitudinal myoblast fusion. It has been proposed that the activation of Scar/Wave depends on the small Rac-GTPase [64]. The myoblast-fusion-relevant GEF for Rac is Mbc [47]. "
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    ABSTRACT: Background The visceral musculature of Drosophila larvae comprises circular visceral muscles tightly interwoven with longitudinal visceral muscles. During myogenesis, the circular muscles arise by one-to-one fusion of a circular visceral founder cell (FC) with a visceral fusion-competent myoblast (FCM) from the trunk visceral mesoderm, and longitudinal muscles arise from FCs of the caudal visceral mesoderm. Longitudinal FCs migrate anteriorly under guidance of fibroblast growth factors during embryogenesis; it is proposed that they fuse with FCMs from the trunk visceral mesoderm to give rise to syncytia containing up to six nuclei. Results Using fluorescence in situ hybridization and immunochemical analyses, we investigated whether these fusion events during migration use the same molecular repertoire and cellular components as fusion-restricted myogenic adhesive structure (FuRMAS), the adhesive signaling center that mediates myoblast fusion in the somatic mesoderm. Longitudinal muscles were formed by the fusion of one FC with Sns-positive FCMs, and defects in FCM specification led to defects in longitudinal muscle formation. At the fusion sites, Duf/Kirre and the adaptor protein Rols7 accumulated in longitudinal FCs, and Blow and F-actin accumulated in FCMs. The accumulation of these four proteins at the fusion sites argues for FuRMAS-like adhesion and signaling centers. Longitudinal fusion was disturbed in rols and blow single, and scar wip double mutants. Mutants of wasp or its interaction partner wip had no defects in longitudinal fusion. Conclusions Our results indicated that all embryonic fusion events depend on the same cell-adhesion molecules, but that the need for Rols7 and regulators of F-actin distinctly differs. Rols7 was required for longitudinal visceral and somatic myoblast fusion but not for circular visceral fusion. Importantly, longitudinal fusion depended on Kette and SCAR/Wave but was independent of WASp-dependent Arp2/3 activation. Thus, the complexity of the players involved in muscle formation increases from binucleated circular muscles to longitudinal visceral muscles to somatic muscles.
    BMC Cell Biology 07/2014; 15(1):27. DOI:10.1186/1471-2121-15-27 · 2.34 Impact Factor
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    • "After incubation and washing, the bound proteins were eluted with reduced glutathione or maltose, respectively. Pyrene-actin assembly assays were performed as previously described (Ismail et al., 2009). Coimmunoprecipitation was performed by mixing mouse brain lysate with anti-WAVE1 monoclonal antibody (Sigma) and protein A/G beads in the presence of different competitors. "
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    ABSTRACT: The WAVE regulatory complex (WRC) controls actin cytoskeletal dynamics throughout the cell by stimulating the actin-nucleating activity of the Arp2/3 complex at distinct membrane sites. However, the factors that recruit the WRC to specific locations remain poorly understood. Here, we have identified a large family of potential WRC ligands, consisting of ∼120 diverse membrane proteins, including protocadherins, ROBOs, netrin receptors, neuroligins, GPCRs, and channels. Structural, biochemical, and cellular studies reveal that a sequence motif that defines these ligands binds to a highly conserved interaction surface of the WRC formed by the Sra and Abi subunits. Mutating this binding surface in flies resulted in defects in actin cytoskeletal organization and egg morphology during oogenesis, leading to female sterility. Our findings directly link diverse membrane proteins to the WRC and actin cytoskeleton and have broad physiological and pathological ramifications in metazoans.
    Cell 01/2014; 156(1-2):195-207. DOI:10.1016/j.cell.2013.11.048 · 32.24 Impact Factor
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    • "Furthermore, biochemical assays of Arabidopsis W/SRC (Basu et al., 2004; El-Assal et al., 2004; Frank et al., 2004; Le et al., 2006; Uhrig et al., 2007) and ARP2/ 3 assembly (Kotchoni et al., 2009) have shown that the binary interactions among W/SRC subunits and ARP2/3 complex assembly mechanisms are indistinguishable from those that have been observed for human W/SRC (Gautreau et al., 2004) and yeast ARP2/3 (Winter et al., 1999). After an initial period of controversy concerning the biochemical control of W/SRC, it is now apparent that vertebrate W/SRC (Derivery et al., 2009; Ismail et al., 2009), like the ARP2/3 complex (Machesky et al., 1999), is intrinsically inactive and requires positive regulation by Rac and other factors to fully activate ARP2/3 (Ismail et al., 2009; Lebensohn and Kirschner, 2009; Chen et al., 2010). Although overexpression of dominant negative ROP mutants causes trichome swelling and a reduced trichome branch number (Fu et al., 2002), the involvement of ROPs in trichome morphogenesis has been difficult to prove with a loss-offunction ROP allele because so many ROPs are expressed in this cell type (Marks et al., 2009). "
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    ABSTRACT: During plant cell morphogenesis signal transduction and cytoskeletal dynamics interact to locally organize the cytoplasm and define the geometry of cell expansion. The WAVE (WASP-family verprolin-homologous)/SCAR (Suppressor of Cyclic AMP Receptor) regulatory complex (W/SRC) is an evolutionarily conserved heteromeric protein complex. Within the plant kingdom W/SRC is a broadly used effector that converts ROP/RAC small GTPase signals into actin-related protein (ARP) 2/3 and actin-dependent growth responses. Although the components and biochemistry of the W/SRC pathway are well understood, a basic understanding of how cells partition W/SRC into active and inactive pools is lacking. In this paper we report that the endoplasmic reticulum (ER) is an important organelle for W/SRC regulation. We determined that a large intracellular pool of the core W/SRC subunit NAP1, like the known positive regulator of W/SRC, the DOCK family guanine nucleotide exchange factor SPIKE1 (SPK1), localizes to the surface of the ER. The ER-associated NAP1 is inactive because it displays little colocalization with the actin network, and the ER localization requires neither activating signals from SPK1 nor a physical association with its W/SRC binding partner, SRA1. Our results indicate that in leaf pavement cells and trichomes, the ER is a reservoir for W/SRC signaling and may have a key role in the early steps of W/SRC assembly and/or activation.
    Plant physiology 04/2013; 162(2). DOI:10.1104/pp.113.217422 · 6.84 Impact Factor
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