Wash functions downstream of Rho and links linear and branched actin nucleation factors

Development, Aug 2009

Raymond Liu, Maria Teresa Abreu-Blanco, Kevin C. Barry, Elena V. Linardopoulou, Gregory E. Osborn, Susan M. Parkhurst

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Wash functions downstream of Rho and links linear and branched actin nucleation factors

Raymond Liu 0 Maria Teresa Abreu-Blanco 0 Kevin C. Barry 0 Elena V. Linardopoulou 0 Gregory E. Osborn 0 Susan M. Parkhurst ) 0 0 Division of Basic Sciences, Fred Hutchinson Cancer Research Center , 1100 Fairview Avenue North, Seattle, WA 98109 , USA Wiskott-Aldrich Syndrome (WAS) family proteins are Arp2/3 activators that mediate the branched-actin network formation required for cytoskeletal remodeling, intracellular transport and cell locomotion. Wasp and Scar/WAVE, the two founding members of the family, are regulated by the GTPases Cdc42 and Rac, respectively. By contrast, linear actin nucleators, such as Spire and formins, are regulated by the GTPase Rho. We recently identified a third WAS family member, called Wash, with Arp2/3-mediated actin nucleation activity. We show that Drosophila Wash interacts genetically with Arp2/3, and also functions downstream of Rho1 with Spire and the formin Cappuccino to control actin and microtubule dynamics during Drosophila oogenesis. Wash bundles and crosslinks F-actin and microtubules, is regulated by Rho1, Spire and Arp2/3, and is essential for actin cytoskeleton organization in the egg chamber. Our results establish Wash and Rho as regulators of both linear- and branched-actin networks, and suggest an Arp2/3-mediated mechanism for how cells might coordinately regulate these structures. INTRODUCTION The actin cytoskeleton consists of linear and branched filament networks required for processes ranging from cell division to migration (Chhabra and Higgs, 2007; Faix and Grosse, 2006; Goley and Welch, 2006). How these two networks function and are coordinated is of major interest, as their misregulation results in infertility, immunodeficiency, and tumor metastasis in humans (Bione et al., 1998; Burns et al., 2004; Yamaguchi and Condeelis, 2007). Linear actin filament networks, required for cytokinesis and filopodia formation, are regulated by nucleators and bundling proteins, which enhance filament formation rates and control filament organization, respectively (Chhabra and Higgs, 2007; Faix and Grosse, 2006; Goode and Eck, 2007; Wallar and Alberts, 2003). Examples include Spire and the formin Cappuccino (Capu), which exhibit both nucleation and bundling activities and are essential for oocyte development during Drosophila oogenesis (Chhabra and Higgs, 2007; Kerkhoff, 2006; Manseau and Schupbach, 1989; Quinlan et al., 2005; Quinlan et al., 2007; Rosales-Nieves et al., 2006; Theurkauf, 1994; Wang and Riechmann, 2008). Both Spire and Capu are regulated by the GTPase Rho1 of the Rho family of small GTPases, which is upstream of other linear nucleators, such as Diaphanous, and is considered a key regulator of linear filament formation (Goode and Eck, 2007; Wallar and Alberts, 2003). Branched or dendritic actin filament networks, which are required for phagocytosis and lamellipodia formation, are primarily regulated by the Arp2/3 complex and by nucleationpromoting factors that associate with Arp2/3 and actin monomers to nucleate daughter filaments off of existing mother filaments (Goley and Welch, 2006; Takenawa and Suetsugu, 2007). Like Spire and Capu, Arp2/3 is essential for Drosophila oogenesis, specifically for maintaining proper nurse cell cyto-architecture and function (Hudson and Cooley, 2002). One family of Arp2/3 activators, the Wiskott-Aldrich Syndrome (WAS) protein family, has been shown to function downstream of Rho GTPases to mediate the branched-actin network formation required for cytoskeletal remodeling, intracellular transport and cell locomotion (Ben-Yaacov et al., 2001; Campellone et al., 2008; Linardopoulou et al., 2007; Stradal et al., 2004; Takenawa and Suetsugu, 2007; Zallen et al., 2002). WASP and SCAR/WAVE, the two founding subclasses of the family, are activated by the GTPases Cdc42 and Rac, respectively (Stradal et al., 2004; Takenawa and Suetsugu, 2007). Two new WAS subclasses, WASH and WHAMM, have recently been reported (Campellone et al., 2008; Linardopoulou et al., 2007) and have been shown to exhibit Arp2/3-mediated branched nucleation activity. Which GTPases might regulate them, however, is not known. Here, we report that Drosophila Wash functions downstream of Rho1 and interacts with Spire and Capu to regulate actin and microtubule organization during Drosophila oogenesis. We show that Wash nucleates actin in an Arp2/3-dependent manner, and exhibits F-actin and microtubule bundling and crosslinking activity that is regulated by a pathway involving Rho1, Spire and Arp2/3. We find that Wash genetically interacts with Rho1, Capu, Spire and Arp2/3, and is essential for actin cytoskeleton organization during oogenesis. Our results establish Wash and Rho as regulators of both linear- and branched-actin networks, and suggest an Arp2/3-mediated mechanism of cytoskeletal control through which cells might coordinately regulate linear and branched architectures. MATERIALS AND METHODS Fly strains and genetics Flies were cultured and crossed on (...truncated)


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Raymond Liu, Maria Teresa Abreu-Blanco, Kevin C. Barry, Elena V. Linardopoulou, Gregory E. Osborn, Susan M. Parkhurst. Wash functions downstream of Rho and links linear and branched actin nucleation factors, Development, 2009, pp. 2849-2860, 136/16, DOI: 10.1242/dev.035246