Wash functions downstream of Rho and links linear and branched actin nucleation factors
Raymond Liu
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Maria Teresa Abreu-Blanco
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Kevin C. Barry
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Elena V. Linardopoulou
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Gregory E. Osborn
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Susan M. Parkhurst
)
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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)