Forward Chemical Genetic Screen Identifies Wiskostatin a Small Molecule Inhibitor of NWASP

In order to identify small molecules that perturb Rho GTPase-dependent signaling, we conducted a high-throughput screen for compounds that inhibit Cdc42-dependent actin filament assembly in cytoplasmic extracts of Xenopus laevis eggs. These extracts are prepared by simple mechanical disruption of intact eggs by gentle centrifugation rather than through detergent extraction, resulting in essentially undiluted, detergent-free cytoplasm containing proteins at physiological concentrations (Lebensohn et al. 2006). Consequently, these extracts are widely used as a model system to study diverse cellular signaling and regulatory networks (Chen and Murray 1997; de la Barre et al. 1999; Salic et al. 2000; Castro et al. 2006; Chan and Forbes 2006; Deming and Kornbluth 2006; Lebensohn et al. 2006; Moon et al. 2006; Shennan 2006; Tutter and Walter 2006). High-speed centrifuga-tion depletes the extract of endogenous lipid membranes, resulting in a quiescent extract termed HSS (high-speed supernatant) in which actin monomers do not spontaneously polymerize. Subsequent stimulation of the extract by the addition of lipid vesicles (liposomes) containing small amounts of phosphatidylinositol 4,5-bisphosphate (PIP2) results in the polymerization of endogenous actin onto the surface of the liposomes (Ma et al. 1998a; Peterson et al. 2001). Although this activity may recapitulate the localized polymerization of actin at the plasma membrane (Ma et al. 1998a), recent studies suggest that this pathway may also be used physiologically to mediate endocytosis and the intracellular motility of endosomally derived vesicles (Taunton et al. 2000; Sokac et al. 2003). As described below, actin assembly by PIP2-stimulated HSS has proven a robust assay for identifying components of the actin regulatory machinery that are critical for cell motility and morphology in many systems.

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