The theme of this proposal is the interface between cellular signaling systems and the actin cytoskeleton. The actin cytoskeleton determines the shape, mechanical properties, and motility of most eukaryotic cells. We know a lot about the structures and functions of proteins that comprise the actin cytoskeleton. What we do not understand is how a signaling event like the activation of a small G-protein is converted into a specific structural change in the cytoskeleton. To understand how cellular signals are converted into structures we will focus on one recently identified pathway that links activation of Rho-family G-proteins to polymerization and crosslinking of actin filaments. In this pathway, the activated Rho-family G-protein Cdc42 binds and activates the adaptor protein WASP. WASP, in turn binds the Arp2/3 complex and stimulates its ability to nucleate actin filament formation. Stimulation requires the presence of an actin binding domain in WASP, called a WH2 domain. WASP also binds multiple molecules of profilin and monomeric actin and may act as a scaffold for a large heteromeric nucleation complex. Activated Arp2/3 remains bound to the newly formed actin filaments and crosslinks them into rigid networks. To recycle the Arp2/3 complex and actin monomers, these actin networks must eventually be disassembled. We address the following questions: 1) What is the conformational change induced in WASP by activated Cdc42? 2) Why are WH2-domains required for activation of the Arp2/3 complex? 3) What are the structures of multisubunit complex built from Arp2/3, WASP, profilin and actin? 4) Is ATP hydrolysis a timer regulating Arp2/3 function?
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