The dynamic assembly and disassembly of actin filaments underlies diverse processes including cell migrations during development and phagocytosis of invading microorganisms by inunune cells. Mechanisms that control actin dynamics in vivo are largely unknown. A central unsolved question about the actin filament nucleating Arp2/3 complex concerns roles for ATPs bound to Arp2 and Arp3. Based on studies of actin, nucleotide states could influence Arp2/3 complex activation, branching and de-branching activities, and interactions with regulatory factors. Because Arp2 and Arp3 are closely related to actin, and because actin nucleotide pocket mutants have provided valuable insights into cytoskeletal dynamics, similar mutants will be constructed in Arp2 and Arp3. Effects of these mutants will be tested in vivo and in in vitro tests for nucleation activity, interactions with different activators, and branching and debranching reactions. To elucidate more fully the in vivo functional relationships and regulatory networks that regulate the actin cytoskeleton in vivo, the large collection of existing yeast actin cytoskeleton mutants created in the Drubin laboratory will be used in functional genomics studies. Systematic, genome-wide synthetic lethal and suppressor screens using recently developed procedures designed to achieve comprehensive genome coverage will be employed to uncover these complex relationships. Finally, having successfully translated studies of actin-mediated endocytosis in budding yeast to mammalian cells, studies of yeast actin cytoskeleton dynamics will now be translated to the problem of tumor cell invasion in mammalian cells. Cytoskeletal constituents of invadopodia responsible for tumor cell invasion will be systematically identified, then RNAi and dominant negative mutants will be used to knock down expression of these proteins. In total, studies proposed here will increase our understanding of fundamental mechanisms for regulating actin cytoskeleton dynamics, and principles derived from studies of yeast actin dynamics will be used to develop a deeper understanding of the molecular mechanisms responsible for mammalian tumor cell invasion. Novel therapeutic targets may be identified.
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