Changes in cell shape, movement and vesicle traffic all require remodelling of the actin cytoskeleton. While the actin filament remains chemically the same, the filaments are assembled differently for different functions, e.g. long, bundled, parallel filaments in filopodia and short meshworks in endocytosis. The membrane appears to function as an organising surface where proteins come together to form multiprotein complexes to generate distinct actin structures. The proposed research is aimed at understanding the roles contributed by the membrane and the identities of the signalling and effector proteins that form distinct actin structures. I have formulated in vitro reconstitution systems, using artificial membranes and extracts, that mimic the formation of two uses of actin: filopodia and endocytosis. I propose to use these reconstitution systems to identify proteins important for actin polymerisation during filopodia formation and endocytosis and then investigate their functions in vivo in Xenopus embryos. The first objective is to use biochemical fractionation to identify actin filament elongation factors to understand how filopodial length and rate of growth are regulated in cells during morphogenesis, with the ultimate goal of finding a minimal set of proteins that can make a filopodium. My second objective is to elucidate the molecular pathway that dictates how phosphoinositides and membrane curvature stimulate actin polymerisation during endocytosis, find how different actin regulators contribute to trafficking pathways in vivo, and the contribution of these endocytic pathways to early development. Solving the molecular basis of how the membrane-cytosol interface signals to actin polymerisation will reveal how changes in cellular organisation are orchestrated to achieve cell shape and movement during morphogenesis.
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