The long-term goal of this work is to understand how cellular responses mediated by the cytoskeleton are regulated and carried out during development. The experimental system is Drosophila oogenesis, during which nurse cells feed growing oocytes maternal components including mRNA, protein and organelles. The transport of cytoplasm from nurse cell to the oocyte requires precisely timed rearrangements of the actin cytoskeleton and the execution of programmed cell death (apoptosis) in nurse cells. Late in oogenesis, dense cytoplasmic actin bundles form in nurse cells, just at the onset of nurse cell apoptosis. Several genes encoding actin binding proteins will be studied using a combination of genetics, biochemistry and cell biology to elucidate their function during oogenesis. In addition, regulatory proteins involved in actin organization will be investigated. This work is relevant to understanding the contribution of a dynamic actin cytoskeleton to motility, cell shape or subcellular localization of molecules in both normal cells and during disease progression such as metastasis.
The specific aims of the proposal are: (1) to study the function in vivo of Profilin, a small actin monomer binding protein important for modulating the rate of actin filament polymerization; (2) to perform genetic and biochemical analyses of Profilin interactions with the Arp2/3 complex and FH domain proteins; (3) to carry out functional analyses on proteins that crosslink actin filaments into highly organized bundles within cytoplasm; and (4) to study the role of the steroid hormone ecdysone in regulating nurse cell apoptosis. In Drosophila nurse cells, Profilin is required for the polymerization of the cytoplasmic actin filaments that form during stage 10 of oogenesis. Specific mutations in the genes encoding Profilin and Profilin binding proteins will be made and tested in nurse cells using a germline expression vector and P element- mediated transformation. A similar experimental strategy will be used to test the function of Drosophila Fascin, an actin filament bundling protein also required at stage 10. The possible coordinate regulation of both Fascin and a Villin-like actin bundling protein called Quail will be studied by examining their phosphorylation and ability to bind Armadillo (beta-catenin). Profilin, Quail and Fascin proteins function in cells that are undergoing apoptosis. The role of the steroid hormone ecdysone in signaling nurse cell apoptosis will be studied by using mutants in the ecdysone response pathway, and cloning a new gene called midway.
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