This is a proposal to continue study of three Drosophila genes, chickadee, singed and quail, that are necessary for transport of nurse cell components into the developing oocyte. The products of each of these genes act to regulate the actin cytoskeleton: chickadee encodes a profilin; quail encodes a villin; singed encodes a fascin. Phenotypic analysis from the Cooley lab has shown that these mutants block cytoplasmic transport because the nurse cell nuclei in the mutants are not held in position by an actin support system and, once flow begins, the nuclei block further cytoplasmic flow. The proposed experiments will exploit this aspect of oogenesis to elucidate the in vivo structure/function relationships of these actin- binding proteins. For chickadee (profilin) extensive in vitro mutagenesis and transformation experiments will explore how profilin might regulate actin polymerization and how it might mediate signaling effects on the cytoskeleton. Studies on singed (fascin) will test what part of the protein is necessary for binding to actin in vitro. EMS- induced alleles of singed will be isolated and protein-positive alleles that affect actin functioning in nurse cells will be sequenced and their ability to bind and/or bundle actin will be tested in vitro. Similarly for quail (villin) 12 existing EMS-induced alleles will be characterized, and 6 protein-positive alleles will be sequenced. Deletion mutants of quail that should remove G and R actin binding sites will be tested for function in vivo. Genetic screens for other proteins that interact with these gene products will be carried out. Dominant suppressors of particular chickadee, singed and quail mutant alleles will be carried out. For each gene, additional studies will depend on the particular activity expected of the gene product. For instance, because interaction of vertebrate villin with actin is calcium dependent in vitro, experiments will test whether calcium levels affect actin bundling in cultured egg chambers. The final section of the proposal explores two different aspects of oogenesis. First, because members of the rho family of GTPases appear to be involved in actin-based cell shape changes in mammalian cells, a modification of the GAL4 system for expressing activated or dominant negative Rho proteins specifically in the germ line will be developed. Second, because nurse cell regression shares some features of apoptosis, regressing nurse cells will be analyzed for the characteristic signs of apoptosis and the role of the Drosophila reaper gene in oogenesis will be investigated.
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