The early Drosophila embryo is being used to study the role of proteins that cooperate with actin to generate an organized cytoplasm in this large and highly dynamic syncytial cell. Actin filament structures participate in both the organization of the cytoplasm and the dynamic events required during normal development. Thus, these studies will provide insight into the actin-mediated process that contribute to the formation of a properly organized cellular blastoderm as a substrate of differentiation. Since actin structures and processes similar to those important in the embryo undoubtedly play important roles in the function of other cells, the studies of this model system will likely uncover general principles of actin organization and function that will apply to all cells. Two actin-associated proteins, identified in early embryos using F-actin affinity chromatography and located in vivo using monoclonal antibodies, will be studied in detail. Injections of either monoclonal antibody into early embryos causes disruption of normal development. The mechanism of disruption is being studied using fluorescently-labeled cytoskeletal proteins, histones and antibodies, allowing the visualization of many major embryonic components during the period when the antibody acts. The proteins will be purified from early embryos using standard biochemical methods and characterized for in vitro activities involving actin filaments. Expression libraries will be screened using the antibodies to detect cDNA clone that express these proteins. Once cloned coding sequences will be altered to determine which regions of the protein are important for function. Both in vitro and in vivo assays will be attempted to assess the impact of sequence changes. Fluorescent-labeling and injection of the altered protein will allow function and localization of mutant proteins to be assayed in vivo. Using overexpressed protein, affinity matricies will be constructed to identify componants that interact with these proteins in the macromolecular assemblies that are thought control actin polymerization sites and times in vivo. Study of these and other proteins that control the cell-cycle coordinated rearrangements of actin that occur during early development of the Drosophila embryo could provide insight into the coordination of actin rearrangements with the other events during the cell cycle and the mechanism by which actin rearrangements take place. Since rearrangements of actin are critical in all cells, these experiments should contribute to our understanding of how actin filament structures are positioned in space and regulated to polymerize at appropriate times.
