The ability of cells to properly change shape, migrate and divide, within the embryo, can determine the success or failure of embryonic development. Many of the motile events during morphogenesis are directed by the cell's actin cytoskeleton. The actin cytoskeleton of eukaryotic cells is a highly dynamic assembly of assorted structures. Fortunately, there exists a functional universality linking the actin regulatory proteins from broadly diverse cell types. The specific goals of this project are: a. Characterize the effects of sea urchin egg llOK, 95K, 50K, and 13K (profilin?) actin binding proteins on actin filament assembly and structure. In particular, the modulatory effects of second messengers such as Ca , pH, phospholipid and the nucleotide bound to the actin subunit will be analysed; b. Investigate the mechanism of Ca -sensitive regulation of egg spectrin-actin crosslinking: c. Generate and characterize polyclonal and/or monoclonal antibodies that monospecifically crossreact with the isolated and characterized actin binding proteins; d. Employ the anti-actin binding protein antibodies for immunocytochemical studies determining the spatio-temporal association of the regulatory proteins with the various actin cytoskeleton domains in oocytes, eggs and embryos; e. Examine the synthesis of the actin binding proteins during oogenesis and embryogenesis using in situ labeling and immunoprecipitation and/or autoradiography. Successful completion of the above aims will provide the necessary correlations between the in vitro protein interactions and the in vivo actin cytoskeletal localization and synthesis to generate hypotheses describing the potential molecular mechanisms modulating the actin cytoskeleton. Additionally, the production of anti-actin binding protein antibodies will provide the necessary probes to test the hypothetical molecules mechanisms in vivo and to begin examining differential expression of the actin binding proteins during embryogenesis.
