The aim of this investigation is to elucidate the factors which control the formation and dissociation of actin-containing cables in a non-muscle cell. From these studies, a deeper understanding of the molecular controls which govern cell to substrate adhesion during cell division and the events resulting in cell locomotion along a substratum should result. Initially, we will purify the protein(s) from Dictyostelium amoebae that causes bundling of actin filaments in vitro, biochemically characterize the isolated bundling factor, and compare it to the known bundling proteins from sea urchin oocytes (fascin) and intestinal microvilli (villin). Furthermore, by a fluorescence energy transfer assay recently employed by the applicant to follow filament formation, filament subunit exchange, and formation of actin paracrystals, we will quantitate the kinetics, extent, and regulation of bundle formation in vitro by salts, pH, ATP, and cAMP. The effects of severin (a 40 Kd actin filament severing protein from Dictyostelium) on bundle disruption will also be tested. By fluorescence energy transfer, it should be possible to determine the site of severin interaction with bundles and the relative positions of actin, bundle protein, and severin within reconstituted bundles. The second goal of this investigation is to develop a biotechnology suitable for observing the biochemical regulation of actin cables in situ. Living Dictyostelium amoebae will be immobilized on spherical beads, and adhesion conditions optimized with respect to time, temperature, and media conditions. Actin cables on the floor of these substrate-adhered cells will be exposed by a bulk method for shearing off the cell tops. Exposed in situ cables will subsequently be analysed for protein components and regulation of cable dissociation and reformation by metabolites, severin, and protein phosphorylation. By virtue of the isolation of bulk quantities of in situ actin cables, a complete evaluation of the biochemical regulation of cable association to membrane and the resulting adherence characteristics of cell to substratum can be made.
