Dynamic changes in cell motility and in the actin-based membrane skeleton occur during immune cell functioning, neuronal outgrowth, wound healing, and the transformation of cells into invasive cancers. Although these changes are thought to involve rearrangements of the proteins at the actin-membrane interface, the relevant interacting proteins are largely unknown. The proposed research will continue the characterization of membrane skeletons in amoeboid cells, e.g., cells such as unactivated neutrophils and Dictyostelium amoebae. Although these cells lack stable attachments to other cells or to extracellular matrices, their membranes contain cytoskeletal linkages responsible for the spatial and temporal control of cell shape and/or the regulation of intracellular responses to extracellular signals. These 'amoeboid' membrane skeletons appear to be structurally distinct from the membrane skeletons that characterize sites of cell anchorage. Two types of membrane-associated actin-binding proteins will be investigated: (a) transmembrane proteins that provide direct connections between the cell surface and the actin cytoskeleton, and (b) peripheral membrane proteins that can be recruited from the cytoplasm to the membrane skeleton where they may regulate the assembly or stability of membrane-associated actin filaments. The specific aims of the proposed research are: (1) to generate mutant cell lines for ongoing functional analyses of Dictyostelium ponticulin, the first integral membrane protein shown to bind directly to actin and the only integral membrane protein with demonstrated actin nucleation activity; (2) to determine the molecular mechanism by which diacylglycerols mediate increases in the actin nucleation activity of purified Dictyostelium plasma membranes; and (3) to ascertain whether membrane-associated actin- binding proteins identified in neutrophils and cervical carcinoma cells are structurally or functionally similar to ponticulin. This research will lead to a better understanding of the molecular mechanisms involved in pseudopod formation and stabilization, in the regionalization of the plasma membrane, and in cell detachment from surfaces or other cells. This research thus will both increase our knowledge of normal motile processes and shed light on defects underlying pathological conditions, including birth defects, cancer cell metastasis, and dementia caused by transit of HIV-laden macrophages across the blood-brain barrier.