Dynamic changes in cell motility and in the actin-based membrane skeleton occur during leukocyte migration, 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 uncharacterized. The proposed research will continue the characterization of Dictyostelium ponticulin, the only integral membrane protein known that binds actin and nucleates actin filament assembly. A number of techniques, including those of molecular genetics, biochemistry, cell biology, and lipid chemistry, will be used to determine the primary structure of ponticulin and to ascertain how and when it functions in vivo. The biochemical mechanisms by which ponticulin activity is regulated also will be elucidated.
The specific aims of the proposed research are: (1) to clone and sequence the cDNA and genomic DNA for Dictyostelium ponticulin; (2) to prepare domain- specific and conformation-specific antibodies against Dictyostelium ponticulin and structurally-related proteins; (3) to examine the role of Dictyostelium ponticulin in living cells by generating loss-of-function mutants using homologous recombination, anti-sense RNA, and/or overexpression of defective or truncated ponticulin; (4) to screen existing motility mutants for defects in ponticulin; (5) to examine mutant cell lines for alterations in stimulus-mediated actin polymerization, pseudopod extension, cell translocation, chemotaxis, phagocytosis, and cell-cell cohesion; (6) to explore the mechanisms regulating ponticulin activity by looking for Dictyostelium proteins associated with ponticulin and by examining the regulatory roles of diacylglycerols, phosphorylation, oligomerization, disulfide reduction, and transmembrane pH and/or membrane potential; and (7) to characterize the actin-binding and nucleating activities of a 16-kD integral membrane protein found in bovine granulocyte membranes that may be bovine ponticulin. Eventually, we hope to use the tissue distribution of this protein as a guide to the generality of ponticulin function in higher organisms. The long-term goals of this project are to understand the molecular basis and control of the actin- membrane interactions involved in motile processes and to apply this knowledge to the understanding of pathological conditions such as developmental abnormalities, cancer cell invasion, and HIV-induced dementia.
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