The long term goal of this proposal is to understand the structure and function of gelsolin, a major vertebrate actin filament capping/severing protein. We have made progress in understanding the organization of this protein into serving and capping domains and have localized two Ca(2+) regulatory sites and the C-terminal half of the molecule. Peptides with unregulated severing activity have been isolated and shown to be active both in vivo and in vitro. We want to continue to clone gelsolin cDNAs and to use recombinant DNA techniques to construct expression vectors and to sequence this DNA. We will use these vectors, monoclonal antibodies, biochemical methods and light and fluorescence microscopy to study the biological effects of modulating the levels of human gelsolin and of over- expressing this molecule and its Ca(2+) insensitive fragments in cells in order to decide whether function of this protein is to sever filaments or to regulate growth by capping filament ends. Specifically we propose: 1) To clone cDNAs for gelsolin and a closely related plasma protein, brevin, and determine the sequence of both. 2) To use the protein sequence data to locate more precisely actin and Ca(2+) binding sites on gelsolin and determine the structural properties of both molecules. 3) To construct inducible expression vectors carrying coding sequences for the entire gelsolin molecule and for the fragments including the N-terminal half with Ca(2+) insensitive constitutive severing activity. 4) To construct inducible expression vectors carrying the 5'- end of gelsolin DNA in an anti-sense orientation. 5) To transfect cells with each of these vectors, establish that gelsolin or gelsolin fragments are produced or reduced and then study the biological effects of these changes in expression on cell division, cell morphology and motility.
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