CYTOCHALASIN & PROTEINS THAT AFFECT ENDS OF F-ACTIN

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The long term goal of this project is to understand the regulation of cytoskeletal and contractile functions by studying the control of the assembly and disassembly of actin filaments (F-actin). The approach we have been taking is to use cytochalasins (fungal metabolites with anti-motility activity) as tools to identify cellular components that affect actin polymerization. We previously found that these compounds bind with high affinity to the fast growing end of F-actin, thereby strongly affecting the rates of filament assembly and disassembly as well as filament length distribution in vitro. Moreover, we were able to isolate from skeletal muscle a protein (designated as capactin) that affects actin assembly and disassembly in a manner that closely resembles that of the cytochalasins. In the proposed research, we plan to study the structure and function of capactins in muscle and nonmuscle cells in the following ways. (A) We will determine the biochemical structure of the protein by analyzing its primary and subunit structures, active site, isoelectric isoforms, etc. cDNA encoding capactin will be isolated and sequenced. Monoclonal antibodies against different epitopes on the protein will be prepared. We will compare the properties of capactins from muscle and nonmuscle cells, and between this class of proteins and other actin modulating proteins such as gelsolin. (B) We will study the localization of capactins in adult and developing muscle and in nonmuscle tissues by immunofluorescence and by incorporation of labelled capactin into cellular and cell-free systems. The appearance of the protein and its messengers will also be studied under a variety of physiological conditions with the use of Western and Northern blots. (C) The interaction of capactin with actin and with other cellular components will be studied with biophysical and pharmacological techniques.