The purpose of this project is to determine the role played by tropomyosin in the organization of the actin cytoskeleton in normal and transformed rat kidney cells in culture. The normal cells contain five isoforms of subunit tropmyosin: two of 30 kilodalton apparent molecular weight (as determined on two-dimensional polyacrylamide gels, one of 35 kilodalton, one of 37 kilodalton, and one of 40 kilodalton. Transformed cells contain the 30 kilodalton isoforms but have only trace amounts of the 40 kilodalton isoform and are lacking the 35 and 37 kilodalton isoforms. Experiments are being conducted on the effects of microinjecting complete mixtures of isoforms of tropomyosin purified from normal cells into transformed cells in order to determine if the transformed cells can acquire the ability to organize stress fibers. Using a rabbit polyclonal antibody that binds to the 35, 37, and 40 kilodalton isoforms but not to the 30 kilodalton isoforms, we have found them to be localized in stress fibers in fully spread cells. In freshly plated cells, we have found that the antibody stains the ruffles at the peripheries of the cells, in contrast to previous reports that ruffles lacked tropomyosin. The same antibody also stains ruffles at the edges of transformed cells, which never contain stress fibers. Injection of an affinity-purified derivative of this antibody into normal cells disrupts stress fibers in the cells and alters cell shape. Another antibody has been raised in rabbits to a 30 kilodalton protein from transformed cells that appears to be an isoform of tropomyosin. This antibody stains a reticular network just beneath the plasmalemma of normal and transformed cells but does not stain stress fibers in normal cells. The possibility that this is a membrane-associated isoform of tropomyosin is being investigated. Monoclonal antibodies to individual tropomyosin isoforms are now being raised. Actin binding studies indicate that the 30 kilodalton tropomyosin isoforms bind less tightly to actin than the other three isoforms. (L)
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