The purpose of our work is to examine the role played by the cytoskeletal protein tropomyosin in stress fiber formation in cultured mammalian cells. In our previous work we have shown that normal rat kidney cells, which spread over the substrate and adhere tightly to it, contain extensive basal networks of actin filament bundles termed "stress fibers." We can demonstrate biochemically and immunologically that tropomyosin is associated with the actin filaments in these bundles. A virally transformed NRK cell, termed 442, neither spreads upon nor adheres well to the substrate, and lacks stress fibers. These cells also appear to lack two of the tropomyosin subunits found in nonmuscle cells. Since it is known that tropomyosin stabilizes actin filaments and is required for the organization of actin filaments in developing muscle, we hypothesize that a virally induced modification of tropomyosin in 442 cells is responsible in part for the absence of stress fibers in 442 cells. We have begun to test this hypothesis by microinjecting FITC-labeled tropomyosin (purified from chicken gizzard) into normal and transformed cells. We have shown that this tropomyosin binds to stress fibers in live NRK cells. It also localizes in leading edge ruffles of moving cells. This same protein, however, induces neither cell spreading nor stress fiber formation when injected into live 442 cells. We are now purifying tropomyosin from NRK cells to see if it is more effective in stimulating actin reorganization in 442 cells. We now have affinitypurified antitropomyosin and have shown that it binds to stress fibers but does not disrupt them after injection into well-spread, nonmoving NRK cells. We are currently assessing the effect of injection of antibody into cells that are just beginning to spread after plating, to see if the assembly of stress fibers is blocked by antibody.