Tropomyosin is a major contractile protein which is an integral component of the muscle thin filament and the actin containing microfilaments of the cellular cytoskeleton. Recent research using two dimensional gels and in vitro synthesis has observed that several different tropomyosin proteins, which represent different gene products (isoforms), are synthesized within most cells. Each tropomyosin isoform may vary in its affinity to troponin and actin, where tropomyosin apparently increases the rigidity of F-actin. Furthermore, during myogenesis in culture, there is a developmentally regulated progression of at least four different tropomyosin genes being synthesized. The expression of a family of tropomyosin genes suggests an important functional or structural specialization of this molecule. We will examine the tropomyosin isoforms in adult striated muscle, myogenic cells in culture, and fibroblasts in order to observe the possible specialization of these proteins. In order to recognize the various isoforms, we have successfully prepared antitropomyosin hybridoma antibodies. Three of these antibodies specifically recognizes the 30,000 molecular weight tropomyosin isoform, while other hybridoma antibodies recognize all tropomyosins. When used in conjunction with two dimensional gel electrophoresis and western blotting, these antibodies will allow an important cataloging of the tropomyosin isoforms present in each of the cell types being examined. Immunofluorescence using these antibodies will examine the location of the recognized isoform and possible specificity to a particular microfilament or sarcomere population. Affinity chromatography can then be used to prepare large quantities of each recognized isoform to allow for the examination of its physical chemistry and protein interactions. Additional hybridoma antibodies against tropomyosin isoforms present in each of the cell types being examined will also be prepared. This research will describe the localization, functional specialization, and structural diversity of this tropomyosin gene family.
