The purpose of this project is to determine the differential function and subcellular localization of actin isozymes. Experiments will exploit variation in actin primary sequence at the N-terminus among the three isozymes to be studies (Alpha, Beta and Gamma). N-terminal peptides of the three isozymes will be purified from tryptic digests, using high pressure liquid chromatography. Peptides will be coupled to hemocyanin and this complex will be used as the immunogen in order to raise rabbit polyclonal and rat monoclonal antibodies. Antibodies will be tested for their actin isozyme specificity. Peptide antibodies specific for skeletal muscle (Alpha) actin will be used in immunolocalization at the electron microscope level of muscle-specific Alpha actin during myogenesis in human cultured myoblasts. Those specific for Beta and Gamma will be used in immunolocalizations in human muscle cells, monkey spleen fibroblasts, and rat neuronal cells. In addition antibodies specific for the N-terminus of actin isozymes will be used to determine tha role of the N-terminal domain of actin in several of its functions: microfilament polymerization, actomyosin ATPase, and profilin and DNAse binding. Since abnormalities in organization and function of actin are symptomatic of muscular dystrophy, cancer and many other diseases, investigations of normal actin organization and function might be useful in understanding these disease conditions.
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| Otey, C A; Kalnoski, M H; Lessard, J L et al. (1986) Immunolocalization of the gamma isoform of nonmuscle actin in cultured cells. J Cell Biol 102:1726-37 |
