The focus of this research is the focal contacts of cultured fibroblasts in normal and transformed cells. Focal contacts are discrete plaque-like regions on the periphery of the ventral cell surface where that surface most closely approaches the substratum and are the sites of strongest cell-substratum adhesion. Inside the cell, focal contacts are the sites where microfilament bundles terminate. Following transformation of oncogenic viruses, microfilaments are disrupted, peripheral focal contacts are lost, and their protein components redistributed. Transformation by Rous sarcoma and related viruses involves phosphorylation of host cell proteins on tyrosine residues. These molecular alterations probably are responsible for the typical transformed cell phenotype. Several pieces of evidence indicate that the fascia adherens of cardiac muscle cell intercalated disk membrane is a structure closely homologous to the fibroblast focal contact. Since the fascia is a stable structure unlike focal contacts, and a much richer source of microfilament-membrane attachment sites, it can be used to identify components of fibroblast focal contacts. Thus, fascia have been isolated from adult chicken hearts and several of several of the protein components identified, purified, and used to generate polyclonal and monoclonal antibodies. Immunocytochemical methods have been used to look for the same or similar components in fibroblast focal contacts. Two of these fascia components, with molecular weights of 200 kilodalton and 245 kilodalton, have been found in focal contacts. The next step has been to study the effect of transformation on these two proteins which are localized to focal contacts. The distribution of the focal contact proteins in cells transformed by Rous sarcoma and related viruses has been examined by light microscopic immunofluorescent methods and found to be altered compared to normal cells. In addition, the degree of phosphorylation on tyrosine residues of each protein, in both normal and transformed cells, is under investigation using antibodies to phosphotyrosine. For each transforming agent, these two properties of the focal contact proteins are being compared with the cell shape in order to determine if a correlation exists between the molecular features of the protein and transformed cell morphology. Thus, by focusing on the areas of the cell responsible for cell-substrate adhesion, this research should provide a better understanding of how that process can be altered by transformation. (A)
