The desmosome is an intercellular junction which plays an important role in adhesion of epithelial cells. The hemidesmosome appears to effect adherence of basal cells in certain epithelia to the basement zone (BMZ). The program described in this application is aimed at characterizing the adhesion molecules of the desmosome and the hemidesmosome. Desmosomes will be prepared from bovine epithelial tissues by standard procedures. The 9.5M urea insoluble fraction of desmosomes is enriched is desmosomal glycoprotein components i.e. polypeptides with a possible role in cell adhesion. Monoclonal and polyclonal antibodies will be generated against the components of this urea insoluble fraction. These antibody preparations will be used in immunofluorescence assays to determine the distribution of desmosomal components in a variety of epithelial tissues. In addition, these antibody preparations will be localized ultrastructurally by the immungold procedure. Furthermore, using a keratinocyte culture system in which desmosome formation can be manipulated by the extracellular Ca2+ levels, incorporation of components into assembling desmosomes will be monitored by immunofluorescence and by immunoelectron microscopy. Fab' fragments derived from these desmosomal antibodies will be used in cell adhesion assays using cultured keratinocytes and organ cultures of skin. A technique is described for the enrichment of the components of hemidesmosomes i.e. the removal of the epidermis from the underlying connective tissue following EDTA treatment results in the retention of hemidesmosomes along the BMZ. The hemidesmosomes will be removed from the BMZ by solubilization in urea/SDS and monoclonal antibody preparations directed against hemidesmosomal components will be raised. The distribution of hemidesmosomal antigens in a variety of tissue types will be determined by indirect immunofluorescence. The binding sites of these hemidesmosomal antigens will be determined at the ultrastructural level be immunogold localization. Attempts to monitor the formation of hemidesmosomes by immunofluorescence and immunoelectron microscopy will involve the use of cultured keratinocytes. Fab' fragments of the hemidesmosomal antibodies will also by assayed for their ability to inhibit the adhesion of cultured cells to substrata and/or inhibit the formation of hemidesmosomes in an in vitro organ system. These studies may support a hypothesis that these two organelles play a role in mediating the kinds of cell interactions which are essential for tissue morphogenesis and the maintenance of tissue integrity.
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