In epithelial tissues the hemidesmosome is considered to play an important role in epithelial-connective tissue adherence. Its reassembly during wound healing in epithelia appears essential for the establishment of firm attachment of epithelial cells to the underlying connective tissue. The hemidesmosome acts as the structural link between components of the extracellular matrix in the connective tissue and the cytoskeleton of the basal epithelial cell. The hemidesmosomal plaque is the anchorage site of bundles of keratin containing intermediate filaments along the basal surface of the epithelial cell. It is the goal of Dr. Jones to investigate the molecular structure and assembly of the hemidesmosome. He is able to undertake such analyses since he has derived a number of antibody preparations directed against the hemidesmosome and also molecular probes for certain hemidesmosomal plaque elements during the previous grant period. In addition, he recently identified a cell line, termed 804G, which readily assembles hemidesmosomes in culture. The specific aims are: (1) He will continue studies of the isolation of hemidesmosomal polypeptides using a combination of chromatographic and immunopurification procedures. (2) He will monitor hemidesmosome assembly at the ultrastructural level in his recently described epithelial explant wound healing model. (3) He will clone and sequence hemidesmosomal components. The deduced amino acid sequences and secondary structural predictions that are derived from such analyses may indicate homologies with known polypeptides and may also suggest functions for hemidesmosomal polypeptides. (4) He will monitor hemidesmosome assembly in 804G cells at the morphological, biochemical and molecular levels. (5) He will investigate hemidesmosomal protein turnover in 804G cells. (6) He will identify glycosylated hemidesmosomal elements involved in the initiation of hemidesmosomal assembly and/or hemidesmosome-substratum adherence by adhesion assays. This will involve the use of monoclonal and polyclonal antibodies generated against both tissue hemidesmosomal components and the hemidesmosomes of 804G cells. (7) He will use transfection techniques to identify sequences of hemidesmosomal proteins necessary for the incorporation of elements into the hemidesmosome and assembly of the hemidesmosome complex.