The cell-cell adhesion structures, desmosomes, are intimately involved in the morphogenesis of epithelial tissues. Transmembrane desmosomal glycoproteins, desmocollin (Dsc) and desmoglein (Dsg), belong to a multi- gene family of cadherins and play an important role in desmosome function. The intracellular regions of desmosomal cadherins are integrated in the cytoplasmic plaque, serving as an anchorage site for intermediate filaments. Little is known about the mechanisms of desmosome assembly and function. No data has been reported concerning desmosomal dynamics. Our preliminary results indicate a direct calcium-dependent interaction between the extracellular regions of Dsg and Dsc. We hypothesize that these interactions not only mediate cell-cell adhesion, but also trigger plakoglobin and desmoplakin dependent assembly of Dsg/Dsc complexes into desmosomes. The broad goal of the present proposal is to evaluate this hypothesis. To this end we will define amino acid residues within extracellular regions of the desmosomal cadherins involved in Dsg-Dsc interactions and determine the effect of this interaction on desmosome assembly. We will also study whether desmosomal cadherins are able to form this interaction on desmosome assembly. We will study whether desmosomal cadherins are able to form homodimers. In conjunction with this investigation we will examine the function of the plakoglobin in desmosomes. While our previous experiments demonstrated a critical role of this protein in desmosome assembly, its function is not well understood. Recently a novel model of desmosome reconstitution in plakoglobin-negative HIT-1080 cells was established in my laboratory. This model provides us with an opportunity to investigate the function of plakoglobin using wild type and mutant forms of the protein expressed in HT-1080 cells. The final part of this proposal is focused on elucidating desmosome dynamics in epithelial cells, in particular, during their migration. To accomplish this, we have constructed a PgGFP chimeric protein consisting of intact plakoglobin and Green Fluorescent protein. We have shown that this chimera is integrated properly into desmosomes that can be visualized in living cells by time-lapse fluorescence microscopy. Using this chimera we will investigate the dynamics of assembly and disassembly of desmosomes in response to injury of an epithelial sheet and to different cellular environments in culture. The experiments described in this proposal will provide data indispensable for understanding the molecular mechanisms responsible for remodeling of epithelial tissue in development and wound healing.
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