Plakoglobin is a major structural component of cell adherence/cytoskeleton-attachment junctions. It also appears to play a key role in inductive signaling mediated by the Wnt family of proto-oncogenes and may interact with the APC tumor suppressor gene product. We propose to exploit the experimental accessibility of the Xenopus embryo to study further the role of plakoglobin in epithelial behavior and cellular differentiation. Expression of exogenous plakoglobin leads to anterior axis duplication in Xenopus. Using the injection of RNA, together with immunocytochemical methods, we will determine which regions of the embryo are most sensitive to the effects of exogenous plakoglobin and which regions of the plakoglobin molecule are required to produce axis duplication. The cytoplasmic tail of the desmosomal cadherin desmoglein-l (DsgTail) interacts with plakoglobin and suppresses its axis duplication effect. DsgTail also suppresses the effects of exogenous Xwnt8, presumably through its interaction with plakoglobin. Immunoprecipitation and RNA co- injection will be used to define the specificity of the DsgTail- plakoglobin interaction; DsgTail will also be used to study the role of endogenous plakoglobin in Wnt-mediate signaling. High levels of plakoglobin induce a gastrulation defect. This gastrulation defect is not suppressed, and may be enhanced, by the co-expression of DsgTail. Using whole-mount immunocytochemistry and in vitro studies, we will examine whether the over-expression of plakoglobin leads to changes in: i) cellular adhesion; ii) the number or distribution of adherence junctions; or iii) convergent extension, a major morphogenetic engine of vertebrate morphogenesis. At adhesion junctions or as a part of the Wnt signaling pathway, we assume that plakoglobin acts through its interactions with other proteins. To identify these proteins, we will use the yeast two- hybrid system. Co-immunoprecipitation will be used to identify plakoglobin-binding proteins (PBPs) that interact strongly with plakoglobin. Immunocytochemistry will be used to identify PBPs associated with adhesion junctions. Exogenous plakoglobin is localized to the nuclei of embryonic cells; putative PBPs that are also localized to the nucleus may be involved in the signaling functions of plakoglobin. The plakoglobin-binding region of a putative PBP may act as a dominant """"""""interfering"""""""" mutation; we will examine the effects of expressing putative PBPs on cell adherence and axis formation. This analysis should identify PBPs involved in the structure and regulation of cellular adhesion junctions and in the processes of mesoderm induction.
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