Analysis of Rho GEF function in epithelial cell morphogenesis

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DESCRIPTION (provided by applicant): Epithelia are sheets of cells that comprise an essential feature of many human tissues which rely on their intrinsic barrier and transport functions. Epithelial cell morphogenesis is complex and little is known about the mechanisms regulating the actions of single cells during this process. Additionally, it is difficult to study these mechanisms in vivo. However, three-dimensional (3D) culture of epithelial cells can serve as an intermediate between basic cell biology and in vivo development in the study of epithelial morphogenesis. Analysis of the mammalian MDCK epithelial cell line in such a 3D environment has revealed a number of important insights regarding cellular mechanisms regulting epithelial cyst formation and tubulogensis. Specifically, cytoskeletal rearrangements mediated by Rho-family GTPases were shown to be required for several distinct features of epithelial morphogenesis. In order to more fully understand the function and regulation of Rho GTPases in epithelial morphogenesis, experiments are proposed to systematically analyze the roles of a family of Rho GTPase regulators, the Rho-family GEFs. All identified Rho GEFs in the genome will be individually disrupted through RNAi and any phenotypes on epithelial cyst formation and tubulogenesis will be confirmed and characterized. The subcellular localization of Rho GEFs functioning in these processes will be determined. Additionally, the downstream Rho GTPase specificity of each Rho GEF affecting morphogenesis will be determined. Genetic mutations in both Rho family GTPases and their regulatory GEFs have been identified in human cancers and developmental disorders. Analysis of these proteins in a model system for epithelial development should increase our understanding of their role in development and disease. The long term aim in obtaining such knowledge is to identify targets for drugs that can be used to treat human disease.