Analysis of Rho GEF function in epithelial cell morphogenesis

描述（由申请人提供）：上皮细胞是由许多人体组织的基本特征组成的细胞片，其依赖于其固有的屏障和运输功能。上皮细胞的形态发生是复杂的，在这一过程中单细胞的调控机制尚不清楚。此外，很难在体内研究这些机制。然而，在上皮细胞形态发生的研究中，上皮细胞的三维培养可以作为基础细胞生物学和体内发育之间的中介。在这种3D环境下对哺乳动物MDCK上皮细胞系的分析揭示了许多关于调节上皮囊肿形成和小管形成的细胞机制的重要见解。具体来说，rho家族gtpase介导的细胞骨架重排被证明是上皮形态发生的几个不同特征所必需的。为了更全面地了解Rho GTPase在上皮细胞形态发生中的功能和调控，我们提出了系统分析Rho GTPase调控家族Rho-family GEFs的作用的实验。基因组中所有已鉴定的Rho gef将通过RNAi单独中断，并且将确认和表征上皮囊肿形成和小管形成的任何表型。Rho gef在这些过程中的亚细胞定位将被确定。此外，还将确定影响形态发生的每种Rho GEF的下游Rho GTPase特异性。已经在人类癌症和发育障碍中发现了Rho家族gtpase及其调控gef的基因突变。在上皮细胞发育的模型系统中分析这些蛋白质应该增加我们对它们在发育和疾病中的作用的理解。获得此类知识的长期目标是确定可用于治疗人类疾病的药物靶点。

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.