Analysis of Rho GEF function in epithelial cell morphogenesis

描述（由申请人提供）：上皮细胞是细胞片，其包括依赖于其内在屏障和转运功能的许多人体组织的基本特征。上皮细胞的形态发生是复杂的，在这一过程中，对调节单细胞作用的机制知之甚少。此外，很难在体内研究这些机制。然而，上皮细胞的三维（3D）培养可以作为基础细胞生物学和上皮形态发生研究中的体内发育之间的中间体。哺乳动物MDCK上皮细胞系在这样的3D环境中的分析揭示了许多重要的见解，关于细胞机制调节上皮囊肿形成和tubulogensis。具体而言，细胞骨架重排Rho家族GTP酶介导的上皮形态发生的几个不同的功能所需的。为了更全面地了解Rho GTPases在上皮形态发生中的功能和调节，提出了实验来系统地分析Rho GTPases调节剂家族，Rho家族GEFs的作用。基因组中所有鉴定的Rho GEF将通过RNAi单独破坏，并且将确认和表征上皮囊肿形成和小管发生的任何表型。将确定在这些过程中发挥作用的Rho GEFs的亚细胞定位。此外，将确定影响形态发生的每个Rho GEF的下游Rho GTdR特异性。Rho家族GTP酶及其调节GEF的基因突变已在人类癌症和发育障碍中被鉴定。在上皮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。获得这些知识的长期目标是确定可用于治疗人类疾病的药物的靶点。

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.