Analysis of Rho GEF function in epithelial cell morphogenesis

描述(申请人提供)：上皮细胞是构成许多人体组织的基本特征的细胞片，这些组织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，对单个细胞在这一过程中的调控机制知之甚少。此外，在体内研究这些机制也是困难的。然而，在上皮形态发生的研究中，三维培养的上皮细胞可以作为基础细胞生物学和活体发育之间的中介。在这样的3D环境中对哺乳动物MDCK上皮细胞系的分析揭示了许多关于调控上皮囊形成和小管形成的细胞机制的重要见解。具体地说，Rho家族GTP酶介导的细胞骨架重排被证明是上皮形态发生的几个明显特征所必需的。为了更全面地了解Rho GTP酶在上皮形态发生中的功能和调控，本实验拟系统地分析Rho GTP酶调节家族--Rho家族GEF的作用。基因组中所有已识别的Rho GEF都将通过RNAi单独干扰，并将确认和表征上皮性包囊形成和小管形成的任何表型。在这些过程中发挥作用的Rho GEF的亚细胞定位将被确定。此外，还将确定每个影响形态发生的Rho GTP酶下游Rho GTP酶的特异性。已在人类癌症和发育障碍中发现了Rho家族GTP酶及其调控的GEF的基因突变。在上皮发育的模型系统中分析这些蛋白质应该会增加我们对它们在发育和疾病中的作用的理解。获取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标。

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.