Crumbs3 in epithelial polarity and ciliogenesis

描述(由申请人提供)：上皮极性的产生和维持对正常的肾功能至关重要。肾上皮细胞必须完全极化到基底面和根尖表面，然后将适当的转运蛋白分类到每个区域，以促进液体重吸收和电解质维持。根尖表面还含有原始的不活动的纤毛，这可能在多囊肾病等肾脏疾病中发挥作用。这项建议将集中在Crumbs家族的跨膜蛋白上，这些蛋白被认为在细胞极化中很重要。Crumbs最先在果蝇中被鉴定为一种上皮极性蛋白，它在果蝇眼睛的发育过程中也很重要。哺乳动物的同源基因Crums1也在哺乳动物的眼睛中表达，该基因的突变会导致视网膜色素变性。Crums3是哺乳动物上皮细胞中存在的异构体，以顶端跨膜蛋白的形式表达，与紧密连接蛋白PALS1和PATJ结合。我们认为Crums3对顶膜的形成、细胞的极性以及紧密连接的形成都是重要的。我们还假设，Crums3对于根尖表面非运动纤毛的正确形成是必要的。第一个具体目标将研究Crums3如何定位到根尖表面，以更好地理解它是如何标记这一区域的。下一个具体目标将研究Crums3在MDCK细胞的细胞极化和紧密连接形成中的作用。这些研究将在通过siRNA降低Crums3表达的MDCK细胞中进行。我们将通过拯救含有Crums3野生型和突变型的Crums3 siRNA细胞株来进行Crums3的结构/功能分析。使用类似的技术，我们将把我们的研究扩展到Crums3在初级静止纤毛的形成中的作用。这一特定目标的延伸将是研究Crums3与微管和马达蛋白的相互作用，作为顶膜和初级纤毛形成的关键事件。最后，我们将使用Crums3的小胞内域作为亲和矩阵，寻找能够与Crums3结合的其他蛋白质。这项工作将为细胞极性的产生提供新的见解，并将对Crumbs蛋白在与睫状体功能障碍相关的疾病中的作用产生广泛的影响，如视网膜色素变性和多囊肾病。

DESCRIPTION (provided by applicant): The generation and maintenance of epithelial polarity is crucial for proper renal function. Renal epithelial cells must fully polarize into basolateral and apical surfaces then sort the appropriate transporters to each domain to facilitate fluid reabsorption and electrolyte maintenance. The apical surface also contains primary non-motile cilia, which likely plays a role in kidney disorders such as polycystic kidney disease. This proposal will focus on the Crumbs family of transmembrane proteins that are believed to be important in cell polarization. Crumbs was first identified in Drosophila as an epithelial polarity protein that is also important in the development of the Drosophila eye. The mammalian orthologue, Crumbs1, is also expressed in mammalian eye and mutations in this gene lead to retinitis pigmentosa. Crumbs3 is the isoform present in mammalian epithelia and is expressed as an apical transmembrane protein that binds to the tight junction proteins, PALS1 and PATJ. We believe that Crumbs3 is important for apical membrane formation and cell polarity as well as formation of the tight junction. We also hypothesize that Crumbs3 is necessary for proper formation of the non-motile cilia of the apical surface. The first specific aim will study how Crumbs3 targets to the apical surface to better understand how it comes to mark this domain. The next specific aim will examine the role of Crumbs3 in cell polarization and tight junction formation in MDCK cells. These studies will be performed in MDCK cells where Crumbs3 expression has been reduced by siRNA. We will perform structure/function analysis of Crumbs3 by rescuing Crumbs3 siRNA cell lines with wild type and mutant forms of Crumbs3. Using similar techniques we will extend our studies to the role of Crumbs3 in the formation of the primary nonmotile cilia. An extension of this specific aim will be to examine the interactions of Crumbs3 with microtubules and motor proteins as a pivotal event in the formation of the apical membrane and the primary cilia. Finally we will search for additional proteins that can bind Crumbs3 by using the small intracellular domain of Crumbs3 as an affinity matrix. This work will provide new insights into the generation of cell polarity and will have broad implications for the role of Crumbs proteins in disorders related to ciliary dysfunction, such as retinitis pigmentosa and polycystic kidney disease.