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肾功能至关重要。肾上皮细胞必须完全极化到基底外侧和根尖表面，然后将适当的转运蛋白分类到每个区域，以促进液体的重吸收和电解质的维持。根尖表面还含有原发的非活动纤毛，这可能在多囊肾病等肾脏疾病中起作用。这一建议将重点放在面包屑家族的跨膜蛋白，被认为是重要的细胞极化。在果蝇中，面包屑首先被发现是一种上皮极性蛋白，在果蝇眼睛的发育中也很重要。哺乳动物的同源基因Crumbs1也在哺乳动物的眼睛中表达，该基因的突变导致视网膜色素变性。crumb3是哺乳动物上皮中存在的异构体，作为一种与紧密连接蛋白PALS1和PATJ结合的顶端跨膜蛋白表达。我们认为，crumb3对顶端膜的形成和细胞极性的形成以及紧密连接的形成起着重要的作用。我们还假设，对于根尖表面非运动纤毛的正常形成，碎屑是必要的。第一个具体目标是研究Crumbs3如何靶向根尖表面，以更好地理解它是如何标记这个区域的。下一个具体的目标是研究crumb3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crumb3表达被siRNA降低的MDCK细胞中进行。我们将通过拯救Crumbs3野生型和突变型的Crumbs3 siRNA细胞系，对Crumbs3进行结构/功能分析。使用类似的技术，我们将把我们的研究扩展到crumb3在初级非运动性纤毛形成中的作用。这一特定目标的延伸将是研究crumb3与微管和运动蛋白的相互作用，作为顶端膜和初级纤毛形成的关键事件。最后，我们将利用Crumbs3的细胞内小结构域作为亲和基质，寻找能够结合Crumbs3的其他蛋白。这项工作将为细胞极性的产生提供新的见解，并将对屑蛋白在睫状体功能障碍相关疾病（如视网膜色素变性和多囊肾病）中的作用具有广泛的意义。

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.