Crumbs3 in epithelial polarity and ciliogenesis

描述（由申请方提供）：上皮极性的产生和维持对于正常的肾功能至关重要。肾上皮细胞必须完全覆盖到基底外侧和顶面，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。顶端表面还含有初级非运动纤毛，这可能在肾脏疾病如多囊肾病中起作用。这项建议将集中在跨膜蛋白的Crumbs家族，被认为是重要的细胞极化。Crumbs首先在果蝇中被鉴定为上皮极性蛋白，其在果蝇眼睛的发育中也是重要的。哺乳动物直系同源基因Crumbs 1也在哺乳动物眼睛中表达，该基因的突变导致视网膜色素变性。Crumbs 3是存在于哺乳动物上皮中的同种型，并表达为与紧密连接蛋白PALS 1和PATJ结合的顶端跨膜蛋白。我们认为Crumbs 3对于顶端膜的形成和细胞极性以及紧密连接的形成是重要的。我们还假设Crumbs 3是必要的正确形成的非运动纤毛的顶端表面。第一个具体目标将研究Crumbs 3如何靶向顶面，以更好地了解它如何标记该域。下一个具体目标将检查Crumbs 3在MDCK细胞中细胞极化和紧密连接形成中的作用。这些研究将在Crumbs 3表达已被siRNA降低的MDCK细胞中进行。我们将通过拯救具有Crumbs 3野生型和突变形式的Crumbs 3 siRNA细胞系来进行Crumbs 3的结构/功能分析。使用类似的技术，我们将扩展我们的研究Crumbs 3在初级非运动纤毛的形成中的作用。这一特定目标的延伸将是研究Crumbs 3与微管和马达蛋白的相互作用，作为顶膜和初级纤毛形成中的关键事件。最后，我们将通过使用Crumbs 3的小细胞内结构域作为亲和基质来寻找可以结合Crumbs 3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（例如色素性视网膜炎和多囊肾病）中的作用产生广泛的影响。

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.