Crumbs3 in epithelial polarity and ciliogenesis

Crumbs3 在上皮极性和纤毛发生中的作用

基本信息

批准号：
7348366
负责人：
BENJAMIN L MARGOLIS
金额：
$ 31.28万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-02-15 至 2010-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7348366
关键词：
Affinity Apical Attention Binding Cell Count Cell Line Cell Polarity Cells Cilia Complex Defect Development Disease Drosophila eye Drosophila genus Electrolytes Epithelial Epithelial Cells Epithelium Event Eye Family Functional disorder Generations Genes Genetic Goals Growth Integral Membrane Protein Kidney Kidney Diseases Lead Liquid substance Localized Localized Disease MDCK cell Maintenance Mammals Methods Microtubules Mind Motor Mutation Peptides Play Polycystic Kidney Diseases Process Protein Binding Protein Isoforms Proteins Recruitment Activity Renal function Retinitis Pigmentosa Role Signal Transduction Small Interfering RNA Sorting - Cell Movement Structure Surface System Techniques Tight Junctions Work apical membrane atypical protein kinase C cilium biogenesis insight link protein mutant protein purification stable cell line

项目摘要

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.

描述（由申请人提供）：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极化到基底外侧和顶端表面，然后将适当的转运蛋白分类到每个域，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，这可能在多囊肾病等肾脏疾病中发挥作用。该提案将重点关注跨膜蛋白 Crumbs 家族，该家族被认为在细胞极化中很重要。 Crumbs 最初在果蝇中被鉴定为一种上皮极性蛋白，对果蝇眼睛的发育也很重要。哺乳动物的同源基因 Crumbs1 也在哺乳动物的眼睛中表达，该基因的突变会导致色素性视网膜炎。 Crumbs3 是存在于哺乳动物上皮细胞中的同种型，表达为顶端跨膜蛋白，与紧密连接蛋白 PALS1 和 PATJ 结合。我们认为 Crumbs3 对于顶膜形成和细胞极性以及紧密连接的形成很重要。我们还假设 Crumbs3 对于顶端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 Crumbs3 如何瞄准顶端表面，以更好地了解它如何标记该域。下一个具体目标是研究 Crumbs3 在 MDCK 细胞极化和紧密连接形成中的作用。这些研究将在 MDCK 细胞中进行，其中 siRNA 降低了 Crumbs3 的表达。我们将通过用野生型和突变型 Crumbs3 拯救 Crumbs3 siRNA 细胞系来进行 Crumbs3 的结构/功能分析。使用类似的技术，我们将研究范围扩展到 Crumbs3 在初级非运动纤毛形成中的作用。这一具体目标的延伸将是检查 Crumbs3 与微管和运动蛋白的相互作用，作为顶膜和初级纤毛形成的关键事件。最后，我们将使用 Crumbs3 的小胞内结构域作为亲和矩阵来寻找可以结合 Crumbs3 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 蛋白在与纤毛功能障碍相关的疾病（如色素性视网膜炎和多囊肾病）中的作用产生广泛的影响。