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. ? ?
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