Crumbs3 and Epithelial Polarity

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DESCRIPTION (provided by applicant): The polarization of renal epithelia is crucial for proper kidney development and function. Recent work from our laboratory and others has pointed to an important role for the Crumbs family of proteins in development and maintenance of epithelial cell polarity. Our work has focused on Crumbs3 which is the predominant form in renal epithelia. Crumbs3 is a small apical transmembrane protein that exists as two different splice isoforms. One isoform, Crumbs3a, uses an evolutionarily conserved carboxy-terminal tail to organize two important polarity complexes, the Crumbs/PALS/PATJ complex and the PAR3/PAR6/aPKC complex. Loss of Crumbs3 expression or overexpression of Crumbs3 can lead to a loss of cell polarity and tight junction formation. It is felt that Crumbs3 functions as a transmembrane anchor that localizes signaling molecules such as aPKC to define the apical domain via reciprocal phosphorylation steps with kinases localized on the basolateral membrane. However a fundamental question is how is Crumbs3 initially localized to instruct creation of the apical membrane. Our newest studies in three-dimensional culture demonstrate that Crumbs3 initiates apical membrane and lumen formation very early in cell division during the cytokinetic process. We hypothesize that the ability of Crumbs3 to target to the mid-body during the cytokinetic process is a crucial step in initial apical membrane and lumen formation. This hypothesis will be tested in the first specific aim where we will examine if factors that regulate membrane trafficking to the midbody during cytokinesis also regulate initial apical membrane formation. In the second specific aim, we will examine the regulation of Crumbs3 expression and trafficking in established polarized epithelia and in epithelia undergoing mesenchymal transition. The last specific aim will examine these processes in vivo by generating a GFP knock in Crumbs3 mouse. This will allow us to determine if our hypothesis generated in tissue culture models holds during kidney development, normal kidney function and during renal recovery from injury. PUBLIC HEALTH RELEVANCE: The goal of this work is to understand the steps that lead to polarized epithelia and kidney tube formation. The proposal focuses on a protein called Crumbs that is crucial in this process. Proper tube formation is important in many diseases including polycystic kidney disease and in recovery from kidney failure. Our data will delineate the basic steps utilized by the Crumbs protein to initiate epithelial polarization.