The kidney consists mainly of polarized epithelial cells. Proper polarization of these epithelial cells is essential for normal kidney function and is deranged in several renal diseases, such as autosomal dominant polycystic kidney disease. The polarization of individual cells is coordinated to form multicellular structures. For instance, the cells that line the tubules of the nephron are all oriented such that their apical surfaces face the central lumen of the tubule, while their basal surface faces towards the periphery. We have found that the establishment of cell polarity and the coordinated orientation of that polarity can be experimentally separated. We use Madin-Darby canine kidney (MDCK) cells grown in three-dimensional collagen gels, where the MDCK cells form hollow cysts lined by a monolayer of polarized epithelial cells. Expression of a dominant negative (DN) form of the small GTPase Racl causes an inversion of the orientation of polarity, so that the apical surface now points toward the periphery of the cyst, rather than towards the central lumen. DN Racl also causes impaired assembly of laminin around the cyst. Addition of exogenous laminin rescues the orientation of cell polarity. We also found that inhibition of beta1 integrin, Cdc42, or atypical Protein Kinase C (aPKC); all result in inversion of orientation of polarity. This leads to a hypothesis of a pathway that controls orientation of polarity: Raclr-> beta1-> integrin-> laminin assembly-> laminin receptor (probably beta1 integrin) -> Cdc42-> aPKC/Par3/Par6 complex. The aPKC/Par3/Par6 complex is a conserved module that controls polarization in nearly all-metazoan cells. We will test most aspects of this model in this grant. We will test the hypothesis that laminin assembly is downstream of Racl and upstream of Cdc42. We will test the hypothesis that integrins act both upstream and downstream of laminin assembly. We will use a powerful collection of mutants in Cdc42, aPKC, Par3 and Par6 test the hypothesis that all of these proteins control orientation of polarity. This work will lead to important advances in understanding cell polarization and how the polarization of individual cells is coordinated to organize multicellular structures, such as tubules and cysts. This will be invaluable in understanding the pathogenesis of renal diseases where this polarity is deranged.
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