Establishing epithelial cell polarity and assembling cell-cell junctions are critical steps in tissue morphogenesis. One of the earliest events in epithelial differentiation is the formation of intercellular junctions required for cell-cell adhesion, barrier function and apicobasal polarity. In order to form mature junctions, epithelial cells must (1) respond to polarity cues and localize junction proteins to the site of future junction formation and (2) coalesce these proteins into fully formed junctions. This process is essential for the development of organs such as the kidney, which features a complex epithelial tubule system that relies on the establishment of polarity and maintenance of complex intercellular junctions to regionally segregate functional membrane domains. When renal epithelial cells lose polarity and junctions (for instance, following chronic injury), epithelial cells delaminate and undergo an epithelial-to-mesenchymal transition that leads to tissue fibrosis and impaired kidney function. The proposed research will further our understanding of how epithelial cells polarize and form junctions in vivo by determining the molecular mechanisms of localizing proteins to junctions and coalescing junction proteins into fully-formed junctions. This information will be instrumental in learning how to prevent pathologic EMTs that occur in kidney disease. Mechanisms of epithelial polarization have been studied predominantly in cultured MDCK cells, where the initial polarity cue is generated by E-cadherin-mediated cell adhesion. However, studies examining epithelial polarization in Drosophila, C. elegans and some types of human cultured cells have shown that polarization can occur in the absence of E-cadherin, indicating that epithelial polarization may occur through different mechanisms in vivo. C. elegans is an excellent model system for studying in vivo mechanisms of epithelial polarization and junction assembly during MET as it offers the possibility to watch polarization as it occurs in wild-type and mutant embryos. The specific goal of the proposed research is to determine how homologues of two conserved polarity regulators (Crumbs/EAT-20 and aPKC/PKC-3) regulate C. elegans epithelial cell polarization and junction assembly, respectively. We have shown that EAT-20 functions redundantly with the scaffolding protein PAR-3 to polarize the C. elegans epidermis. In my first aim, I will determine how EAT-20 localizes and functions using genetics and live imaging. To address mechanisms of junction assembly by aPKC/PKC-3, we have performed a genetic suppressor screen to identify genes that function with pkc-3 to regulate epithelial junctions. In my second aim, I will clone two pkc-3 suppressors and determine how they regulate epithelial junctions. Together, I anticipate that my findings will provide new insights into the basic mechanisms of epithelial polarization and junction formation as they occur in vivo, providing a foundation for understanding the molecular mechanisms responsible for junction loss in kidney disease and cancer.
Establishing epithelial cell polarity and assembling cell-cell junctions are critical steps in tissue morphogenesis and kidney development. Defects in this process can lead to loss of tissue structure, and have been shown to result in renal fibrosis secondary to chronic kidney disease. Determining the mechanisms epithelial cells use to develop polarity as they differentiate in vivo will be instrumental in learning how to prevent undesired loss of cell polarity that contributes to kidney disease and cancer.
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|Armenti, Stephen T; Lohmer, Lauren L; Sherwood, David R et al. (2014) Repurposing an endogenous degradation system for rapid and targeted depletion of C. elegans proteins. Development 141:4640-7|