Ubiquitin system regulation of epithelial cell morphogenesis.

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The establishment and maintenance of cell polarity is essential for the formation and functional epithelial tissues. Epithelial cells have a characteristic apical-basal axis of polarity established by cortical protein complexes that define specific membrane domains. Epithelial cells also form extensive cell-cell contacts through adherens and tight junctions that create a barrier function. In addition, the actomyosin network in polarized epithelial cells regulates the size of apical, lateral, and basal membrane domains which in turn controls epithelial cell shape. The protein complexes that control apical-basal polarity, junction formation and cell morphology are regulated by post-translational modifications such as phosphorylation that control their localization and activity. Our analysis of protein complexes in polarized epithelial cells revealed a previously unrecognized function for the E3 ubiquitin ligase Mindbomb 1 (Mib1) in epithelial morphology. We found that Mib1 forms a complex with EPB41L5, a FERM domain protein that forms part of a lateral membrane polarity complex. We have also found that Mib1 depletion in polarized MDCK cells leads to a loss of polarity and altered cell morphology. These data point to a role for ubiquitin modification of proteins by Mib1 in the regulation of epithelial cell polarity and morphology. The objective of my research program is to understand how protein interactions and protein modification by ubiquitin regulates cell polarity, signaling and membrane protein trafficking. To achieve this, our short-term objective is to define the function of Mib1 in the regulation of epithelial cell polarity and morphology. Towards this objective we propose the following specific aims: AIM 1: To define the Mib1 domains involved in regulation of A-B polarity. We will determine the contributions of the multiple RING and CC domain to Mib1 ubiquitin ligase activity using mutagenesis and in vitro ubiquitination assays. To define the role of Mib1 protein interaction domains in cell polarity we will generate a series of mutations in the full length FLAG tagged-Mib1 that specifically disrupt Mib/HERC, ZZ domain, Ankyrin repeats, as well as each of the RING domains and the CC region. The effect of these mutations on Mib1 function will be examined in polarized MDCKII cells. AIM2: Identification of Mib1 substrates and interacting proteins that regulate cell polarity. We have identified MIB substrates and interacting proteins using an in-cell proximity biotinylation method followed by mass spectrometry (called BioID). Proteins identified will be tested for direct binding to Mib1 and we will determine whether they are Mib1 substrates. The role of identified Mib1 interacting proteins and substrates in epithelial morphology will be determined in MDCK cells. AIM3: Determine the role of Mib1 in epithelial morphogenesis. To determine the Mib1 dependent dynamic events that occur during reshaping of epithelial sheets to form more complex structures we will use through 3-dimentional (3D) cell culture and live cell imaging. In addition, we will study the dynamic localization of Mib1 during epithelial morphogenesis. This research program will impact our understanding of the mechanisms that regulate cell polarity and epithelial morphogenesis, and provide new knowledge on the role of ubiquitin modification in regulation of cell polarity. This program will also contribute to the training of HQP in the fields of molecular and cell biology.