Regulation of epithelial cell polarity by ubiquitin ligase signalling networks.

点击翻译按钮获取中文摘要

Epithelial cells have a characteristic apical-basal axis of polarity essential for the formation and function of epithelial tissues. Apical-basal polarity is defined by specific apical, lateral, and basal membrane domains, and the formation extensive cell-cell contacts that together control cell shape, assembly of signaling complexes and formation of cell structures such as cilia and microvilli. The protein complexes that control apical-basal polarity, junction formation and cell morphology are regulated by post-translational modifications. We have used proteomic approaches to define the interaction network of the E3 ubiquitin ligase Mindbomb 1 (Mib1). While Mib1 has a well characterized role in the regulation of the Notch signaling pathway in mammals our recent analysis suggests a novel function for Mib1 in cell polarity. Polarity complex protein EPB41L5 and the apical membrane determinant Crumbs (CRB) were identified as Mib1 interactors and substrates. Our data also suggest that ubiquitin modification of EPB41L5 and CRB by Mib1 could serve as a mechanism to coordinate epithelial cell polarity and Notch signaling. The objective of our research program is to understand how protein modification by ubiquitin regulates apical-basal polarity, signaling, as well as epithelial cell morphogenesis and function. We hypothesize that Mib1 regulates epithelial cell polarity, morphology and signaling through ubiquitin modification of EPB41L5 and CRB. To address this hypothesis we propose the following specific aims: Aim 1. Determine the role of EPB41L5-Mib1 on CRB ubiquitination and endocytosis. We will precisely map the Mib1 domains and substrate recognition motifs required for Mib1 ubiquitination of EPB41L5 and CRB using in vitro protein binding assays and mutagenesis. In addition, the role of  Mib1 in ubiquitination and endocytosis of CRB will be investigated in polarized MDCKII and ARPE19 cell models. Aim 2. Characterization of EPB41L5-Mib1 function in epithelial morphogenesis. To investigate the function of Mib1 in epithelial polarity and morphogenesis we will use 3D culture of MDCKII and intestinal organoids. The effects of Mib1 and EPB41L5 depletion on polarity, lumen formation, morphogenesis, cell extrusion and differentiation will be determined. AIM3: Determine the role of Mib1-EPB41L5 in Notch and Crumbs signaling cross-talk. We will investigate EPB41L5 effects on Notch signaling by testing how EPB41L5 expression alters Notch ligand binding to, and ubiquitination by Mib1. Complimentary experiments will test effects of Notch ligand expression on endocytosis and activity of CRB.   This research program will contribute to our understanding of the mechanisms that regulate cell polarity, signaling and epithelial morphogenesis, and provide new knowledge of how ubiquitin ligases and their substrates function in these important in these processes. This program will contribute to the training of HQP in the fields of molecular and cell biology.