Regulation of epithelial cell polarity by ubiquitin ligase signalling networks.

上皮细胞有一个特有的顶端-基底轴的极轴，对上皮组织的形成和功能至关重要。顶端-基底端的极性由特定的顶端、侧端和基底膜区域定义，形成广泛的细胞-细胞接触，共同控制细胞形状、信号复合体的组装和细胞结构的形成，如纤毛和微绒毛。控制顶端-基底端极性、连接形成和细胞形态的蛋白质复合体受翻译后修饰的调节。我们使用蛋白质组学的方法来定义E3泛素连接酶Mind Bomb 1(Mib1)的相互作用网络。虽然Mib1在哺乳动物Notch信号通路的调控中发挥了很好的作用，但我们最近的分析表明，Mib1在细胞极性中具有新的功能。极性复杂蛋白EPB41L5和顶膜决定簇(CRB)被鉴定为Mib1的相互作用和底物。我们的数据还表明，Mib1对EPB41L5和CRB的泛素修饰可能是一种协调上皮细胞极性和Notch信号的机制。我们的研究计划的目的是了解泛素修饰的蛋白质如何调节顶端-基底端的极性、信号以及上皮细胞的形态发生和功能。我们假设Mib1通过EPB41L5和CRB的泛素修饰来调节上皮细胞的极性、形态和信号转导。为了解决这一假设，我们提出了以下具体目标：目的1.确定EPB41L5-Mib1在CRB泛素化和内吞作用中的作用。我们将使用体外蛋白结合分析和突变技术精确地定位EPB41L5和CRB的Mib1泛素化所需的Mib1结构域和底物识别基序。此外，还将在极化的MDCKII和ARPE19细胞模型中研究MIb1在CRB泛素化和内吞作用中的作用。目的2.EPB41L5-Mib1在上皮形态发生中的作用特征。为了研究Mib1在上皮细胞极性和形态发生中的作用，我们将使用MDCKII和肠道器官的3D培养。Mib1和EPB41L5缺失对极性、管腔形成、形态发生、细胞挤出和分化的影响将被确定。目的：确定Mib1-EPB41L5在Notch和Crumbs信号串扰中的作用。我们将通过测试EPB41L5的表达如何改变Notch配体与Mib1的结合和泛素化来研究EPB41L5对Notch信号的影响。免费实验将测试Notch配体表达对CRB内吞作用和活性的影响。该研究计划将有助于我们了解调控细胞极性、信号和上皮形态发生的机制，并提供泛素连接酶及其底物如何在这些过程中发挥重要作用的新知识。该项目将有助于HQP在分子和细胞生物学领域的培训。

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.