Regulation of epithelial cell polarity by ubiquitin ligase signalling networks.

上皮细胞具有对上皮组织的形成和功能必不可少的特征性顶-底极性轴。顶-底极性由特定的顶、侧和基底膜结构域定义，并且形成广泛的细胞-细胞接触，其共同控制细胞形状、信号复合物的组装和细胞结构如纤毛和微绒毛的形成。控制顶基极性、连接形成和细胞形态的蛋白质复合物受翻译后修饰的调节。我们使用蛋白质组学方法来定义E3泛素连接酶Mindbomb 1（Mib 1）的相互作用网络。虽然Mib 1在哺乳动物Notch信号通路的调节中具有良好的特征化作用，但我们最近的分析表明Mib 1在细胞极性中具有新的功能。极性复合物蛋白EPB 41 L5和顶端膜决定簇（CRB）被确定为Mib 1的相互作用和底物。我们的数据还表明，Mib 1对EPB 41 L5和CRB的泛素修饰可以作为协调上皮细胞极性和Notch信号传导的机制。我们的研究计划的目的是了解蛋白质修饰泛素调节顶基极性，信号，以及上皮细胞的形态和功能。我们推测Mib 1通过泛素修饰EPB 41 L5和CRB调节上皮细胞极性、形态和信号传导。 为了解决这一假设，我们提出了以下具体目标：目标1。确定EPB 41 L5-Mib 1在CRB泛素化和内吞作用中的作用。我们将精确地映射Mib 1结构域和底物识别基序所需的Mib 1泛素化的EPB 41 L5和CRB使用体外蛋白结合试验和诱变。此外，还将在极化MDCKII和ARPE 19细胞模型中研究Mib 1在CRB遍在蛋白化和内吞中的作用。目标二。EPB 41 L5-Mib 1在上皮形态发生中功能的表征。为了研究Mib 1在上皮极性和形态发生中的功能，我们将使用MDCK II和肠类器官的3D培养。将确定Mib 1和EPB 41 L5缺失对极性、管腔形成、形态发生、细胞挤出和分化的影响。目标3：确定Mib 1-EPB 41 L5在Notch和Crumbs信号串扰中的作用。我们将通过测试EPB 41 L5表达如何改变Notch配体与Mib 1的结合以及Mib 1的泛素化来研究EPB 41 L5对Notch信号传导的影响。补充实验将测试Notch配体表达对CRB的内吞作用和活性的影响。 这项研究计划将有助于我们了解调节细胞极性，信号传导和上皮形态发生的机制，并提供有关泛素连接酶及其底物在这些重要过程中如何发挥作用的新知识。该计划将有助于培养HQP在分子和细胞生物学领域。

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.