Regulation of epithelial cell polarity by ubiquitin ligase signalling networks.

通过泛素连接酶信号网络调节上皮细胞极性。

• 批准号: RGPIN-2019-06485
• 负责人: McGlade, Catherine
• 金额: $ 3.64万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760728
• 关键词: Regulation; epithelial; cell; polarity; ubiquitin

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.

上皮细胞具有典型的顶基极性轴，这对上皮组织的形成和功能至关重要。顶基极性由特定的顶、侧、基膜结构域和广泛的细胞-细胞接触的形成决定，这些细胞-细胞接触共同控制细胞形状、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是通过翻译后修饰来调节的。我们使用蛋白质组学方法来定义E3泛素连接酶Mindbomb 1 （Mib1）的相互作用网络。虽然Mib1在哺乳动物Notch信号通路的调控中具有很好的特征，但我们最近的分析表明，Mib1在细胞极性中具有新的功能。极性复合物蛋白EPB41L5和顶膜决定因子crumb （CRB）被确定为Mib1相互作用物和底物。我们的数据还表明，Mib1对EPB41L5和CRB的泛素修饰可能是协调上皮细胞极性和Notch信号传导的机制。我们的研究目标是了解泛素修饰蛋白如何调节顶基极性、信号传导以及上皮细胞的形态发生和功能。我们假设Mib1通过泛素修饰EPB41L5和CRB调控上皮细胞极性、形态和信号传导。为了解决这一假设，我们提出以下具体目标：目标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试验和诱变技术精确绘制EPB41L5和CRB的Mib1泛素化所需的Mib1结构域和底物识别基序。此外，将在极化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。目标2。EPB41L5-Mib1在上皮形态发生中的功能表征。为了研究Mib1在上皮极性和形态发生中的功能，我们将使用MDCKII和肠道类器官的3D培养。将确定Mib1和EPB41L5缺失对极性、管腔形成、形态发生、细胞挤压和分化的影响。AIM3：确定Mib1-EPB41L5在Notch和crumb信号串扰中的作用。我们将通过测试EPB41L5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。补充实验将测试Notch配体表达对CRB内吞作用和活性的影响。该研究项目将有助于我们理解细胞极性、信号和上皮形态发生的调节机制，并为泛素连接酶及其底物在这些重要过程中的作用提供新的知识。该计划将有助于培养分子和细胞生物学领域的HQP。