Ubiquitin system regulation of epithelial cell morphogenesis.

泛素系统调节上皮细胞形态发生。

• 批准号: RGPIN-2014-03649
• 负责人: McGlade, Catherine
• 金额: $ 3.86万
• 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: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=610566
• 关键词: Ubiquitin; system; regulation; epithelial; cell

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.

细胞极性的建立和维持对于形成和功能上皮组织至关重要。上皮细胞具有由定义特定膜结构域的皮质蛋白复合物建立的极性的特征顶基轴。上皮细胞还通过粘附和创建屏障功能的紧密连接剂形成广泛的细胞接触。此外，极化上皮细胞中的肌动球蛋白网络调节顶端，外侧和基底膜结构域的大小，后者又控制上皮细胞的形状。控制顶端极性，结形成和细胞形态的蛋白质复合物受到翻译后修饰（例如控制其定位和活性的磷酸化）的调节。我们对极化上皮细胞中蛋白质复合物的分析揭示了上皮形态中E3泛素连接酶Mindbomb 1（MIB1）的先前未识别的功能。我们发现MIB1与EPB41L5（一种FERM结构域蛋白质，构成侧膜极性复合物的一部分）形成复合物。我们还发现，极化MDCK细胞中的MIB1耗竭会导致极性丧失和细胞形态改变。这些数据表明，MIB1在调节上皮细胞极性和形态中对蛋白质的泛素修饰的作用。 我的研究计划的目的是了解泛素通过蛋白质相互作用和蛋白质修饰如何调节细胞极性，信号传导和膜蛋白运输。为此，我们的短期目标是定义MIB1在调节上皮细胞极性和形态学中的功能。 为了实现这一目标，我们提出以下具体目的： 目标1：定义参与A-B极性调节的MIB1域。我们将使用诱变和体外泛素化测定法确定多环和CC结构域对MIB1泛素连接酶活性的贡献。为了定义MIB1蛋白相互作用结构域在细胞极性中的作用，我们将在全长标记为标记的MIB1中生成一系列突变，这些突变特异性破坏了MIB/HERC，Zz域，aNkyrin重复，以及每个环域和CC区域。这些突变对MIB1功能的影响将在极化MDCKII细胞中检查。 AIM2：鉴定MIB1底物和调节细胞极性的相互作用蛋白。我们已经使用质谱法（称为Bioid）鉴定了MIB底物和相互作用的蛋白质。确定的蛋白质将进行测试以直接与MIB1结合，我们将确定它们是否是MIB1底物。鉴定的MIB1相互作用蛋白和底物在上皮形态中的作用将在MDCK细胞中确定。 AIM3：确定MIB1在上皮形态发生中的作用。为了确定上皮床单重塑过程中发生的MIB1依赖性动态事件，以形成更复杂的结构，我们将通过三维（3D）细胞培养和活细胞成像使用。此外，我们将研究上皮形态发生过程中MIB1的动态定位。 该研究计划将影响我们对调节细胞极性和上皮形态发生机制的理解，并就泛素修饰在调节细胞极性调节中的作用提供新的知识。该程序还将有助于在分子和细胞生物学领域的HQP培训。