Ubiquitin system regulation of epithelial cell morphogenesis.

细胞极性的建立和维持对上皮组织的形成和功能发挥至关重要。上皮细胞具有由皮质蛋白复合物建立的特征性顶-底极性轴，皮质蛋白复合物定义特定的膜结构域。上皮细胞还通过粘附和紧密连接形成广泛的细胞-细胞接触，从而产生屏障功能。此外，极化上皮细胞中的肌动球蛋白网络调节顶膜、侧膜和基底膜结构域的大小，从而控制上皮细胞的形状。控制顶端-基底极性、连接形成和细胞形态的蛋白质复合物通过翻译后修饰（例如控制其定位和活性的磷酸化）来调节。我们对极化上皮细胞中蛋白复合物的分析揭示了E3泛素连接酶Mindbomb 1（Mib 1）在上皮形态中的一种以前未被认识的功能。我们发现Mib 1与EPB 41 L5形成复合物，EPB 41 L5是一种FERM结构域蛋白，形成侧膜极性复合物的一部分。我们还发现，极化MDCK细胞中的Mib 1缺失导致极性丧失和细胞形态改变。这些数据表明，泛素修饰的蛋白质Mib 1在调节上皮细胞的极性和形态的作用。 我的研究项目的目标是了解蛋白质相互作用和蛋白质修饰的泛素调节细胞极性，信号和膜蛋白运输。为了实现这一目标，我们的短期目标是确定Mib 1在上皮细胞极性和形态调节中的功能。 为实现这一目标，我们提出以下具体目标： 目的1：确定Mib 1蛋白中参与A-B极性调控的结构域。我们将使用诱变和体外泛素化测定来确定多个RING和CC结构域对Mib 1泛素连接酶活性的贡献。为了确定Mib 1蛋白相互作用结构域在细胞极性中的作用，我们将在全长FLAG标记的Mib 1中产生一系列突变，这些突变特异性地破坏Mib/HERC、ZZ结构域、锚蛋白重复序列以及每个RING结构域和CC区域。将在极化MDCKII细胞中检查这些突变对Mib 1功能的影响。 AIM 2：Mib 1底物和调节细胞极性的相互作用蛋白的鉴定。我们已经确定了MIB基板和相互作用的蛋白质，使用细胞内接近生物素化方法，然后通过质谱（称为BioID）。将检测鉴定的蛋白质与Mib 1的直接结合，并确定它们是否为Mib 1底物。将在MDCK细胞中确定鉴定的Mib 1相互作用蛋白和底物在上皮形态中的作用。 目的3：确定Mib 1在上皮形态发生中的作用。为了确定Mib 1依赖的动态事件发生在重塑上皮片形成更复杂的结构，我们将使用通过三维（3D）细胞培养和活细胞成像。此外，我们将研究Mib 1在上皮形态发生过程中的动态定位。 这项研究计划将影响我们对调节细胞极性和上皮形态发生的机制的理解，并提供有关泛素修饰在调节细胞极性中的作用的新知识。该计划还将有助于HQP在分子和细胞生物学领域的培训。

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.