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至关重要。上皮细胞有一个特殊的顶端-基底轴，由皮质蛋白复合体建立，该复合体定义了特定的膜域。上皮细胞还通过黏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端、侧端和基底膜区域的大小，进而控制上皮细胞的形状。控制顶端-基底端的极性、连接形成和细胞形态的蛋白质复合体受到翻译后修饰的调节，例如控制它们的定位和活性的磷酸化。我们对极化上皮细胞中蛋白质复合体的分析揭示了E3泛素连接酶思维炸弹1(Mib1)在上皮形态中的一个先前未知的功能。我们发现Mib1与EPB41L5形成复合体，EPB41L5是一种费米结构域蛋白，形成侧膜极性复合体的一部分。我们还发现，在极化的MDCK细胞中，Mib1的缺失会导致极性的丧失和细胞形态的改变。这些数据表明，Mib1对蛋白质的泛素修饰在调节上皮细胞的极性和形态方面发挥了作用。 我的研究计划的目的是了解蛋白质相互作用和泛素对蛋白质修饰如何调节细胞极性、信号和膜蛋白运输。为了实现这一点，我们的短期目标是确定Mib1在调节上皮细胞极性和形态中的功能。 为此，我们提出以下具体目标： 目的1：确定参与A-B极性调节的Mib1结构域。我们将使用诱变和体外泛素化试验来确定多环和CC结构域对Mib1泛素连接酶活性的贡献。为了确定Mib1蛋白相互作用结构域在细胞极性中的作用，我们将在全长标记的-Mib1中产生一系列突变，这些突变专门破坏Mib/HERC、ZZ结构域、Ankyrin重复序列，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的MDCKII细胞中进行检测。 目的：鉴定调节细胞极性的Mib1底物和相互作用蛋白。我们已经使用细胞内邻近生物素化方法和质谱仪(称为BioID)来鉴定MIB底物和相互作用的蛋白质。鉴定出的蛋白质将被测试是否直接与Mib1结合，我们将确定它们是否为Mib1底物。已确定的Mib1相互作用蛋白和底物在MDCK细胞的上皮形态中的作用将被确定。 目的：探讨Mib1在上皮细胞形态发生中的作用。为了确定在上皮片重塑过程中发生的Mib1依赖的动态事件，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来使用。此外，我们还将研究Mib1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细胞极性和上皮形态发生调控机制的理解，并为泛素修饰在细胞极性调控中的作用提供新的知识。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.