Defining the molecular mechanisms underlying apical-basal polarity establishment and morphogenesis

摘要 我们身体和其他动物体内的大部分组织都是上皮细胞。适当的组织结构和完整性 需要建立和维持根尖-基底极性，细胞-细胞和细胞-基质黏附，以及连接 附着在肌动蛋白细胞骨架上。这些也调节了力的产生，允许细胞改变形状和 移动。大多数癌症是上皮起源的，在许多不同的癌症中，突变或改变的表达 极性和连接蛋白导致细胞极性的改变，促进细胞迁移和细胞侵袭。 钙粘附素结合的细胞-细胞黏附连接与肌动蛋白细胞骨架调控动态细胞 发育中的行为和癌症中的行为。在两者中整合组织粘附性和极性的关键角色 哺乳动物和果蝇是非洲独木舟(CNO)。Cno是一种在果蝇中起关键作用的多结构域蛋白 在从心尖-基底极性建立到维持AJ-细胞骨架连接的过程中的作用 在中胚层内陷期间，生殖带延伸，以及在背部闭合期间集体细胞迁移。在这里，我 解决该领域的两个悬而未决的问题：CNO通过什么机制控制组织 架构和动态细胞行为，以及上游输入如何调节CNO的功能？我通过两个途径做到这一点 目标：1)确定CNO将AJ与肌动蛋白细胞骨架联系起来的潜在机制 形态发生，以及2)定义RAP1活性调节因子Dizzy如何协调CNO的定位和功能 在胚胎发育过程中，并确定活性RAP1的定位。我在目标1中的假设是 CNO的PDZ和F-肌动蛋白结合(Fab)结构域在CNO的定位和功能的多个阶段起着关键作用 但并不是所有角色都是必需的。使用CRISPR/Cas9，我设计了CNO的轨迹，重新引入了一系列 CNO的PDZ和FAB结构域的突变体，以确定它们如何影响CNO的定位和功能 在整个发育过程中，从而提供了对Afadin在哺乳动物中的作用的见解。同时，我将表演 体内外CNO突变蛋白的蛋白质相互作用分析。目标2是建立在假设的基础上的 RAP1的活性池调节CNO活性，而Dizzy是主要的RAP1环境基金调节因子 CNO在胚胎发育过程中的定位和功能。为了测试这一点，我将探索Rap1，一个上游 CNO的调节者，协调CNO的定位和功能。我会比较CNO基因敲除的表型 通过Rap1，以及Dizzy和RapGAP1基因敲除，已知的Rap1活性调节因子。同时，我会 开发工具，揭示Active Rap1的本地化位置。这项研究将定义RAP1活性调节器，RAP1， 和CNO致力于调节动态的细胞行为，并将提供关键信息来理解它们的作用 在疾病中。通过这次培训，我将获得分子生物学、生物化学、遗传学和 显微镜，学习蛋白质结构与结构域功能之间的桥梁，并发展批判性陈述和写作 技能。这些都将推动我成为一名教授和首席研究员的长期职业目标。

Abstract Most tissues in our bodies and those of other animals are epithelia. Proper tissue architecture and integrity require apical-basal polarity establishment and maintenance, cell-cell and cell-matrix adhesion, and linkage of adhesions to the actin cytoskeleton. These also mediate force generation, allowing cells to change shape and move. Most cancers are of epithelial origin, and in many different cancers mutation or altered expression of polarity and junctional proteins leads to changes in cell polarity, promoting cell migration and cell invasion. Linkage of cadherin-based cell-cell adherens junction (AJs) with the actin cytoskeleton regulate dynamic cell behaviors during development and in cancer. A critical player integrating tissue adhesion and polarity in both mammals and Drosophila is Afadin/Canoe (Cno). Cno is a multidomain protein that in Drosophila plays key roles in processes ranging from apical-basal polarity establishment to maintaining AJ-cytoskeleton linkage during mesoderm invagination, germ-band extension, and collective cell migration during dorsal closure. Here I address two outstanding questions in the field: by what mechanisms does Cno work to control tissue architecture and dynamic cell behaviors, and how do upstream inputs regulate Cno's function? I do so via two Aims: 1) Define mechanisms underlying Cno's ability to link the AJs with the actin cytoskeleton during morphogenesis, and 2) Define how Dizzy, a Rap1 activity regulator, coordinates Cno localization and function during embryonic development, and determine where active Rap1 localizes. My hypothesis in Aim 1 is that Cno's PDZ and F-actin binding (FAB) domains play key roles in Cno's localization and function at many stages but may not be essential for all roles. Using CRISPR/Cas9, I engineered cno's locus to reintroduce a series of mutants of Cno's PDZ and FAB domains, to define how they contribute to Cno localization and function throughout development, thus providing insights into Afadin's roles in mammals. In parallel I will perform protein-protein interaction analysis of Cno mutant proteins in vitro and in vivo. Aim 2 is built on the hypothesis that an active pool of Rap1 regulates Cno activity, and that Dizzy is the predominant Rap1 GEF regulating Cno's localization and thus function during embryogenesis. To test this, I will explore how Rap1, an upstream regulator of Cno, coordinates Cno localization and function. I will compare the phenotype of Cno knockdown with that of Rap1, and with Dizzy and RapGAP1 knockdown, known regulators of Rap1 activity. In parallel I will develop tools revealing where active Rap1 localizes. This study will define how Rap1 activity regulators, Rap1, and Cno work to regulate dynamic cell behaviors and will provide critical information to understand their roles in disease. Through this training, I will gain cutting edge skills in molecular biology, biochemistry, genetics and microscopy, learn to bridge protein structure with domain function, and develop critical presentation and writing skills. These will all further my long-term career goal of becoming a professor and a principal investigator.