Mechanisms of Junctional Actin Recruitment in C. elegans

描述(申请人提供)：了解是什么调节钙粘附素介导的黏附连接(AJs)对于理解和治疗胚胎发育过程中的缺陷以及诊断和治疗转移肿瘤具有广泛的意义。该领域的一个关键悬而未决的问题是连环蛋白如何招募并通过其C端连接到肌动蛋白细胞骨架。另一个是连环蛋白依赖和非依赖肌动蛋白招募通路如何相互作用来增强AJ。线虫是目前唯一能快速分析活体胚胎中连环蛋白功能的遗传模型系统，为解决这两个悬而未决的问题提供了关键的优势。我们将使用体外分析、遗传学和创新的体内成像来解决以下具体目标：目标1：HMP-1/catenin的C末端在招募肌动蛋白到钙粘附素基础上的作用。我们的数据表明，连接素C末端绝对保守区的突变增加了它对肌动蛋白的亲和力，并且它受到邻近区域的调控。我们将使用生化分析和高时间分辨率的体内成像来验证这一假说。我们还将采用结构-功能的方法来鉴定连环蛋白中的其他重要结构域，并确定连环蛋白的C末端作为直接连接物是否可以在体内发挥其功能。目的2：连环蛋白在钙粘附素粘连的MAGI募集中的作用。我们对弱连环蛋白突变体HMP-1(FE4)的全基因组增强子筛选确定了MAUK，MAGI-1。我们将使用生物化学、遗传学和MAGI-1结构的体内成像来测试HMP-2/Catenin是否在物理上将MAGI-1招募到AJs。我们还将测试MAGI-1是否通过直接物理相互作用将Rap-Global，PXF-1/PDZ-Global招募到基于钙粘素的连接。这些实验是首次对活胚胎中基于钙粘附素的MAGI进行分析。目的3：RAPs和AFD-1/AF-6在依赖MAGI-1的钙粘连成熟中的作用。Rap和AFD-1/AF-6功能的丧失也会增强HMP-1(FE4)。我们将确定MAGI-1是否导致AJ的RAP激活。我们将使用生物化学、遗传学和Rap活性的体内创新评估来测试这个模型。我们还将测试AFD-1/AF-6是否与MAGI-1一起充当Rap效应器。这种分析是第一次在活胚胎中对RAPS和AF-6进行动态分析。作为这些研究的结果，我们将阐明钙粘附素/连环蛋白复合体如何在活体的上皮形态发生过程中将肌动蛋白招募到新生的细胞-细胞接触中，并将对人类发育和肿瘤发生过程中的一个基本过程有全新的见解。公共卫生相关性 了解细胞是如何相互粘连的，对于了解许多常见的出生缺陷，以及癌细胞如何失去彼此的联系并入侵人体非常重要。这项建议研究了一种名为连环蛋白的关键蛋白质，它调节细胞的粘附性，以及这种蛋白质如何与其他蛋白质一起工作，以确保细胞在体内建立适当的连接。通过研究这种蛋白质如何在活胚胎中发挥作用，我们将获得可用于理解和治疗人类疾病的重要信息。

DESCRIPTION (provided by applicant): Understanding what modulates cadherin-mediated adhesion at adherens junctions (AJs) has widespread implications for understanding and treating defects during embryonic development, and for diagnosing and treating metastatic tumors. One key unresolved issue in the field is how catenin recruits and connects to the actin cytoskeleton via its C terminus. Another is how catenin- dependent and -independent actin recruitment pathways interact to strengthen AJs. C. elegans is currently the only genetic model system for rapid functional analysis of catenin in a living embryo, and provides key advantages for addressing these two unresolved issues. We will use in vitro assays, genetics, and innovative in vivo imaging to address the following specific aims: Aim 1: Role of the C terminus of HMP-1/catenin in recruiting actin to cadherin-based adhesions. Our data suggest that a mutation in an absolutely conserved region in the C terminus of catenin increases its affinity for actin, and that it is regulated by adjacent regions. We will test this hypothesis using biochemical assays and highly time-resolved imaging in vivo. We will also take a structure-function approach to identify other important domains in catenin, and to determine whether the C terminus of catenin, acting as a direct linker, can carry out its functions in vivo. Aim 2: Role of catenin in recruitment of MAGI to cadherin-based adhesions. Our genome- wide screen for enhancers of a weak catenin mutant, hmp-1(fe4), identified the MAGUK, MAGI-1. We will test whether HMP-2/catenin physically recruits MAGI-1 to AJs, using biochemistry, genetics, and in vivo imaging of MAGI-1 constructs. We will also test whether MAGI-1 recruits the Rap-GEF, PXF-1/PDZ-GEF, to cadherin-based junctions via direct physical interaction. These experiments represent the first analysis of a MAGI in cadherin-based adhesion in a living embryo. Aim 3: Role of Raps and AFD-1/AF-6 in MAGI-1-dependent maturation of cadherin-based adhesions. Loss of Rap and AFD-1/AF-6 function also enhances hmp-1(fe4). We will determine whether MAGI-1 leads to Rap activation at AJs. We will test this model using biochemistry, genetics, and innovative in vivo assessment of Rap activity. We will also test whether AFD-1/AF-6 acts as a Rap effector in concert with MAGI-1. Such analysis represents the first dynamic analysis of Raps and AF-6 in a living embryo. As a result of these studies, we will clarify how the cadherin/catenin complex recruits actin to nascent cell-cell contacts during epithelial morphogenesis in a living organism, and we will gain fundamental new insights into a process fundamental for events during human development and oncogenesis. PUBLIC HEALTH RELEVANCE Understanding how cells stick to one another is important for understanding many common birth defects, and how cancer cells lose their connections to one another and invade the body. This proposal examines a key protein, called catenin that regulates cell adhesiveness, and how this protein works together with other proteins to ensure that cells make proper connections in the body. By studying how this protein works in living embryos, we will gain important information that can be used to understand and treat human disease.