Mechanisms of Junctional Actin Recruitment in C. elegans

描述（由申请人提供）：了解是什么调节钙粘蛋白介导的粘附连接（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性肿瘤具有广泛的意义。该领域尚未解决的一个关键问题是连环蛋白如何通过其C端招募并连接到肌动蛋白细胞骨架。另一个是连环蛋白依赖和独立的肌动蛋白募集途径如何相互作用以加强AJs。秀丽隐杆线虫是目前唯一能够在活胚胎中快速分析连环蛋白功能的遗传模型系统，为解决这两个尚未解决的问题提供了关键优势。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端在钙粘蛋白粘连中招募肌动蛋白的作用。我们的数据表明，在catenin的C端绝对保守区域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节。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设。我们还将采用结构-功能的方法来鉴定catenin的其他重要结构域，并确定catenin的C端作为直接连接体是否可以在体内发挥其功能。目的2：连环蛋白在钙粘蛋白粘连中招募MAGI的作用。我们在全基因组筛选弱连环蛋白突变体hmp-1（fe4）的增强子，鉴定出MAGUK， MAGI-1。我们将使用生物化学、遗传学和MAGI-1构建体的体内成像来测试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-GEF， PXF-1/PDZ-GEF招募到基于钙粘蛋白的连接中。这些实验首次对活胚胎中钙粘蛋白黏附的MAGI进行了分析。目的3:Raps和AFD-1/AF-6在magi -1依赖性钙粘蛋白粘连成熟中的作用。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是否会导致AJs的Rap激活。我们将使用生物化学、遗传学和创新的Rap活性体内评估来测试该模型。我们还将测试AFD-1/AF-6是否与MAGI-1一起作为Rap效应器。这是首次在活胚胎中对Raps和AF-6进行动态分析。作为这些研究的结果，我们将阐明钙粘蛋白/连环蛋白复合物是如何在活体组织上皮形态发生过程中招募肌动蛋白进行新生细胞-细胞接触的，并且我们将对人类发育和肿瘤发生过程的基本事件获得新的见解。公共卫生相关性

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.