Mechanisms of Junctional Actin Recruitment in C. elegans

描述（由申请人提供）：了解是什么调节了粘附连接处（AJs）的钙粘蛋白介导的粘附，对于了解和治疗胚胎发育期间的缺陷以及诊断和治疗转移性肿瘤具有广泛的意义。该领域一个尚未解决的关键问题是连环蛋白如何通过其C末端招募并连接到肌动蛋白细胞骨架。另一个是如何连环蛋白依赖性和非依赖性肌动蛋白募集途径相互作用，以加强AJs。C. elegans是目前唯一的用于活胚胎中连环蛋白的快速功能分析的遗传模型系统，并且为解决这两个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，以解决以下具体目标：目的1：在招募肌动蛋白钙粘蛋白为基础的粘附HMP-1/catenin的C末端的作用。我们的数据表明，在一个绝对保守的区域中的C端的连环蛋白的突变增加其对肌动蛋白的亲和力，它是由相邻区域的调节。我们将使用生物化学测定和体内高时间分辨成像来测试这一假设。我们还将采取结构-功能的方法来确定其他重要的结构域的连环蛋白，并确定是否C末端的连环蛋白，作为一个直接的连接器，可以执行其功能在体内。目的2：连环蛋白在MAGI募集至钙粘蛋白粘连中的作用。我们对弱连环蛋白突变体hmp-1（fe 4）的增强子进行了全基因组筛选，鉴定出MAGUK，MAGI-1.我们将测试是否HMP-2/连环蛋白物理招募MAGI-1到AJs，使用生物化学，遗传学和体内成像的MAGI-1结构。我们还将测试MAGI-1是否通过直接的物理相互作用将Rap-GEF，PXF-1/PDZ-GEF招募到基于钙粘蛋白的连接处。这些实验代表了在活胚胎中基于钙粘蛋白的粘附中的MAGI的第一次分析。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（fe 4）。我们将确定MAGI-1是否导致AJs的Rap激活。我们将使用生物化学，遗传学和Rap活性的创新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。这种分析代表了Raps和AF-6在活胚胎中的第一次动态分析。作为这些研究的结果，我们将阐明钙粘蛋白/连环蛋白复合物如何招募肌动蛋白在活生物体上皮形态发生过程中新生细胞-细胞接触，我们将获得基本的新见解，在人类发育和肿瘤发生过程中的事件的基础。公共卫生相关性 了解细胞如何相互粘附对于了解许多常见的出生缺陷以及癌细胞如何失去彼此之间的连接并侵入身体非常重要。该提案研究了一种名为连环蛋白的关键蛋白质，它调节细胞增殖，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正确的连接。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.