Mechanisms of Junctional Actin Recruitment in C. elegans

线虫中连接肌动蛋白招募的机制

• 批准号: 7647211
• 负责人: Jeffrey D Hardin
• 金额: $ 33.8万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7647211
• 关键词: Actins; Address; Adherens Junction; Adhesions; Adhesiveness; Affinity; BAIAP1 gene; Binding; Biochemical; Biochemistry; Biological Assay; Biological Models; C-terminal; Cadherins; Caenorhabditis elegans; Cells; Complex; Congenital Abnormality; Cytoskeleton; Data; Defect; Diagnosis; Dominant-Negative Mutation; Embryo; Embryonic Development; Enhancers; Ensure; Epithelial; Event; Genetic; Genetic Epistasis; Genetic Models; Human Development; Image; Invaded; Life; MLLT4 gene; Mediating; Modeling; Morphogenesis; Mutation; Neoplasm Metastasis; Organism; Pathway interactions; Process; Proteins; Recruitment Activity; Role; Structure; System; Testing; Time; Tissue Engineering; Vertebrates; Work; base; cancer cell; cell behavior; genetic analysis; genome wide association study; human disease; in vitro Assay; in vivo; innovation; insight; mutant; public health relevance; research study; tumorigenesis

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.

描述（由申请人提供）：了解什么是在粘附连接处（AJS）调节钙粘蛋白介导的粘附，这对在胚胎发育过程中的理解和治疗缺陷以及诊断和治疗转移性肿瘤具有广泛的影响。该领域的一个关键尚未解决的问题是Catenin如何通过其C末端招募并连接到肌动蛋白细胞骨架。另一个是依赖性依赖性和独立的肌动蛋白募集途径如何相互作用以增强AJ。秀丽隐杆线虫目前是唯一的遗传模型系统，用于在活胚胎中快速功能分析Catenin，并为解决这两个未解决的问题提供了关键优势。我们将使用体外化测定，遗传学和创新成像来解决以下特定目的：AIM 1：HMP-1/Catenin的C末端在将肌动蛋白招募至基于钙粘着蛋白的粘附方面的作用。我们的数据表明，Catenin C末端中绝对保守的区域的突变增加了其对肌动蛋白的亲和力，并且受相邻区域的调节。我们将在体内使用生化测定和高度时间分辨的成像检验这一假设。我们还将采用一种结构功能的方法来识别链氨酸蛋白酶中的其他重要领域，并确定Catenin的C末端是否充当直接接头，可以在体内执行其功能。 AIM 2：Catenin在招募MAGI到基于cadherin的粘附中的作用。我们的基因组宽屏幕，用于弱链蛋白酶突变体HMP-1（FE4）的增强子，确定了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物理将MAGI-1招募到AJS。我们还将通过直接的物理相互作用来测试MAGI-1是否招募RAP-GEF PXF-1/PDZ-GEF为基于钙粘蛋白的交界。这些实验代表了生命胚胎中基于钙粘着蛋白的粘附的魔法的首次分析。 AIM 3：RAPS和AFD-1/AF-6在基于钙粘蛋白的粘附的MAGI-1依赖性成熟中的作用。 RAP和AFD-1/AF-6功能的损失也增强了HMP-1（FE4）。我们将确定MAGI-1是否会导致AJS的说唱激活。我们将使用生物化学，遗传学和RAP活动的创新评估测试该模型。我们还将测试AFD-1/AF-6是否与MAGI-1共同充当说唱效应器。这种分析代表了在活胚胎中对RAP和AF-6的第一个动态分析。这些研究的结果是，我们将阐明在生物体中上皮形态发生过程中，钙粘蛋白/catenin复合物在上皮形态发生过程中如何募集肌动蛋白，并如何获得对人类发育和发育生成期间事件的过程的基本新见解。公共卫生相关性 了解细胞如何彼此坚持对于理解许多常见的先天缺陷以及癌细胞如何彼此之间的联系并侵入身体很重要。该提案检查了一种关键蛋白质，称为Catenin，可调节细胞粘附性，以及该蛋白如何与其他蛋白质一起起作用，以确保细胞在体内建立适当的连接。通过研究该蛋白在生物胚胎中的作用，我们将获得可用于理解和治疗人类疾病的重要信息。