Actin Assembly at cadherin dependent adherens junctions

肌动蛋白在钙粘蛋白依赖性粘附连接处的组装

• 批准号: 8483248
• 负责人: William Brieher
• 金额: $ 28.47万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8483248
• 关键词: Accounting; Actin-Binding Protein; Actinin; Actins; Actomyosin; Adherens Junction; Adhesions; Adult; Architecture; Binding; Biochemical; Biological Process; Bundling; Cadherins; Cell Adhesion; Cell Adhesion Molecules; Cell membrane; Cell-Cell Adhesion; Cells; Complex; Congenital Abnormality; Cytoskeletal Modeling; Cytoskeleton; Data; Defect; Development; Disease; Embryo; Embryonic Development; F-Actin; Focal Segmental Glomerulosclerosis; Health; Human Development; In Vitro; Inherited; Intercellular Junctions; Kidney Diseases; Kinetics; Life; Light; Link; Mediating; Methods; Modeling; Molecular; Movement; Mutation; Neoplasm Metastasis; Organ; Pathology; Peripheral; Play; Process; Proteins; Recruitment Activity; Resolution; Role; Site; Structural Models; Structure; System; Techniques; Time; Tissues; Work; cell motility; cellular imaging; crosslink; mutant; novel; protein protein interaction; public health relevance; receptor; reconstitution; research study

DESCRIPTION (provided by applicant): Adherens junctions are specialized sites of strong cell-cell adhesion built around cadherin adhesion molecules that organize an actin cytoskeleton to mechanically couple adjacent cells. Adherens junctions can therefore harness actomyosin generated forces and integrate them across entire sheets of interconnected cells to drive the morphogenetic movements that fashion tissues, organs, and entire early embryos. Despite its importance, little is known as to how actin is assembled at adherens junctions. A previous model proposed a linkage between cadherins and actin via the catenins, which would have provided a simple mechanism for recruiting actin to adherens junctions. This model was seriously challenged however when it was demonstrated that ¿-catenin couldn't bind simultaneously to both ¿-catenin and F-actin. Therefore, additional factors peripheral to the core cadherin-catenin complex are necessary to organize the actin cytoskeleton at adherens junctions. We developed an in vitro system to reconstitute actin assembly at adherens junctions to identify such factors and have already used this system to identify ¿-actinin as an essential factor necessary for arp2/3 mediated junctional actin assembly. ¿-Actinin and arp2/3 alone however cannot account for actin organization at adherens junctions. We reasoned that junctional proteins in close proximity to ¿-actinin would also be involved in junctional actin assembly and used photoactivatable crosslinking to identify six factors in close proximity to ¿-actinin in cadherin enriched plasma membrane sheets. We have confirmed that all six of these proteins localize to adherens junctions, all of them are thought to regulate the actin cytoskeleton, and mutations in two of them result in the same inherited kidney disease as mutant forms of ¿-actinin known as FSGS suggesting a common cell biological function. We therefore hypothesize that these new factors are important for organizing actin at adherens junctions. In the first aim we determine which of ¿-actinin's neighbors at the junction are necessary for recruiting it to adherens junctions and use biochemical methods to identify direct binding interactions between these factors and ¿-actinin. In the second aim, we determine the roles of these newly identified factors in organizing actin at adherens junctions in cells and use biochemical methods to determine the effects of these factors on actin assembly in vitro. In the third aim we determine the role of ¿-actinin and its junctional neighbors in cadherin mediated cell adhesion and adherens junction assembly and maturation. RELEVANCE TO HUMAN HEALTH: Cadherins and adherens junctions are implicated in numerous developmental defects and diseases, and a number of the factors that we have identified in our biochemical reconstitution are implicated in the inherited kidney disease, Focal Segmental Glomerulosclereosis. Defining the molecular mechanisms underlying actin assembly at adherens junctions will not only reveal core mechanisms necessary for human development but might also shed light on the pathology of certain diseases in adults.

描述（由适用提供）：粘附连接是围绕钙粘着蛋白粘附分子建立的强细胞粘附的专门部位，该分子组织了肌动蛋白细胞骨架与机械耦合的相邻细胞。因此，粘附连接可以利用肌动蛋白产生力并将其整合在整个相互连接的细胞中，以驱动时尚组织，器官和整个早期胚胎的形态发生运动。尽管它很重要，但对于在粘附连接处如何组装肌动蛋白的鲜为人知。先前的模型提出了通过蛋白茶碱与钙粘蛋白和肌动蛋白之间的联系，这将为粘附连接提供一种简单的机制。但是，当证明` - 蛋白蛋白都可以与 - 蛋白酶和F-肌动蛋白都结合时，该模型受到了严重的挑战。因此，对于在粘附连接处组织肌动蛋白细胞骨架是必要的，需要其他因素的外围因素。我们开发了一种体外系统，以重新建立粘附连接处的肌动蛋白组件来识别此类因素，并已经使用该系统识别 - 肌动蛋白是ARP2/3介导的连接肌动蛋白组装所必需的重要因素。 � -Actinin和ARP2/3仅在粘附连接处无法说明肌动蛋白组织。我们认为，与 - 肌动蛋白接近 - 肌动蛋白的连接蛋白也将参与连接肌动蛋白组件，并使用可光活化的交联，以识别富含钙粘着蛋白的质量质量质膜的六个因素。我们已经证实，所有这些蛋白质中的所有六个都定位在粘附连接处，所有这些蛋白都被认为调节肌动蛋白细胞骨架，其中两个突变导致与称为FSG的突变形式的遗传性肾脏疾病相同的肾脏疾病，表明称为FSGS，表明一种常见的细胞生物学功能。因此，我们假设这些新因素对于在粘附连接处组织肌动蛋白很重要。在第一个目的中，我们确定连接处的哪种 - 肌动蛋白的邻居对于将其募集到粘附连接处，并使用生化方法来识别这些因素与�-肌动蛋白之间的直接结合相互作用。在第二个目标中，我们确定了这些新确定的因素在细胞中粘附连接蛋白组织肌动蛋白组织肌动蛋白中的作用，并使用生化方法来确定这些因素在体外对肌动蛋白组装的影响。在第三个目的中，我们确定了 - 肌动蛋白及其交叉邻居在钙粘蛋白介导的细胞粘附和粘附结的结合和成熟中的作用。与人类健康的相关性：在许多发育缺陷和疾病中实施了钙粘蛋白和粘附连接，并且我们在生物化学重构中确定的许多因素在遗传性的肾脏疾病，局灶性节段性段落肾小球乳细胞血球症中实施。定义粘附连接处肌动蛋白组件的分子机制不仅会揭示人类发育所需的核心机制，而且还可能阐明成人某些疾病的病理。