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（由申请人提供）：粘附连接是围绕钙粘蛋白粘附分子构建的强细胞间粘附的特殊位点，其组织肌动蛋白细胞骨架以机械耦合相邻细胞，因此粘附连接可以利用肌动球蛋白产生的力并将其整合到整个互连片上。尽管肌动蛋白很重要，但人们对肌动蛋白如何在粘附体上组装却知之甚少。先前的模型提出了钙粘蛋白和肌动蛋白之间通过连环蛋白连接，这为将肌动蛋白募集到粘附连接提供了一种简单的机制，但是当它被证明时，该模型受到了严重的挑战。 -连环蛋白不能同时与两个 ¿因此，核心钙粘蛋白-连环蛋白复合物的外围因子对于在粘附连接处组织肌动蛋白细胞骨架是必要的，我们开发了一种体外系统来重建粘附连接处的肌动蛋白组装，以识别这些因子。使用该系统来识别-肌动蛋白作为arp2/3介导的连接肌动蛋白组装所必需的重要因子。然而，单独的肌动蛋白和 arp2/3 不能解释粘附连接处的肌动蛋白组织，我们推断连接蛋白与 ¿ -肌动蛋白还参与连接肌动蛋白组装，并使用光活化交联来识别与 ¿ 接近的六个因子我们已经证实，所有这六种蛋白质都定位于粘附连接，所有这些蛋白质都被认为可以调节肌动蛋白细胞骨架，其中两种蛋白质的突变会导致与突变形式相同的遗传性肾病。的 -肌动蛋白（称为 FSGS）表明具有共同的细胞生物学功能，因此我们发现这些新因子对于在粘附连接处组织肌动蛋白非常重要，我们的第一个目标是确定其中的哪一个。 -肌动蛋白在连接处的邻居对于将其募集到粘附连接处是必要的，并使用生化方法来识别这些因子和 ¿在第二个目标中，我们确定这些新发现的因子在细胞粘附连接处组织肌动蛋白中的作用，并使用生化方法确定这些因子对体外肌动蛋白组装的影响。的 -肌动蛋白及其在钙粘蛋白介导的细胞粘附和粘附连接组装和成熟中的邻近连接与人类健康的相关性：钙粘蛋白和粘附连接与许多发育缺陷和疾病以及我们在生化重建中确定的许多因素有关。与遗传性肾病、局灶节段性肾小球硬化症有关，定义粘附连接处肌动蛋白组装的分子机制。不仅揭示了人类发展所必需的核心机制，而且还可能揭示成人某些疾病的病理学。