Actin Assembly at Cadherin Dependent Adherens Junctions

钙粘蛋白依赖性粘附连接处的肌动蛋白组装

基本信息

批准号：
10410514
负责人：
William Brieher
金额：
$ 28.77万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10410514
关键词：
1-Phosphatidylinositol 3-Kinase Actins Actomyosin Adherens Junction Adhesions Adhesives Apical Back Binding Proteins Biochemical Biological Assay Bulla Cadherins Cell Adhesion Molecules Cell-Cell Adhesion Cells Clathrin Complex Consensus Coupled Cytoskeleton Data Defect Drops E-Cadherin Endocytosis Environment Epithelial Epithelial Cells Funding Human In Vitro Inherited Intercellular Junctions Kidney Diseases Knock-out Lateral Lead MDCK cell Maintenance Mediating Membrane Methods Microfilaments Modeling Mutate Mutation Myosin ATPase Myosin Type II Pharmaceutical Preparations Polymers Population Proteins Research Rupture Scaffolding Protein Signal Pathway Spontaneous Rupture System Testing Textbooks Tissues Tube Vesicle cohesion deletion analysis experimental study fluorescence imaging interest knock-down live cell imaging monolayer novel polymerization predictive modeling prevent recruit response

项目摘要

Project Summary. We are interested in understanding how the actin cytoskeleton maintains cell-cell adhesion and epithelial barrier function. Epithelial tissue is characterized by extensive cell-cell adhesive contacts that organize the cells into cohesive sheets or tubes that separate two different environments. E-cadherin is a cell-cell adhesion molecule necessary for the formation and maintenance of many different epithelial sheets. Cadherin mediated cell-cell adhesion is actin dependent, but we do not fully understand how actin contributes to cadherin adhesive function. The prevailing model is that actomyosin dependent tensile forces stabilize cadherin dependent cell-cell junctions, but this system by itself is not robust because small defects in adhesion would tend to propagate and expand if myosin continued to pull on a ruptured junction. Cells must have a back-up plan to maintain cell-cell adhesion and the epithelial barrier whenever adhesive junctions break. We have identified CD2AP, EVL, and CRMP1 as three factors necessary for assembling the actin cytoskeleton at cell-cell junctions. Depleting any of these factors results in a precipitous drop in the amount of junctional actin and a loss of cell-cell adhesion such that the epithelial sheet becomes perforated with holes. CD2AP, EVL, and CRMP1 all modulate actin filament (+) end polymerization in vitro, and they are all necessary for protrusive activity in migrating cells. Therefore, I hypothesize that the three factors direct actin polymerization towards junctions to prevent them from spontaneously rupturing and to quickly reseal them if the junctions should break. Consistent with our hypothesis, we have identified novel populations of actin dependent protruding microspikes and small lamellipodia that form continually at cell junctions in mature epithelial sheets that are no longer moving. Therefore, actin polymerization continues to drive protrusive activity at cell-cell borders long after the cells made contact, stopped moving, and built cell-cell adhesive junctions. The specific aims are to 1) identify the factors necessary for generating the protrusions at cell-cell junctions and identify the underlying mechanisms, 2) test whether loss of cell-cell adhesion triggers actin assembly, and 3) determine why loss of actin polymerization at cell-cell junctions leads to disruption of E- cadherin organization and adhesive function. Results from this proposal could fundamentally alter our current view of how actin helps maintain cell-cell adhesion while providing new information as to why mutations in CD2AP lead to inherited kidney disease.

项目摘要。我们有兴趣了解肌动蛋白细胞骨架如何保持细胞 - 细胞粘附和上皮屏障功能。上皮组织的特征是广泛的细胞 - 细胞粘合剂将细胞组织成粘板或管的触点，分离两个不同环境。电子钙粘蛋白是形成所需的细胞细胞粘附分子维护许多不同的上皮床单。钙粘蛋白介导的细胞 - 细胞粘附是肌动蛋白依赖性，但我们不完全了解肌动蛋白如何对钙粘蛋白的粘附功能贡献。盛行的模型是肌球蛋白依赖性拉伸力稳定钙粘蛋白依赖性细胞电池连接，但是该系统本身并不强大，因为粘附的小缺陷如果肌球蛋白继续拉出破裂的交界处，将倾向于繁殖和扩展。细胞必须有一个备用计划，以维持细胞细胞粘附和上皮屏障粘合连接处断裂。我们已经将CD2AP，EVL和CRMP1确定为三个因素在细胞 - 细胞连接处组装肌动蛋白细胞骨架所必需的。耗尽其中的任何一个因素导致连接肌动蛋白的量急剧下降和细胞细胞损失粘附使上皮片被孔穿孔。 CD2AP，EVL和 CRMP1所有调节肌动蛋白丝（+）末端聚合在体外，都是必要的迁移细胞中的突出活性。因此，我假设这三个因素直接肌动蛋白聚合到连接处的聚合，以防止它们自发破裂并快速地破裂如果连接应断开，请重新密封它们。与我们的假设一致，我们已经确定肌动蛋白依赖性突出的微刺和小薄片的新型种群形成不再移动的成熟上皮床单的细胞连接处。所以，肌动蛋白聚合在细胞后很长一段时间内继续在细胞细胞边界驱动伸出活性进行了接触，停止移动并建立了细胞 - 细胞粘合剂连接。具体目的是 1）确定在细胞 - 细胞连接处产生突起所必需的因素并确定基本机制，2）测试细胞细胞粘附触发肌动蛋白的损失， 3）确定为什么在细胞 - 细胞连接处肌动蛋白聚合的丧失会导致E-破坏钙粘蛋白的组织和粘合剂功能。该提议的结果从根本上可以改变我们当前对肌动蛋白如何帮助维持细胞细胞粘附的看法有关为什么CD2AP突变导致遗传肾脏疾病的信息。