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：
10165738
负责人：
William Brieher
金额：
$ 28.86万
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 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10165738
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维持细胞间粘附和上皮屏障功能。上皮组织的特点是广泛的细胞间粘附将细胞组织成粘性片或管的接触，将两个不同的细胞分开环境。 E-钙粘蛋白是细胞间粘附分子的形成和形成所必需的维持许多不同的上皮层。钙粘蛋白介导的细胞间粘附是肌动蛋白依赖，但我们不完全了解肌动蛋白如何促进钙粘蛋白粘附功能。普遍的模型是肌动球蛋白依赖性张力稳定钙粘蛋白依赖性细胞-细胞连接，但该系统本身并不稳健，因为粘附存在小缺陷如果肌球蛋白继续拉扯破裂的连接处，就会倾向于传播和扩张。细胞必须有一个后备计划来维持细胞间粘附和上皮屏障粘合接头断裂。我们将 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。耗尽其中任何一个因素导致连接肌动蛋白数量急剧下降和细胞间损失粘附使得上皮片变得有孔。 CD2AP、EVL 和 CRMP1 均在体外调节肌动蛋白丝 (+) 末端聚合，并且它们都是肌动蛋白丝 (+) 末端聚合所必需的迁移细胞的突出活动。因此，我假设这三个因素直接影响肌动蛋白向连接处聚合，以防止它们自发破裂并快速如果连接处破裂，请重新密封它们。与我们的假设一致，我们已经确定形成肌动蛋白依赖的突出微刺和小片状伪足的新群体不断地在不再移动的成熟上皮片的细胞连接处。所以，在细胞形成后很久，肌动蛋白聚合继续驱动细胞-细胞边界的突出活动接触，停止移动，并建立细胞与细胞的粘合连接。具体目标是 1) 确定在细胞-细胞连接处产生突起所需的因素并确定潜在机制，2) 测试细胞与细胞粘附的丧失是否会触发肌动蛋白组装， 3) 确定为什么细胞-细胞连接处肌动蛋白聚合的丧失会导致 E- 的破坏钙粘蛋白组织和粘附功能。该提案的结果可以从根本上改变我们目前对肌动蛋白如何帮助维持细胞间粘附同时提供新的观点关于为什么 CD2AP 突变会导致遗传性肾病的信息。