Molecular mechanisms underlying force sensing at intercellular junctions

细胞间连接处力传感的分子机制

基本信息

批准号：
9281753
负责人：
Alexander R Dunn
金额：
$ 36.79万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-01 至 2020-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9281753
关键词：
Actins Address Adherens Junction Adhesions Affinity Apical Behavior Binding Biochemical Biological Biological Assay Biological Models Biophysics Cadherins Cardiomyopathies Cell physiology Cells Collaborations Complex Cues Cytoskeleton Data Defect Desmosomes Development Developmental Biology Disease Disseminated Malignant Neoplasm E-Cadherin Epithelial Focal Adhesions Foundations Goals Homeostasis In Vitro Integrins Intercellular Junctions Intermediate Filaments Link Magnetism Maintenance Malignant Neoplasms Measurement Mechanics Modeling Molecular Molecular Conformation Nature Neoplasm Metastasis Neoplasms Normal tissue morphology Organ Play Positioning Attribute Protein Fragment Proteins Recruitment Activity Rest Role Scaffolding Protein Signal Transduction Signaling Protein Solid Structure System Techniques Testing Tight Junctions Tissues Transducers Vinculin Work afadin alpha catenin base beta catenin biophysical properties desmoplakin experience experimental study in vivo innovation insight mechanical force mechanical load mechanotransduction mutant optical traps plakoglobin plakophilins protein complex protein protein interaction public health relevance reconstitution response sensor single molecule skin barrier skin disorder

项目摘要

DESCRIPTION (provided by applicant): The purpose of this project is to elucidate the molecular mechanisms by which intercellular adhesion complexes form and remodel in response to mechanical load. Recent evidence demonstrates that mechanically initiated signaling at cell-cell junctions is a fundamental aspect of cell and developmental biology. Aberrant assembly and remodeling of intercellular junctions has likewise emerged as a defining feature of diseases including metastatic cancers, cardiomyopathies, and skin barrier defects. However, at present very little is known about how the complex protein assemblies present at cell-cell contacts convert molecule-scale forces into biochemical signals, or how mechanical cues govern the complex junctional dynamics that typify multicellular tissues. Previous work from our collaboration showed that a complex of E-cadherin, β-catenin, and αE-catenin forms a minimal force-sensing unit at adherens junctions (AJs). Separate work suggests that αE-catenin additionally plays a central role in organizing epithelial tissues based on its interactions with vinculin, Epithelial Protein Lost in Neoplasm (EPLIN), Zonula Occludens (ZO)-1, and afadin, all of which bind actin and recruit other scaffolding and signaling proteins. In Aim 1 we will test the hypothesis that force-sensitive, cooperative actin binding by αE-catenin and vinculin leads to dramatic increases in actin affinity over a very small range in force. This idea, if correct, would explain how a four-protein system amplifies small changes in force into dramatic alterations in adhesion stability and downstream signal transduction. Further, we will perform the first detailed biochemical and biophysical characterization of the interaction of the cadherin-catenin complex with EPLIN, ZO-1, and afadin. These studies lay the foundation for a quantitative understanding for how the AJ functions as an integrated, multifunctional force-sensing assembly. In Aim 2 we will examine force sensitivity in desmosomes. These junctions link desmosomal cadherins to the intermediate filament (IF) cytoskeleton, and are essential for tissue integrity. However, while cel biological data suggest a role of desmosomes in transmitting force between cells, there is currently no direct evidence for when, where, and even whether desmosomal cadherins transmit tension between neighboring cells in the absence of externally applied force. To address this gap, we will use genetically encoded molecular tension sensors to determine when and where desmosomal cadherins transduce force between neighboring cells. We will then critically evaluate the role of desmoplakin in transmitting force at desmosomes, analogous to the role established for αE-catenin at AJs. Finally, we will use a single-molecule magnetic tweezers assay to test the innovative hypothesis that recruitment of plakoglobin, plakophilin, or both to desmoplakin is inherently force sensitive. These experiments will dramatically enhance our basic understanding of how desmosomes function as a mechanical linkage between cells.

描述（由适用提供）：该项目的目的是阐明细胞间粘合剂复合物响应机械负载而形成和重塑的分子机制。最近的证据表明，在细胞细胞连接处机械启动的信号传导是细胞和发育生物学的基本方面。异常的组装和重塑细胞间连接处同样成为疾病的定义特征，包括转移性癌症，心肌病和皮肤屏障缺陷。然而，目前，关于在细胞 - 细胞接触中存在的复杂蛋白质组件如何将分子尺度力转化为生化信号，或者机械提示如何控制代表多细胞组织的复杂连接动力学的情况很少知道。我们合作的先前工作表明，E-钙粘着蛋白，β-catenin和αE-catenin的复合物在粘附连接处形成最小的力传感单元（AJS）。单独的工作表明，αe-catenin在组织上皮组织中的核心作用，其与杂种蛋白的相互作用，在肿瘤（Eplin）（eplin），Zonula occludens（ZO）-1和Afadin中丢失的上皮蛋白和Afadin，所有这些都结合了肌动蛋白和肌动蛋白和招募其他信号蛋白质。在AIM 1中，我们将测试假设通过αe-catenin和vinculin的力敏感，合作肌动蛋白结合会导致肌动蛋白亲和力在极小范围内急剧增加。如果正确的话，这个想法将解释四蛋白系统放大器如何将力的小变化变为粘合剂稳定性和下游信号转导的急剧变化。此外，我们将对钙粘蛋白 - 钙蛋白复合物与Eplin，ZO-1和Afadin的相互作用进行第一个详细的生化和生物物理表征。这些研究为AJ如何充当综合的多功能力传感组件的定量理解奠定了基础。在AIM 2中，我们将这些连接连接到中间细丝（IF）细胞骨架，对组织完整性至关重要。但是，尽管CEL生物学数据表明脱染色体在细胞之间传输力中的作用，但目前尚无直接证据表明何时，何时何地，甚至在没有外部施加力的情况下是否在邻近细胞之间发出张力。为了解决这一差距，我们将使用遗传编码的分子张力传感器来确定邻近细胞之间的脱骨钙蛋白何时何地转导力。然后，我们将批判性地评估脱莫普拉金在脱糖体中发射力中的作用，类似于AJSαe-catenin的作用。最后，我们将使用单分子磁性镊子测定法来检验创新的假设，即plakoglobin，plakophilin或两者都募集到脱莫普拉金蛋白固有地敏感。这些实验将极大地增强我们对脱糖体如何充当细胞之间的机械连接的基本理解。