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（由申请人提供）：该项目的目的是阐明细胞间粘附复合物响应机械负荷而形成和重塑的分子机制，最近的证据表明，细胞-细胞连接处机械启动的信号传导是细胞和细胞的基本方面。细胞间连接的异常组装和重塑同样已成为转移性癌症、心肌病和皮肤屏障缺陷等疾病的一个决定性特征。细胞与细胞接触处存在的复杂蛋白质组装体将分子尺度的力转化为生化信号，或者机械信号如何控制典型的多细胞组织的复杂连接动力学。 αE-连环蛋白在粘附连接（AJ）处形成一个最小的力传感单元，另外的研究表明，αE-连环蛋白基于其与上皮组织的相互作用，在组织上皮组织中也发挥着核心作用。纽蛋白、肿瘤中丢失的上皮蛋白 (EPLIN)、闭塞带 (ZO)-1 和 afadin，所有这些都与肌动蛋白结合并招募其他支架和信号蛋白。在目标 1 中，我们将测试假设αE-连环蛋白和纽蛋白对力敏感、协同的肌动蛋白结合会导致肌动蛋白亲和力在很小的力范围内急剧增加，如果这个想法正确的话，将会。解释四蛋白系统如何将力的微小变化放大为粘附稳定性和下游信号转导的巨大变化此外，我们将对钙粘蛋白-连环蛋白复合物与 EPLIN、ZO-1 的相互作用进行首次详细的生化和生物物理表征。这些研究为定量理解 AJ 作为集成的多功能力传感组件的功能奠定了基础。在目标 2 中，我们将研究桥粒中的力敏感性。连接将桥粒钙粘蛋白与中间丝 (IF) 细胞骨架连接起来，对于组织完整性至关重要。然而，虽然细胞生物学数据表明桥粒在细胞之间传递力方面发挥着作用，但目前还没有直接证据表明桥粒钙粘蛋白在何时、何地甚至细胞间传递。在没有外部施加力的情况下，桥粒钙粘蛋白是否在相邻细胞之间传递张力为了解决这一差距，我们将使用基因编码的分子传感器张力来确定桥粒钙粘蛋白何时何地传递。然后，我们将批判性地评估桥粒斑蛋白在桥粒传递力中的作用，类似于 αE-连环蛋白在 AJ 上的作用。最后，我们将使用单分子磁性镊子测定来测试创新假设。这些实验将极大地增强我们对桥粒如何作为细胞之间的机械连接发挥作用的基本理解。