Molecular mechanisms underlying force sensing at intercellular junctions

 描述（由申请人提供）：本项目的目的是阐明细胞间粘附复合物形成和重塑的分子机制，以响应机械负荷。最近的证据表明，在细胞-细胞连接处的机械启动信号是细胞和发育生物学的一个基本方面。细胞间连接的异常组装和重塑同样已经成为包括转移性癌症、心肌病和皮肤屏障缺陷在内的疾病的定义特征。然而，目前很少有人知道复杂的蛋白质组装存在于细胞-细胞接触如何将分子尺度的力量转化为生化信号，或机械线索如何管理复杂的连接动力学，典型的多细胞组织。 我们合作的先前工作表明，E-cadherin，β-catenin和αE-catenin的复合物在粘附连接（AJs）处形成最小的力感应单元。单独的工作表明，α E-连环蛋白还在组织上皮组织中发挥核心作用，这是基于其与黏着斑蛋白、肿瘤中丢失的上皮蛋白（EPLIN）、闭锁小带（ZO）-1和afadin的相互作用，所有这些蛋白都结合肌动蛋白并招募其他支架和信号蛋白。在目标1中，我们将测试 假设α E-连环蛋白和黏着斑蛋白对肌动蛋白的力敏性、协同性结合导致肌动蛋白亲和力在很小的力范围内显著增加。如果这个想法是正确的， 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显著变化。此外，我们将进行第一个详细的生物化学和生物物理特性的相互作用的钙粘蛋白-连环蛋白复合物与EPLIN，ZO-1，和afadin。这些研究奠定了定量的了解AJ如何作为一个集成的，多功能的力传感组件的功能的基础。 在目标2中，我们将检查桥粒中的力敏感性。这些连接将桥粒钙粘蛋白连接到中间丝（IF）细胞骨架，并且对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的作用，但目前还没有直接证据表明桥粒钙粘蛋白在没有外力的情况下何时、何地甚至是否在相邻细胞之间传递张力。为了解决这一问题，我们将使用遗传编码的分子张力传感器来确定桥粒钙粘蛋白在相邻细胞之间的作用时间和位置。然后，我们将严格评估桥粒斑蛋白在桥粒传递力中的作用，类似于在AJs建立的α E-连环蛋白的作用。最后，我们将使用单分子磁镊试验来测试的创新假设，即招募斑珠蛋白，斑嗜蛋白，或两者桥斑蛋白是固有的力敏感。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。

 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.