Understanding force-dependent binding of alpha-catenin to actin

 描述（由申请人提供）：细胞-细胞粘附定义实体组织，粘附失调是癌细胞转移的重要步骤。蛋白质aE-连环蛋白通过将钙粘蛋白细胞粘附分子和肌动蛋白细胞骨架之间的机械张力转换成生物化学信号而在细胞和组织发育中具有关键作用。我们将研究aE-连环蛋白结构如何响应力而变化的分子基础，以及其分子行为如何有助于细胞-细胞接触的形成和解离。我们的方法是使用严格的生物化学表征（Weis）和创新的单分子光学捕获测定（Dunn）的组合来发现蛋白质α-连环蛋白如何加强细胞-细胞连接并触发下游信号转导以响应机械应力。这个问题具有深刻的生物医学意义，因为已知α-连环蛋白是形成多细胞组织所必需的，并且是器官发生和癌症转移的核心参与者。 细胞生物学数据表明，α-连环蛋白及其结合伴侣β-连环蛋白是将细胞内粘附蛋白E-钙粘蛋白（上皮钙粘蛋白）连接到肌动蛋白细胞骨架所必需的。然而，α-连环蛋白/β-连环蛋白/E-钙粘蛋白在批量生物化学测定中不结合肌动蛋白。在初步工作中，我们使用了一种新的单分子光阱分析，以表明钙粘蛋白/连环蛋白三元复合物确实可以结合肌动蛋白，但只有在机械负荷的存在下。此外，我们发现α-连环蛋白-肌动蛋白键的强度随着机械负荷而增加，并且钙粘蛋白/连环蛋白复合物与肌动蛋白丝的结合是高度合作的。这些发现的含义是，α-连环蛋白作为一种力 敏感的连接体，可以响应机械负荷而加强细胞-细胞接触。这种机制提供了一种优雅的手段，以保持组织的完整性，在机械应变的存在下，并提供了一个解释细胞如何可以感觉到紧张的细胞连接，一个强烈的当前感兴趣的话题。然而，α-连环蛋白究竟如何感知机械张力尚不清楚。 我们将使用生物化学和单分子生物物理方法的组合：1）确定α-连环蛋白形成钙粘蛋白和肌动蛋白细胞骨架之间的力敏感连接的分子机制; 2）发现α-连环蛋白，肌动蛋白或两者的合作结构转化如何调节钙粘蛋白/连环蛋白复合物和丝状肌动蛋白之间的结合。这些测量将揭示α-连环蛋白在细胞-细胞连接处感知力的分子机制。此外，这项工作将提供一个机制的基础，了解如何组钙粘蛋白-连环蛋白复合物协同工作，重塑细胞-细胞连接，以响应机械负荷的变化，与潜在的广泛影响，我们的理解上皮重塑和形态发生。

 DESCRIPTION (provided by applicant): Cell-cell adhesion defines solid tissues, and dysregulation of adhesion is an essential step in cancer cell metastasis. The protein aE-catenin has critical roles in cell and tissue development by transducing mechanical tension between cadherin cell adhesion molecules and the actin cytoskeleton into biochemical signals. We will investigate the molecular basis of how aE-catenin structure changes in response to force, and how its molecular behavior contributes to the formation and dissociation of cell-cell contacts. Our approach is to use a combination of rigorous biochemical characterization (Weis) and innovative single-molecule optical trapping assays (Dunn) to discover how the protein a-catenin both reinforces cell-cell junctions and triggers downstream signal transduction in response to mechanical stress. This question has deep biomedical significance, since a-catenin is known to be required for the formation of multicellular tissues and is a central player in both organogenesis and cancer metastasis. Cell biological data show that a-catenin and its binding partner ß-catenin are required to link the intracellular adhesion protein E-cadherin (epithelial cadherin) to the actin cytoskeleton. However, the a- catenin/ß-catenin/E-cadherin does not bind actin in bulk biochemical assays. In preliminary work, we used a novel single-molecule optical trap assay to show that the cadherin/catenin ternary complex can indeed bind actin, but only in the presence of mechanical load. Further, we find that the strength of the a-catenin-actin bond increases with mechanical load, and that binding of the cadherin/catenin complex to the actin filament is highly cooperative. The implication of these findings is that a-catenin acts as a force sensitive linker that can reinforce cell-cell contacts in response to mechanical load. This mechanism provides an elegant means to maintain tissue integrity in the presence of mechanical strain, and provides an explanation for how cells may sense tension at cell-cell junctions, a topic of intense current interest. However, how exactly a-catenin senses mechanical tension is not known. We will use a combination of biochemical and single-molecule biophysical approaches to: 1) determine the molecular mechanism by which a-catenin forms a force-sensitive linkage between cadherins and the actin cytoskeleton; and 2) discover how cooperative structural transformations in a-catenin, actin, or both regulate binding between the cadherin/catenin complex and filamentous actin. These measurements will reveal the molecular mechanism by which a-catenin senses force at cell-cell junctions. In addition, this work will provide a mechanistic basis for understanding how groups of cadherin-catenin complexes work in concert to remodel cell-cell junctions in response to changes in mechanical load, with potentially broad implications for our understanding of epithelial remodeling and morphogenesis.