Understanding force-dependent binding of alpha-catenin to actin

 描述(申请人提供)：细胞-细胞黏附定义固体组织，黏附的失调是癌细胞转移的重要步骤。AE-catenin蛋白通过将钙粘蛋白细胞黏附分子和肌动蛋白细胞骨架之间的机械张力转化为生化信号，在细胞和组织的发育中发挥重要作用。我们将研究AE-连环蛋白结构随力的变化的分子基础，以及它的分子行为如何有助于细胞-细胞接触的形成和解离。我们的方法是结合使用严格的生化特征(WEIS)和创新的单分子光学捕获分析(DUNN)来发现蛋白a-catenin如何加强细胞间的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角色。细胞生物学数据表明，细胞内黏附蛋白E-钙粘附素(上皮钙粘附素)与肌动蛋白细胞骨架的连接需要α-连环蛋白及其结合伙伴-连环蛋白。然而，在批量生化分析中，a-catenin/？-catenin/E-cadherin不能与肌动蛋白结合。在前期工作中，我们使用了一种新颖的单分子光学陷阱分析方法来证明钙粘附素/连环蛋白三元复合体确实可以与肌动蛋白结合，但只有在机械载荷存在的情况下。此外，我们还发现，α-连环蛋白-肌动蛋白键的强度随着机械载荷的增加而增加，并且钙粘连蛋白/连环蛋白复合体与肌动蛋白细丝的结合是高度协同的。这些发现的含义是α-连环蛋白作为一种力量。 灵敏的连接物，可以加强细胞与细胞之间的接触，以响应机械负荷。这一机制提供了一种在机械应变存在的情况下保持组织完整性的优雅方法，并解释了细胞如何在细胞-细胞连接处感受到张力，这是当前非常感兴趣的一个主题。然而，α-连环蛋白是如何感受机械张力的还不得而知。我们将使用生化和单分子生物物理方法相结合的方法来：1)确定a-catenin在钙粘连蛋白和肌动蛋白细胞骨架之间形成力敏感连接的分子机制；2)发现a-catenin、肌动蛋白或两者之间的协同结构转换如何调节钙粘连蛋白/catenin复合体和丝状肌动蛋白之间的结合。这些测量将揭示a-连环蛋白在细胞-细胞连接处感知力的分子机制。此外，这项工作将为理解钙粘附素-连接素复合体如何协同工作来重塑细胞-细胞连接以响应机械负荷的变化提供一个机制基础，这可能对我们理解上皮重塑和形态发生具有广泛的意义。

 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.