Understanding force-dependent binding of alpha-catenin to actin

了解 α-连环蛋白与肌动蛋白的力依赖性结合

• 批准号: 8964322
• 负责人: Alexander R Dunn
• 金额: $ 29.14万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-22 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8964322
• 关键词: Actins; Address; Adhesions; Adhesives; Behavior; Binding; Biochemical; Biological; Biological Assay; Cadherins; Cell Adhesion Molecules; Cell-Cell Adhesion; Cells; Characteristics; Complex; Coupled; Cytoskeleton; Data; Defect; Development; Dissociation; E-Cadherin; Epithelial; F-Actin; F-actin-binding proteins; Goals; Homeostasis; Intercellular Junctions; Link; Maintenance; Malignant Neoplasms; Measurement; Mechanical Stress; Mechanics; Microfilaments; Modeling; Molecular; Molecular Conformation; Morphogenesis; Neoplasm Metastasis; Normal tissue morphology; Organ; Organogenesis; Property; Proteins; Role; Signal Transduction; Solid; Solutions; Structure; System; Testing; Tissues; Work; alpha catenin; base; beta catenin; biophysical techniques; cancer cell; driving force; innovation; interest; novel; optical traps; outcome forecast; prevent; public health relevance; research study; response; single molecule

 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.

 描述（由申请人提供）：细胞间粘附定义了实体组织，粘附失调是癌细胞转移的重要步骤。 αE-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化为生化信号，在细胞和组织发育中发挥关键作用。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与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表征 (Weis) 和创新的单分子光学捕获测定 (Dunn) 来发现蛋白质 a-连环蛋白如何增强细胞与细胞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参与者。 细胞生物学数据表明，需要 a-连环蛋白及其结合伴侣 ß-连环蛋白将细胞内粘附蛋白 E-钙粘蛋白（上皮钙粘蛋白）连接到肌动蛋白细胞骨架。然而，在批量生化检测中，α-连环蛋白/β-连环蛋白/E-钙粘蛋白不结合肌动蛋白。在前期工作中，我们使用了一种新颖的单分子光陷阱测定法来证明钙粘蛋白/连环蛋白三元复合物确实可以结合肌动蛋白，但只有在机械负载存在的情况下。此外，我们发现α-连环蛋白-肌动蛋白键的强度随着机械负载的增加而增加，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协同的。这些发现的含义是α-连环蛋白充当一种力量 敏感的连接体可以增强细胞与细胞之间的接触以响应机械负载。这种机制提供了一种在存在机械应变的情况下维持组织完整性的优雅方法，并解释了细胞如何感知细胞-细胞连接处的张力，这是当前人们强烈关注的话题。然而，α-连环蛋白如何准确地感知机械张力尚不清楚。 我们将结合生物化学和单分子生物物理方法来：1）确定α-连环蛋白在钙粘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α-连环蛋白、肌动蛋白或两者的协同结构转变如何调节钙粘蛋白/连环蛋白复合物和丝状肌动蛋白之间的结合。这些测量结果将揭示α-连环蛋白感知细胞与细胞连接处力的分子机制。此外，这项工作将为理解钙粘蛋白-连环蛋白复合物群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机制基础，这对我们对上皮重塑和形态发生的理解具有潜在的广泛影响。