Molecular mechanisms underlying force transduction at cellular adhesion complexes

细胞粘附复合物力传导的分子机制

• 批准号: 9926286
• 负责人: Alexander R Dunn
• 金额: $ 56.25万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-06 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9926286
• 关键词: Adhesions; Architecture; Behavior; Binding; Biochemical; Biological; Cadherins; Cardiomyopathies; Cell Adhesion; Cell-Matrix Junction; Cells; Complex; Cues; Cytoskeleton; Data; Development; E-Cadherin; Embryonic Development; Epithelial; Epithelium; Event; F-Actin; Foundations; Funding; Heart; Human body; Imaging Techniques; Laboratories; Life; Link; Mechanics; Molecular; Neoplasm Metastasis; Physiological; Play; Plus End of the Actin Filament; Proteins; Role; Scaffolding Protein; Signaling Protein; Structure; Testing; Tight Junctions; Tissues; Vinculin; Work; afadin; alpha catenin; beta catenin; biophysical properties; biophysical techniques; mechanical force; mechanical load; protein complex; protein protein interaction; recruit; response; single molecule; skin disorder; virtual

Our objective is to elucidate the molecular mechanisms by which cellular adhesion complexes form and remodel in response to mechanical load. Cell-cell and cell-matrix adhesions are a defining feature of metazoan life and are essential to the physiological function of virtually every tissue in the human body. Despite this central importance, only a few of the protein-protein interactions that make up adhesion complexes have been characterized biochemically, and even less is known about the underlying mechanisms by which these structures respond to mechanical load. This lack of quantitative data presents an unavoidable roadblock in the collective effort to understand how cells build and remodel multicellular tissues. We will use single-molecule biophysical approaches to develop a detailed understanding of how adhesion complexes templated by E-cadherin sense and transduce mechanical cues. Previously, we demonstrated that a complex of E-cadherin, β-catenin, and αE-catenin forms a minimal force-sensing unit at intercellular adhesions. Here, we build on this result to test the hypothesis that this complex lies at the heart of a mechanosensory assembly that converts small changes in input forces into dramatic alterations in adhesion architecture, size, and stability. In parallel work, we will use biophysical techniques unique to our laboratory to determine how directional interactions between proteins within adhesion complexes and filamentous (F)-actin may give rise to long-range organization in the cytoskeleton. Recently, we found that the protein vinculin, which is recruited to both cell- matrix and intercellular adhesions, forms a directionally asymmetric interaction with F-actin that is stabilized ~10- fold when load is oriented toward the pointed (-) vs. barbed (+) end of the actin filament. Preliminary data suggest that force-dependent, asymmetric binding interactions with F-actin are not unique to vinculin, and likely extend to other adhesion proteins. These observations suggest that asymmetric interactions between F-actin and proteins within adhesion complexes may play a central and previously unsuspected role in organizing cells and tissues, a hypothesis that we will test during the next funding period. Cell and developmental biological data indicate that αE-catenin plays a central role in organizing epithelial tissues through its interactions with zonula occludens-1 (ZO-1) and afadin, both of which bind F-actin and recruit other scaffolding and signaling proteins. We will perform the first detailed biochemical and biophysical characterization of the interaction of the cadherin-catenin complex with ZO-1 and afadin, and use cutting-edge imaging techniques to determine how these proteins interact in living cells. These studies will lay the foundation for a quantitative understanding of how intercellular adhesion complexes function as integrated, multifunctional force-sensing assemblies.

我们的目标是阐明细胞粘附复合物形成的分子机制， 根据机械负荷进行改造。细胞-细胞和细胞-基质粘附是后生动物的一个定义性特征 并且对人体内几乎每个组织的生理功能都是必不可少的。尽管中央 重要的是，只有少数的蛋白质-蛋白质相互作用，使粘附复合物已被 生物化学的特点，甚至更少的是知道这些结构的潜在机制， 响应机械负载。这种缺乏定量数据的情况在集体研究中造成了不可避免的障碍。 努力了解细胞如何构建和重塑多细胞组织。 我们将使用单分子生物物理方法来详细了解粘附是如何发生的。 复合物模板的E-钙粘蛋白的感觉和神经机械线索。之前，我们证明了一个 E-cadherin、β-catenin和α-E-catenin的复合物在细胞间粘附处形成最小的力感应单位。 在这里，我们基于这一结果来测试这一假设，即这种复合物位于机械感觉的核心。 将输入力的微小变化转化为粘附结构、尺寸和 稳定 在平行的工作中，我们将使用我们实验室独有的生物物理技术来确定 粘附复合物中的蛋白质与丝状（F）-肌动蛋白之间的相互作用可能引起长距离的 细胞骨架中的组织。最近，我们发现，蛋白粘着斑，这是招募到两个细胞- 基质和细胞间粘附，与F-肌动蛋白形成方向不对称的相互作用，其稳定在~10- 当载荷朝向肌动蛋白丝的尖（-）与倒刺（+）端时折叠。初步数据表明 力依赖性，与F-肌动蛋白的不对称结合相互作用并不是黏着斑蛋白所独有的， 其他粘附蛋白。这些观察结果表明，F-肌动蛋白和 粘附复合物中的蛋白质可能在组织细胞中起中心的和以前未被怀疑的作用， 组织，我们将在下一个资助期内测试这一假设。 细胞和发育生物学数据表明，α E-连环蛋白在组织上皮细胞中起着重要作用， 组织通过其与闭锁小带-1（ZO-1）和afadin的相互作用，两者都结合F-肌动蛋白并招募 其他支架和信号蛋白。我们将进行第一次详细的生物化学和生物物理 表征钙粘蛋白-连环蛋白复合物与ZO-1和afadin的相互作用，并使用尖端的 成像技术，以确定这些蛋白质如何在活细胞中相互作用。这些研究将奠定基础 为了定量地了解细胞间粘附复合物如何作为一种整合的、多功能的 力传感组件。