Molecular mechanisms underlying force transduction at cellular adhesion complexes

我们的目标是阐明细胞黏附复合体形成和形成的分子机制 根据机械载荷进行重塑。细胞-细胞和细胞-基质粘连是后生动物的一个重要特征 它对人体内几乎每一种组织的生理功能都是必不可少的。尽管如此，中央 重要的是，组成黏附复合体的蛋白质-蛋白质相互作用中只有几个是 以生物化学为特征，对这些结构的潜在机制了解更少 对机械负荷作出反应。这种量化数据的缺乏是集体行动中不可避免的障碍 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性 以E-钙粘素为模板的复合体可以感知和传递机械信号。在此之前，我们演示了一个 E-钙粘蛋白、β-连环蛋白和αE-连环蛋白的复合体在细胞间粘连形成一个最小的力敏感单元。 在这里，我们建立在这个结果的基础上，来检验这个复合体位于机械感觉的核心这一假设 一种组件，可将输入力的微小变化转化为附着力体系结构、大小和 稳定性。 在并行工作中，我们将使用我们实验室独有的生物物理技术来确定 黏附复合体中的蛋白质与丝状(F)-肌动蛋白之间的相互作用可能导致长程 细胞骨架中的组织。最近，我们发现纽蛋白，它被招募到两个细胞- 基质和细胞间的粘连，与F-肌动蛋白形成定向不对称的相互作用，稳定在~10- 当载荷朝向肌动蛋白细丝的尖端(-)而不是带刺的(+)端时折叠。初步数据显示 与F-肌动蛋白的力依赖的不对称结合相互作用并不是纽蛋白独有的，而且可能延伸 与其他黏附蛋白的结合。这些观察表明，F-肌动蛋白和肌动蛋白之间的不对称相互作用 黏附复合体中的蛋白质可能在组织细胞和 组织，这是一个假设，我们将在下一个资助期进行测试。 细胞和发育生物学数据表明，αE-连环蛋白在组织上皮细胞中起着核心作用 通过与闭锁带-1(ZO-1)和afadin的相互作用，这两种蛋白都能与F-肌动蛋白结合并招募 其他支架和信号蛋白。我们将进行第一次详细的生化和生物物理实验 钙粘素-连环蛋白复合体与ZO-1和afadin相互作用的表征，并使用尖端技术 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 以定量了解细胞间黏附复合体如何发挥整合、多功能的作用 力感应组件。

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.