Biophysical mechanisms of mechanical tension sensing at cellular integrin complexes

描述(申请人提供)：我们的目标是发现整合素感知和转导机械线索的分子机制。整合素是连接细胞骨架和细胞外基质(ECM)的异源二聚体跨膜蛋白。细胞使用整合素进行迁移，对周围环境施加压力，并感知细胞外基质的物理性质。后一种特性被称为机械转导，对人类健康和疾病特别重要。通过整合素传递的物理张力激活细胞内信号，进而对免疫功能、干细胞分化和癌细胞转移等过程产生深远影响。尽管整合素在生理和医学上具有重要意义，但整合素感知机械力的物理机制尚不清楚。我们的目标是弥合我们对细胞生物学理解上的这一根本差距。在已发表的工作中，我们开发了基于F�Rster共振能量转移(FRET)的分子张力传感器(MTSS)，该传感器报告了单个整合素在活细胞中经历的机械张力。自那以后，我们将mTSS和超分辨率光学显微镜相结合，首次以纳米空间分辨率绘制了整合素粘连内的力传递图。基于MTS成像的新能力使我们能够解决整合素生物学中的两个基本问题，这两个问题到目前为止还不能直接解决。在目标1中，我们将确定整合素感知机械张力的物理机制。特别是，我们将检验最重要的假设，即不同的整合素类别通过根本不同的机制感知张力，并且这些差异允许细胞在广泛的力和时间尺度上感知机械刺激。在目标2中，我们将首次描述微米级整合素组件中的力传递和传感机制，称为焦点粘连(FAs)。具体地说，我们将测试FAs包含高度协调的力敏感微域的假设，这一预测无法使用传统技术进行测试。这项工作将通过揭示细胞感知和传递机械信号的分子组装和生物物理机制来改变我们对细胞机械转导的理解。更广泛地说，FAs的机械反应性和成分复杂性也存在于许多其他细胞结构中。在这个项目中开发的概念和技术方法有能力通过在完整的活细胞的背景下引入强大的单分子生物物理测量来改变多个研究领域。

DESCRIPTION (provided by applicant): Our goal is to discover the molecular mechanisms by which integrins sense and transduce mechanical cues. Integrins are heterodimeric transmembrane proteins that link the cell's cytoskeleton to the extracellular matrix (ECM). Cells use integrins to migrate, exert force on their surroundings, and to sense the physical properties of the ECM. This latter property, termed mechanotransduction, is particularly important in human health and disease. Physical tension transmitted through integrins activates intracellular signaling that in turn exerts profound effects on processes as diverse as immune function, stem cell differentiation, and cancer cell metastasis. Despite this great physiological and medical importance, the physical mechanisms by which integrins sense mechanical force are not known. We aim to close this fundamental gap in our understanding of cell biology. In published work, we have developed F�rster resonance energy transfer (FRET) based molecular tension sensors (MTSs) that report on the mechanical tensions experienced by individual integrins in living cells. We have since combined MTSs and superresolution light microscopy to, for the first time, map force transmission within integrin adhesions with nanometer spatial resolution. The qualitatively new capabilities of MTS-based imaging allow us to tackle two fundamental questions in integrin biology that until now could not be directly addressed. In Aim 1, we will determine the physical mechanisms by which integrins sense mechanical tension. In particular, we will examine the overarching hypothesis that different integrin classes sense tension via fundamentally different mechanisms, and that these differences allow the cell to sense mechanical stimuli over a wide range of forces and timescales. In Aim 2, we will characterize the force transducing and sensing machinery in micron-sized integrin assemblies, termed focal adhesions (FAs), for the first time. Specifically, we will test the hypothesis that FAs contain highly coordinated, force-sensing microdomains, a prediction that cannot be tested using conventional techniques. This work will transform our understanding of cellular mechanotransduction by uncovering the molecular assemblies and biophysical mechanisms by which cells sense and transduce mechanical signals. More broadly, the mechano-responsiveness and compositional complexity that characterize FAs are also present in many other cellular structures. The conceptual and technical approaches developed in this project have the capacity to transform multiple fields of research by introducing powerful new single-molecule biophysical measurements in the context of intact, living cells.