Biophysical mechanisms of mechanical tension sensing at cellular integrin complexes

描述（由申请人提供）：我们的目标是发现整合素感知和转导机械信号的分子机制。整合素是一种异二聚体跨膜蛋白，可将细胞骨架与细胞外基质（ECM）连接起来。细胞利用整合素进行迁移，对周围环境施加力，并感知ECM的物理特性。后一种特性被称为机械转导，在人类健康和疾病中尤为重要。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多种过程产生深远影响。尽管这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，我们开发了基于傅氏共振能量转移（FRET）的分子张力传感器（mts），该传感器可以报告活细胞中单个整合素所经历的机械张力。此后，我们将mts和超分辨率光学显微镜结合起来，首次以纳米空间分辨率绘制了整合素粘附内的力传输。基于mts成像的定性新功能使我们能够解决整合素生物学中的两个基本问题，这些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，我们将研究一个总体假设，即不同的整合素类通过根本不同的机制感知张力，这些差异使细胞能够在大范围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感器和传感机制，称为焦点粘附（FAs）。具体来说，我们将测试FAs包含高度协调的力传感微域的假设，这是一个无法使用传统技术测试的预测。这项工作将通过揭示细胞感知和转导机械信号的分子组装和生物物理机制，改变我们对细胞机械转导的理解。更广泛地说，FAs特征的机械响应性和组成复杂性也存在于许多其他细胞结构中。该项目开发的概念和技术方法有能力通过在完整的活细胞背景下引入强大的新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.