FORCEBIND: Mechanochemical Regulation Of Focal And Fibrillar Adhesion Proteins

细胞感知并适应环境施加的机械力。来自细胞外基质的机械信号在局灶黏附处被检测到，在那里它们被转化为生化信号，引发细胞反应。主要的力转导机制涉及蛋白质结合相互作用的机械和化学调节。从分子角度理解这些过程需要评估这些蛋白质在外力作用下如何相互作用。然而，蛋白质生物化学分析不能对大量分子施加力，而经典的单分子技术通常仅限于使用高力，这就排除了对机械传感蛋白质的研究，因为在细胞-基质粘附中涉及的力很低。我们的目标是实施新的单分子方法来揭示细胞粘附中机械转导过程的分子机制。为此，我们将首先开发一种新的单分子仪器，将荧光与超稳定磁镊子相结合，使我们能够在生理相关力和时间尺度下捕获蛋白质中的结合事件。这项新技术将使我们能够研究关键的机械传感蛋白如何对力做出构象反应，以及这些力如何调节结合相互作用和支撑其功能的翻译后修饰。具体来说，我们将确定磷酸化如何调节蛋白质纳米力学，使用黏附激酶作为模型系统，并深入研究纤维黏附的主要调节剂张力素难以捉摸的机械传感功能。我们预计这些努力将揭示蛋白质动力学、相互作用和化学是如何被机械力精细调节的，并确定细胞将机械信号转化为生化反应的关键分子机制，这与从发育到癌症等多个生物领域至关重要。

Cells sense and adapt to mechanical forces imposed by their environment. Mechanical cues from the extracellular matrix are detected at focal adhesions, where they are converted into biochemical signals to elicit a cellular response. A primary force-transduction mechanism involves the mechanical and chemical regulation of protein-binding interactions. Understanding these processes from a molecular perspective requires evaluating how these proteins interact under force. However, protein biochemistry assays cannot apply force to molecules in bulk, while classic single-molecule techniques are generally limited to using high forces, which precludes the study of mechanosensing proteins given the low forces involved at cell-matrix adhesions. Our objective is to implement novel single-molecule approaches to unravel the molecular mechanisms underpinning mechanotransduction processes at cellular adhesions. To that aim, we will first develop a new single-molecule instrument combining fluorescence with ultra-stable magnetic tweezers, allowing us to capture binding events in proteins under physiologically relevant forces and timescales. This new technology will enable us to examine how key mechanosensing proteins respond conformationally to force and how these forces regulate the binding interactions and post-translational modifications that underpin their function. Specifically, we will determine how phosphorylation regulates protein nanomechanics, using the focal adhesion kinase as a model system, and delve into the elusive mechanosensing function of tensin, the master regulator of fibrillar adhesions. We anticipate that these efforts will unveil how protein dynamics, interactions, and chemistry are finely regulated by mechanical forces, identifying the key molecular mechanisms used by cells to convert mechanical cues into biochemical responses, of critical relevance for multiple biological fields, from development to cancer.