Influences of nanomechanical forces on T cells

描述(申请人提供)：几乎身体中的每个细胞都暴露在通过细胞骨架传递的机械力中，并影响其活动。最近对适应性免疫系统的主要组织细胞T细胞的研究表明，作用于T细胞受体(TCR)并通过细胞骨架的机械力可以触发细胞反应。目前尚不清楚细胞骨架如何发挥这一作用，也不清楚T细胞如何解释受体连接的力量和信号的相互作用。我们发现T细胞 当它们的肌动蛋白细胞骨架不受束缚并且机械地“柔软”时，它们对抗原更敏感(即具有较低的激活阈值)。此外，我们还发现，在自身免疫性糖尿病中，炎症细胞外基质(ECM)的机械力可以激发T细胞自身免疫。我们知识上的差距是由于缺乏能够测量纳米级的力量并将其传递给活细胞的工具。T细胞在细胞骨架结构中编码它们的激活阈值，这为研究细胞如何用一种结合了机械力和受体信号的语言进行交流提供了动力。在这里，我们建议1)在分子水平上确定T细胞受体的机械敏感性，使用先进的原子力显微镜(AFM)将机械力和抗原传递到单分子TCR；2)使用可以测量活细胞中细胞骨架变化的新一代AFM悬臂来确定机械传感所需的细胞骨架网络；以及3)使用自身免疫性糖尿病的小鼠模型和3D仿生基质来模拟发炎的ECM及其对T细胞激活的机械作用，以确定由于炎症ECM而产生的机械力的影响。我们的实验室与我们在斯坦福大学的合作伙伴合作，开创了生物原子力显微镜(AFM)和纳米制造方面的突破，使这些创新研究成为可能，使我们能够精确连接受体并施加微小力量，同时测量活T细胞的机械反应，同时使用实时共聚焦显微镜对细胞及其细胞骨架的变化进行成像。这些研究将帮助我们破译细胞信号中机械力的“语言”，并将对我们理解炎性ECM在自身免疫性疾病中的机械效应产生重大影响。我们希望我们的发现将刺激新的免疫疗法。我们在细胞生物学中使用AFM的新方法有可能彻底改变与许多疾病有关的机械生物学和受体信号通路的研究。

DESCRIPTION (provided by applicant): Virtually every cell in the body is exposed to mechanical forces that are transduced through its cytoskeleton and influence its activity. Recent studies using T cells, the master organizing cells of the adaptive immune system, have shown that mechanical forces acting upon the T cell receptor (TCR) and through the cytoskeleton can trigger cellular responses. It is unclear how the cytoskeleton plays this role, or how T cells interpret the interplay of forces and signals of receptor ligation. We have discovered that T cells are more sensitive to antigen (i.e., have a lower threshold of activation) when their actin cytoskeleton is untethered and mechanically "soft." Furthermore, we have discovered that in autoimmune diabetes, mechanical forces from inflamed extracellular matrix (ECM) can provoke T cell autoimmunity. The gaps in our knowledge are due to a lack of tools that can measure and deliver nanoscale forces to live cells. That T cells encode their threshold of activation in cytoskeletal structures provides impetus to study how cells communicate in a language that combines mechanical forces with receptor signals. Here, we propose to 1) Determine the mechanosensitivity of the T cell receptor at the molecular level, using an advanced atomic force microscope (AFM) to convey mechanical forces and antigens to single-molecule TCRs; 2) Identify the cytoskeletal networks required for mechanosensing, using a new generation of AFM cantilevers that can measure cytoskeletal changes in live cells; and 3) Determine influence of mechanical forces due to inflammatory ECM, using a mouse model of autoimmune diabetes and a 3D biomimetic matrix to emulate the inflamed ECM and its mechanical effects on T cell activation. Our lab, working with our collaborators at Stanford, has pioneered breakthroughs in biological atomic force microscopy (AFM) and nanofabrication that make these innovative studies possible by allowing us to precisely ligate receptors and exert minute forces while measuring mechanical responses in live T cells, all while imaging cells and their cytoskeletal changes using live confocal microscopy. These studies will help us decipher the "language" of mechanical forces in cell signaling, and will have a major impact on our understanding of the mechanical effects of inflammatory ECM in autoimmune diseases. We expect our findings will spur novel immune therapeutics. Our new methods of using AFM in cell biology have the potential to revolutionize studies of mechanobiology and receptor signaling pathways implicated in many diseases.