Mechanobiology of the immune synapse: signal integration via actin dynamics

摘要 机械力对于T细胞活化是必不可少的。它激活TCR信号，并允许T细胞采样 TCR-pMHC相互作用的质量。这极大地扩展了TCR响应的动态范围，并且允许 胸腺选择、T细胞引发和效应子应答过程中的抗原辨别。我们理解 力如何影响TCR-pMHC相互作用已经取得了显着进展，这要归功于生物物理学研究。 单分子水平。然而，在细胞生物学水平上存在巨大的知识差距。这个项目 旨在确定TCR信号转导网络中的生物化学和机械回路， 允许快速翻译TCR-pMHC相互作用的物理特征中的小差异 转化为不同的细胞反应在第一个项目期间，我们发现T细胞肌动蛋白网络发挥作用， 对整联蛋白LFA-1以及TCR的作用力，支持影响激活的机械串扰 这两种分子。有趣的是，这个过程对刺激表面的生物物理特征敏感， 包括配体迁移率和刚度。这些参数是生理相关的，因为它们受到调节 以优化T细胞引发。进一步的分析表明，这种机械生物学也 影响与肌动蛋白细胞骨架相互作用的细胞质信号分子。特别是，我们发现T 细胞硬度反应涉及拉伸敏感性衔接蛋白CasL的磷酸化。的基础上 根据这些发现，我们推测TCR诱导的肌动蛋白聚合使细胞能够感知生物物理信号 由相互作用的APC提供，启动调节力依赖信号的机械反馈回路 与肌动蛋白细胞骨架相互作用的细胞表面受体和细胞内信号分子。测试 这个假设，我们将进行3个具体目标。首先，我们将确定配体迁移率如何影响肌动蛋白 动力学和TCR信号传导。使用刺激玻璃盖玻片，具有不同迁移率的平面双层 性质，和混合流动性图案化的表面，我们将问如何激动剂的强度和流动性的pMHC 复合物和整合素配体影响肌动蛋白动力学和TCR信号传导。作为分析的一部分，我们将 使用TCR张力探针来确定改变TCR和整合素配体的移动性如何影响力 TCR的经历。接下来，我们将进行类似的研究，以了解如何基板刚度 影响T细胞活化。我们将在不同硬度的水凝胶上刺激T细胞，并分析其效果。 对肌动蛋白动力学、TCR张力和完全T细胞活化所需的TCR信号传导事件的影响。最后我们将 研究CasL的作用，CasL是一种原型力敏信号中间体。使用缺乏CasL的T细胞，我们 将研究CasL在T细胞对配体迁移率和底物刚度变化的反应过程中的功能。在 此外，我们还将探索在僵硬反应过程中导致CasL磷酸化的信号通路， 使用质谱法来识别相关的结合伴侣。

ABSTRACT Mechanical force is essential for T cell activation. It activates TCR signaling, and allows the T cell to sample the quality of TCR-pMHC interactions. This greatly expands the dynamic range of TCR responses and permits antigen discrimination during thymic selection, T cell priming, and effector responses. Our understanding of how force influences TCR-pMHC interactions has advanced significantly, thanks to biophysical studies at the single molecule level. However, there are large gaps in knowledge at the cell biological level. This project seeks to identify the biochemical and mechanical circuits within the TCR signal transduction network that permit the rapid translation of small differences in the physical characteristics of the TCR–pMHC interactions into distinct cellular responses. During the first project period, we showed that the T cell actin network exerts force on the integrin LFA-1 as well as the TCR, supporting mechanical crosstalk that influences the activation of both molecules. Interestingly, this process is sensitive to the biophysical features of the stimulatory surface, including ligand mobility and stiffness. These parameters are physiologically relevant, as they are regulated during DC maturation to optimize T cell priming. Further analysis reveals that this mechanobiology also impacts cytoplasmic signaling molecules that interact with the actin cytoskeleton. In particular, we find that T cell stiffness responses involve phosphorylation of the stretch-sensitive adapter protein CasL. On the basis of these findings, we hypothesize that TCR-induced actin polymerization allows the cell to sense biophysical cues provided by the interacting APC, initiating mechanical feedback loops that modulate force-dependent signaling of cell surface receptors and intracellular signaling molecules that interact with the actin cytoskeleton. To test this hypothesis, we will carry out 3 specific aims. First, we will determine how ligand mobility influences actin dynamics and TCR signaling. Using stimulatory glass coverslips, planar bilayers with different mobility properties, and mixed mobility patterned surfaces, we will ask how the agonist strength and mobility of pMHC complexes and integrin ligands influences actin dynamics and TCR signaling. As part of this analysis, we will use TCR tension probes to define how altering the mobility of TCR and integrin ligands influences the forces experienced by the TCR. Next, we will carry out similar studies to understand how substrate stiffness influences T cell activation. We will stimulate T cells on hydrogels of varying stiffness, and analyze the effects on actin dynamics, TCR tension, and TCR signaling events needed for full T cell activation. Finally, we will investigate the role of CasL, a prototypic force-sensitive signaling intermediate. Using T cells lacking CasL, we will study the function of CasL during T cell responses to changes in ligand mobility and substrate stiffness. In addition, we will probe the signaling pathways leading to CasL phosphorylation during stiffness responses, and use mass spectrometry to identify relevant binding partners.