Mechanobiology of the immune synapse: signal integration via actin dynamics

免疫突触的力学生物学：通过肌动蛋白动力学进行信号整合

• 批准号: 10513815
• 负责人: Janis K. Burkhardt
• 金额: $ 42.6万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-11-24 至 2025-10-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10513815
• 关键词: Actins; Adaptor Signaling Protein; Address; Adhesions; Agonist; Antigens; Autoimmune Diseases; Award; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Biophysics; Cell Surface Receptors; Cells; Characteristics; Complex; Consensus; Cues; Cytoplasm; Cytoskeleton; DNA; Data; Dendritic Cells; Discrimination; Elements; Environment; Event; Feedback; Glass; Hydrogels; IL2 gene; Immobilization; Immune response; Immunologic Deficiency Syndromes; Integrins; Kinetics; Knowledge; Lead; Ligands; Lipid Bilayers; Mass Spectrum Analysis; Measures; Mechanics; Microscopy; Modeling; Molecular Genetics; Monitor; Output; Pathway interactions; Patients; Pattern; Peptide/MHC Complex; Phosphorylation; Physiological; Polymers; Process; Property; Role; Sampling; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Stretching; Surface; System; T cell response; T-Cell Activation; T-Lymphocyte; Testing; Thymus Gland; Transcriptional Regulation; Translations; Tyrosine Phosphorylation; Video Microscopy; Work; adaptive immunity; biophysical analysis; biophysical properties; dynamic system; experience; genetic approach; immunological synapse; immunoregulation; mechanical force; mechanical signal; mechanotransduction; pathogen; pharmacologic; polymerization; prototype; response; sensor; single molecule; vaccine development

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.

抽象的 机械力对于T细胞激活至关重要。它激活TCR信号，并允许T细胞采样 TCR-PMHC相互作用的质量。这一伟大扩展了TCR响应和许可的动态范围 胸腺选择，T细胞启动和效应子反应过程中的抗原歧视。我们对 力如何影响TCR-PMHC相互作用已显着发展，感谢您在 单分子水平。但是，在细胞生物学水平上有很大的差距。这个项目 试图识别TCR信号传输网络中的生化和机械电路 允许快速翻译TCR – PMHC相互作用的物理特征的微小差异 变成不同的细胞反应。在第一个项目期间，我们显示T细胞肌动蛋白网络执行 整联蛋白LFA-1和TCR上的力，支持影响激活的机械串扰 两个分子。有趣的是，此过程对刺激表面的生物物理特征敏感， 包括配体的迁移率和刚度。这些参数在物理上是相关的，因为它们受到调节 在直流成熟期间以优化T细胞启动。进一步的分析表明，这种机制生物学也 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 细胞刚度反应涉及拉伸敏感衔接蛋白CASL的磷酸化。基于 这些发现，我们假设TCR诱导的肌动蛋白聚合可以使细胞感知生物物理提示 由相互作用的APC提供的，启动的机械反馈回路调节力依赖性信号传导 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传导分子。测试 这个假设，我们将执行3个具体目标。首先，我们将确定配体动机如何影响肌动蛋白 动力学和TCR信号。使用刺激性玻璃盖玻片，具有不同活动能力的平面双层 属性和混合迁移率图案表面，我们将询问PMHC的激动剂强度和迁移率如何 复合物和整联蛋白配体会影响肌动蛋白动力学和TCR信号传导。作为该分析的一部分，我们将 使用TCR张力问题来定义如何改变TCR和整联蛋白配体的迁移率如何影响力 由TCR体验。接下来，我们将进行类似的研究，以了解底物刚度如何 影响T细胞激活。我们将刺激T细胞在不同刚度的水凝胶上，并分析效果 关于肌动蛋白动力学，TCR张力和TCR信号传导事件，需要全T细胞激活。最后，我们会的 研究CASL的作用，CASL是一种原型力敏感的信号传导中间体。使用缺乏CASL的T细胞，我们 将研究CASL在T细胞对配体迁移率变化和底物刚度变化的响应过程中的功能。在 此外，我们还将探测导致刚度响应期间CASL磷酸化的信号通路，并且 使用质谱识别相关的结合伙伴。