Mechanobiology of [alpha][beta]TCRs

摘要 项目1研究了受体在体外的单分子和单细胞检测中的功能 多肽/MHC(PMHC)配体在体内对力依赖性机械传感器的激活作用 与相应的逆转录基因T细胞进行检测，以解决诸如幼稚T细胞等生物学后果 扩增和记忆T细胞的形成和维持。因此，项目1将TcR分子偶联 机制与功能生物学。从系统的角度来看，项目1与前TCR的项目2相联系 (PT)，在成熟的TCR上执行并行测试。还将测量单个单元格的参数 激活包括激活阈值、激活的力范围和细胞内钙离子分布。项目1 与核磁共振和项目3紧密集成，结构和构象因此发生变化 的突变(加强、减弱、替换)将被定义并映射到测量到的变化中 参数。在单分子水平上，构象变化(S)，力键寿命，延伸幅度， 将测量临界力和过渡能量格局。在单细胞级别，物理和 将描述触发的化学要求(临界力窗口和pMHC密度)。TCR还将 在活体(幼龄小鼠)中评估其在感染后诱导T细胞增殖的能力 病原体，如甲型流感及其形成中枢或组织驻留记忆T细胞的能力。单细胞 将在临界力阈值触发，然后通过微吸管吸入恢复 转录组分析。首要目标是配对目标1的单分子和单细胞测量。 具有AIM的功能生物学影响2.战略上，该项目将执行一轮测量 涉及单分子单细胞、功能生物学、利用核磁共振和分子的结构生物学 动力学允许改进我们的模型并确认或拒绝我们的原子论假设 解释TCR机械生物学。

ABSTRACT Project 1 investigates TCR function spanning in vitro single molecule and single cell assays that interrogate force-dependent mechanosensor activation by peptide/MHC (pMHC) ligands with optical tweezers to in vivo assays with corresponding retrogenic  T cells addressing biological consequences such as naïve T cell expansion and memory T cell formation and maintenance. Thus, Project 1 couples TCR molecular mechanism with functional biology. From a systems perspective, Project 1 links with Project 2 of the preTCR (pT) by executing parallel tests on the mature TCR. Parameters will also be measured for single cell activation including the activation threshold, force range for activation and intracellular calcium profile. Project 1 is tightly integrated with NMR and Project 3, where structural and conformational changes as a consequence of mutations (strengthening, weakening, substitutions) will be defined and mapped onto changes in measured parameters. At the single molecule level, conformational change(s), force bond lifetime, extension magnitude, critical force and transition energy landscape will be measured. At the single cell level, the physical and chemical requirements to trigger (critical force window and pMHC density) will be characterized. TCRs will also be evaluated in vivo (naïve mouse) for their ability to induce T cell proliferation upon exposure to infectious pathogens such as influenza A and their ability to form central or tissue resident memory T cells. Single cells will be triggered at critical force thresholds and then recovered through micropipette aspiration for transcriptome analysis. The overarching goal is to pair single molecule and single cell measurements of Aim 1 with functional biological impact of Aim 2. Strategically, the project will execute a cycle of measurements involving single molecule single cell, functional biology, structure biology utilizing NMR and molecular dynamics to permit refinement of our models and confirmation or rejection of our atomistic hypotheses to explain TCR mechanobiology.