Abstract：
T-cell antigen receptors (TCRs) are mechanosensors, which initiate a signaling cascade upon ligand recognition resulting in T-cell differentiation, homeostasis, effector and regulatory functions. An optical trap combined with fluorescence permits direct monitoring of T-cell triggering in response to force application at various concentrations of peptide-bound major histocompatibility complex molecules (pMHC). The technique mimics physiological shear forces applied as cells crawl across antigen-presenting surfaces during immune surveillance. True single molecule studies performed on single cells profile force-bond lifetime, typically seen as a catch bond, and conformational change at the TCR-pMHC bond on the surface of the cell upon force loading. Together, activation and single molecule single cell studies provide chemical and physical triggering thresholds as well as insight into catch bond formation and quaternary structural changes of single TCRs. The present methods detail assay design, preparation, and execution, as well as data analysis. These methods may be applied to a wide range of pMHC-TCR interactions and have potential for adaptation to other receptor-ligand systems.
摘要：
T细胞抗原受体（TCR）是机械传感器，在配体识别后启动信号级联，导致T细胞分化，稳态，效应和调节功能。与荧光结合的光学陷阱允许响应于在各种浓度的肽结合的主要组织相容性复合物分子（pMHC）下施加的力而直接监测T细胞触发。该技术模拟在免疫监视期间细胞爬行穿过抗原呈递表面时施加的生理剪切力。对单细胞进行的真正的单分子研究轮廓力键寿命，通常被视为捕获键，在力加载时，细胞表面TCR-pMHC键的构象变化。一起,激活和单分子单细胞研究提供了化学和物理触发阈值，以及对单个TCR的捕获键形成和四级结构变化的见解。本方法详细介绍了测定设计，准备,和执行，以及数据分析。这些方法可以应用于广泛的pMHC-TCR相互作用,并且具有适应其他受体-配体系统的潜力。
