Measuring αβ T-Cell Receptor-Mediated Mechanosensing Using Optical Tweezers Combined with Fluorescence Imaging.
Measuring αβ T-Cell Receptor-Mediated Mechanosensing Using Optical Tweezers Combined with Fluorescence Imaging.
复制标题
使用光镊结合荧光成像测量αβ T 细胞受体介导的机械传感。
DOI：
10.1007/978-1-0716-2229-2_26
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Lang,MatthewJ
中科院分区：
文献类型：
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作者：
Stephens,HannahM;Brazin,KristineN;Mallis,RobertJ;Feng,Yinnian;Banik,Debasis;Reinherz,EllisL;Lang,MatthewJ
t细胞抗原受体（TCRs）是机械传感器，在配体识别上启动信号级联，导致t细胞分化、稳态、效应和调节功能。光阱与荧光相结合，可以直接监测t细胞在不同浓度的肽结合主要组织相容性复合体分子（pMHC）施加力时的触发。该技术模拟了免疫监视期间细胞在抗原呈递表面爬行时施加的生理剪切力。在单细胞上进行的真正的单分子研究描述了力键寿命，通常被视为捕获键，以及在力加载时细胞表面的TCR-pMHC键的构象变化。激活和单分子单细胞研究共同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。本方法详细说明了试验设计、制备和执行，以及数据分析。这些方法可以应用于广泛的pMHC-TCR相互作用，并具有适应其他受体-配体系统的潜力。
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.