Ju Lining, Chen Yunfeng, Rushdi Muaz Nik, Chen Wei, Zhu Cheng
Heart Research Institute, The University of Sydney, Camperdown, NSW, 2006, Australia.
Charles Perkins Centre, The University of Sydney, Camperdown, NSW, 2006, Australia.
Methods Mol Biol. 2017;1584:231-258. doi: 10.1007/978-1-4939-6881-7_15.
Upon engagement with a specific ligand, a cell surface receptor transduces intracellular signals to activate various cellular functions. This chapter describes a set of biomechanical methods for analyzing the characteristics of cross-junctional receptor-ligand interactions at the surface of living cells. These methods combine the characterization of kinetics of receptor-ligand binding with real-time imaging of intracellular calcium fluxes, which allow researchers to assess how the signal initiated from single receptor-ligand engagement is transduced across the cell membrane. A major application of these methods is the analysis of antigen recognition by triggering of the T cell receptor (TCR). Three related methods are described in this chapter: (1) the micropipette adhesion assay, (2) the biomembrane force probe (BFP) assay, and (3) combining BFP with fluorescence microscopy (fBFP). In all cases, an ultrasoft human red blood cell (RBC) is used as an ultrasensitive mechanical force probe. The micropipette assay detects binding events visually. The BFP uses a high-speed camera and real-time image tracking techniques to measure mechanical variables on a single molecular bond with up to ~1 pN (10 Newton), ~3 nm (10 m), and ~0.5 ms (10 s) in force, spatial, and temporal resolution, respectively. As an upgrade to the BFP, the fBFP simultaneously images binding-triggered intracellular calcium signals on a single live cell. These technologies can be widely used to study other membrane receptor-ligand interactions and signaling under mechanical regulation.
细胞表面受体与特定配体结合后，会转导细胞内信号以激活各种细胞功能。本章介绍了一组生物力学方法，用于分析活细胞表面跨连接受体 - 配体相互作用的特征。这些方法将受体 - 配体结合动力学的表征与细胞内钙通量的实时成像相结合，使研究人员能够评估单个受体 - 配体结合引发的信号如何跨细胞膜转导。这些方法的一个主要应用是通过触发T细胞受体（TCR）来分析抗原识别。本章描述了三种相关方法：（1）微吸管粘附测定法，（2）生物膜力探针（BFP）测定法，以及（3）将BFP与荧光显微镜（fBFP）相结合。在所有情况下，超软人类红细胞（RBC）都用作超灵敏的机械力探针。微吸管测定法通过视觉检测结合事件。BFP使用高速相机和实时图像跟踪技术，分别以高达约1皮牛（10牛顿）的力、约3纳米（10米）的空间分辨率和约0.5毫秒（10秒）的时间分辨率测量单个分子键上的力学变量。作为BFP的升级版，fBFP可在单个活细胞上同时成像结合触发的细胞内钙信号。这些技术可广泛用于研究其他膜受体 - 配体相互作用以及机械调节下的信号传导。
