Two-Dimensional Analysis of Cross-Junctional Molecular Interaction by Force Probes.

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 (10-12 Newton), ~3 nm (10-9 m), and ~0.5 ms (10-3 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.