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

Abstract

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.

Dual Biomembrane Force Probe Enables Single-Cell Mechanical Analysis of Signal Crosstalk between Multiple Molecular Species

“…LFA-1 dependent lymphocyte adhesion strengthening under antigen or chemokine receptor stimulation has been well demonstrated in population assays, i.e., static cell adhesion assay or flow cytometric analysis with antibodies recognizing activated LFA-1 4,5 . To provide a contrast, we first analyzed the dual receptor crosstalk between TCR and LFA-1 by the published adhesion frequency assay using the existing single-receptor force probe technique 30,31 . A naïve OT1 CD8 + T cell aspirated by LP1 was brought into repeated contact with a RBC (serving as a force probe) coated with a mixture of pMHC and ICAM-1 held by piezo-controlled RP1 ( Fig.…”

“…Ligands or antibodies were coated on glass beads as previously described 13,30,47 . Briefly, proteins were first covalently modified with maleimide-PEG3500-NHS (molecular weight ~3,500 Da; JenKem, TX) in carbonate/bicarbonate buffer (pH 8.5).…”

“…were isolated from venous whole blood of healthy volunteers by a certified venesectionist according to protocols approved by the Institutional Review Board of Georgia Institute of Technology as previously described 13,30 . All human donor blood samples were obtained with informed consent and all experiments were performed in accordance with relevant guidelines and regulations.…”

“…To make adhesion assay probe for dRMP experiment (Fig. 2), RBCs were washed and biotinylated with Biotin-X-NHS 30,48 . To make force probe in dBFP assay (Figs 3 and 4), RBCs were washed, biotinylated with Biotin-PEG3500-NHS (JenKem, TX), swollen with nystatin and stored (up to 2 weeks) for experiments 13,30 .…”

“…3D,E). To observe intraplatelet Ca 2+ signals triggered by GPIb mechanoreception, the platelets were preloaded with a fluorescent dye (Fura-2 AM) and the simultaneous Ca 2+ ratiometric imaging optics were integrated as previously described 13,24,30 . To separate engagements of the two receptors in time, we first drove the platelet to repeatedly touch Probe 1 coated with A1 (or BSA as a control) (Fig.…”