Background

Traditional biochemical methods, such as co-immunoprecipitation, surface plasmon resonance, bio-layer interferometry, or isothermal titration calorimetry, are widely employed to determine the kinetics of receptor–ligand interactions (Cole et al., 2007; Hui et al., 2017; Marasco et al., 2020; Maruhashi et al., 2022). These binding kinetics—on-rate k_on, off-rate k_off, and equilibrium dissociation constant K_D—are detected at a static equilibrium state in solution. Remarkably, however, transmembrane receptors are restricted to the plasma membrane of the cells. Their anchor pattern, orientation, and diffusion rate on the plasma membrane can directly tune the accessibility and association possibility to affect in situ kinetics of receptor–ligand interactions (Dustin et al., 2001; Huang et al., 2004; Hu et al., 2019; Zhang et al., 2021). Therefore, the determination of in situ receptor–ligand binding kinetics on living cells is urgent for understanding the biological functions induced by transmembrane receptor–ligand interactions.

Transmembrane receptors and their ligands also experience mechanical forces on the plasma membrane, which mainly derive from living cell’s movement, cell–cell contact, and dynamic changes to the plasma membrane or cytoplasmic cytoskeleton (Zhu et al., 2019). Many studies have shown that mechanical forces can act on transmembrane receptors and ligands to dynamically regulate their binding kinetics by altering their conformations. For example, SARS-CoV-2 viral invasion induces host cell membrane bending, which generates the tensile force to prolong the bond lifetime of spike/ACE2 interaction, fostering viral entry (Hu et al., 2021). Also, mechanical forces directly enhance the interaction of T-cell receptors (TCR) and agonist peptide-loaded major histocompatibility complex, which can accurately determine TCR antigen recognition (Liu et al., 2014; Wu et al., 2019). From the molecular mechanism, the mechanical force could allosterically regulate the conformation of the transmembrane receptors and ligands, generate force-induced intermediate binding states, and govern dissociation pathway selection to impede their dissociation (Sarangapani et al., 2004; Hong et al., 2015; Wu et al., 2019; Zhu et al., 2019). Also, a sliding-rebinding mechanism is applied to explain force-enhanced transmembrane receptor–ligand binding strength (Lou and Zhu, 2007).

Single-molecule force spectroscopy techniques—such as atomic force microscopy, optical tweezers, magnetic tweezers, or biomembrane force probe (BFP)—have become important for studying the force-dependent kinetics of receptor–ligand interactions (Marshall et al., 2003; Kim et al., 2010; Chen and Zhu, 2013; Kong et al., 2013). Among these, BFP can quantitatively and accurately determine the force-dependent bond lifetime of transmembrane receptor–ligand interactions at the single-molecule level on living cells. The BFP technique uses soft human red blood cells (RBC, which have a proper elasticity for pN-level force detection) attached to microbeads as force sensors (Figure 1). When the transmembrane receptor expressed on the target cell binds to recombinant ligand–coated microbeads, the magnitude of the mechanical force is calculated according to the deformation of RBC, and the bond lifetime is recorded by a high-speed camera. The stiffness, spatial resolution, force, and bond lifetime range of BFP are 0.1–3 pN/nm, 2–3 nm, 1–10³ pN, and 5 × 10^-4–200 s, respectively (Evans et al., 1995; Chen et al., 2008; An et al., 2020). Therefore, BFP has the advantages of ultra-high mechanical detection accuracy of optical tweezers and magnetic tweezers, and also the high dynamic response characteristics of atomic force microscopy, being very suitable for studying transient and weak interactions between transmembrane receptors and their ligands in situ.

Figure 1. Schematic diagram of BFP setup. The left micropipette holds the force probe, which contains a soft RBC attached to a ligand-coated bead. The right micropipette aspirates a transmembrane receptor-expressing target cell.