Measuring Receptor–Ligand Binding Kinetics on Cell Surfaces: From Adhesion Frequency to Thermal Fluctuation Methods
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Interactions between surface-anchored receptors and ligands mediate cell–cell and cell–environment communications in many biological processes. Molecular interactions across two apposing cell membrane are governed by two-dimensional (2D) kinetics, which are physically distinct from and biologically more relevant than three-dimensional (3D) kinetics with at least one interacting molecular species in the fluid phase. Here we review two assays for measuring 2D binding kinetics: the adhesion frequency assay and the thermal fluctuation assay. The former measures the binding frequency as a function of contact duration and extracts the force-free 2D kinetics parameters by nonlinearly fitting the data with a probabilistic model. The latter detects bond formation/dissociation by monitoring the reduction/resumption of thermal fluctuations of a force sensor. Both assays are mechanically based and operate at the level of mostly single molecular interaction, which requires ultrasensitive force techniques. Characterization of one such technique, the biomembrane force probe, is presented.
KeywordsAdhesion frequency assay Thermal fluctuation assay Micropipette Biomembrane force probe Kinetics Receptor–ligand interaction
We thank our coworkers of references Chen et al. 1 and Huang et al. 5 who contributed the original data that are discussed here. This work was supported by National Institutes of Health Grants AI38282, AI44902, and HL091020. W.C. is a Predoctoral Fellowship recipient of the American Heart Association (Greater Southeast Affiliate).
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