Measuring Ligand-Receptor Binding Rates with K-Space Image Correlation Spectroscopy: Theory and Experimental Applications

Abstract

The ability to measure accurate kinetic binding rates of ligand-receptor and receptor-receptor interactions is important in order to study the triggering of signaling events in cell membranes. We apply an image analysis technique, k-space image correlation spectroscopy (kICS), to measure the kinetic binding rates of ligand-receptor interactions from fluorescence video-microscopy data occurring within a thin plane of illumination. We consider two different kinds of experiments where: (1) fluorescently tagged ligands in solution bind and unbind dynamically to unlabeled receptors which are allowed to diffuse in the imaging plane and, (2) fluorescently tagged, diffusing receptors in the imaging plane interact with other (possibly non-fluorescing) receptors and interconvert between two diffusive states. For system (1), by measuring image intensity fluctuations of fluorescently labeled human doublecortin proteins in solution (2.5 nM), binding to microtubules adhering on a sample substrate, the kICS method accurately determines kinetic dissociation rates (kd = 1.6 ± 0.2 s−1) as well as diffusion coefficients (0.38 ± 0.19 μm2/s) of the proteins consistent with single-particle tracking data. For system (2), we perform drug-trial experiments on living COS-7 cells to show that kICS can quantify the degree to which the drugs will affect kinetic binding/unbinding rates. By labeling the glycolipid membrane receptor GM1 with a fluorescent cholera toxin B-subunit, we show that cytoskeleton perturbations with the drug cytochalasin D result in significant changes (ku = 0.013 ± 0.004 s−1 to 0.08 ± 0.02 s−1) to the undocking rate of GM1 to the actin cytoskeleton. With these two proof-of-concept experiments, we pave the way to using kICS as an efficient and reliable method to elucidate kinetic binding rates and transport dynamics parameters for studies in biomembranes.