Optical Tweezers and Fluorescence Recovery After Photo-Bleaching to Measure Molecular Interactions at the Cell Surface

Abstract

This paper describes applications of two optical microscopy techniques, namely laser tweezers and fluorescence recovery after photo-bleaching (FRAP), to the measurement of ligand/receptor/cytoskeleton interactions. These methods are used in combination with ligand-coated microspheres, binding to specific membrane receptors at the dorsal cell surface. A first application exploits the possibility to impose piconewton forces on microspheres. At low ligand density, one can identify the rupture of individual bonds between ligand/receptor complexes and the motile actin cytoskeleton. The second application uses control of the initial contact time by optical tweezers, together with time lapse imaging of GFP-tagged receptor accumulation around the microspheres. Quantification of fluorescence enrichment together with a simple chemical reaction model allows characterization of global ligand/receptor on-rates. These contacts eventually reach steady-state corresponding to continuous formation and dissociation of ligand/receptor bonds. FRAP is then used to probe the equilibrium dynamics of this system, by photo-bleaching GFP-tagged receptors recruited at ligand-coated beads. The recovery curves are fitted by a diffusion/reaction model, to yield turnover rates between adhesion proteins. To illustrate the potential of these techniques, we take examples from our previously published data on neuronal adhesion proteins involved in growth cone locomotion, in particular N-cadherin and immunoglobulin cell adhesion molecules.