Imaging of intermittent lipid-receptor interactions reflects changes in live cell membranes upon agonist-receptor binding

Abstract

AbstractProtein-lipid interactions in cellular membranes modulate central cellular functions, are often transient in character, but occur too intermittently to be readily observable. We introduce transient state imaging (TRAST), combining sensitive fluorescence detection of fluorophore markers with monitoring of their dark triplet state transitions, allowing imaging of such protein-lipid interactions. We first determined the dark state kinetics of the biomembrane fluorophore 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD) in lipid vesicles, and how its triplet state is quenched by spin-labels in the same membranes. We then monitored collisional quenching of NBD-lipid derivatives by spin-labelled stearic acids in live cell plasma membranes, and of NBD-lipid derivatives by spin-labelled G-Protein Coupled Receptors (GPCRs). We could then resolve transient interactions between the GPCRs and different lipids, how these interactions changed upon GPCR activation, thereby demonstrating a widely applicable means to image and characterize transient molecular interactions in live cell membranes in general, not within reach via traditional fluorescence readouts.