Abstract
Protein-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.
Highlights
Protein-lipid interactions in cellular membranes modulate central cellular functions, are often transient in character, but occur too intermittently to be readily observable
Interactions can be studied via altered diffusion behavior of the interacting molecules in the membranes using several different fluorescence techniques, including fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), and single-particle tracking (SPT)
We show that the transient state kinetics of NBD, when labelled to lipids in the membranes of small unilamellar vesicles (SUVs) and live cells, are strongly influenced by presence of spin-labels in the same membranes
Summary
Protein-lipid interactions in cellular membranes modulate central cellular functions, are often transient in character, but occur too intermittently to be readily observable. Fluorescence methods offer a very high sensitivity and high time resolution compared to other biophysical techniques used to study biological membranes, such as NMR, EPR and FTIR, and allow readouts in systems of varying complexity, up to the level of living cells and tissue[10] For these reasons, fluorescence methods are the most extensively used for dynamics and interaction studies of GPCRs and other membrane proteins. In contrast to FCS no particular time resolution in the fluorescence detection is required, and since TRAST measurements do not require single-molecule detection conditions, they can be applied to monitor long-lived dark transient states of fluorescent molecules in a wide range of biological samples[29,30]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
