Lateral mobility of membrane-binding proteins in living cells measured by total internal reflection fluorescence correlation spectroscopy

Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) allows us to measure diffusion constants and the number of fluorescent molecules in a small area of an evanescent field generated on the objective of a microscope. The application of TIR- FCS makes possible the characterization of reversible association and dissociation rates between fluorescent ligands and their receptors in supported phospholipid bilayers. Here, for the first time, we extend TIR- FCS to a cellular application for measuring the lateral diffusion of a membrane-binding fluorescent protein, farnesylated EGFP, on the plasma membranes of cultured HeLa and COS7 cells. We detected two kinds of diffusional motion-fast three-dimensional diffusion (D-1) and much slower two-dimensional diffusion (D-2), simultaneously. Conventional FCS and single-molecule tracking confirmated that D-1 was free diffusion of farnesylated EGFP close to the plasma membrane in cytosol and D-2 was lateral diffusion in the plasma membrane. These results suggest that TIR-FCS is a powerful technique to monitor movement of membrane-localized molecules and membrane dynamics in living cells.