open access

https://hdl.handle.net/2115/16923

Lateral Mobility of Membrane-Binding Proteins in Living Cells Measured by Total Internal Reflection Fluorescence Correlation Spectroscopy

英語

Single-molecule tracking

2006-11

The Biophysical Society

Biophysical Journal

91(9)

3456-3464

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 (D1) and much slower two-dimensional diffusion (D2), simultaneously. Conventional FCS and single-molecule tracking confirmed that D1 was free diffusion of farnesylated EGFP close to the plasma membrane in cytosol and D2 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.

学術雑誌論文

Accepted Manuscript

0006-3495

1542-0086