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Biophys. J. BioFAST: First Published August 4, 2006. doi:10.1529/biophysj.105.074625
© 2006 by the Biophysical Society.

A more recent version of this article appeared on November 1, 2006.
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SPECTROSCOPY, IMAGING, OTHER TECHNIQUES

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

Yu Ohsugi 1, Kenta Saito 1, Mamoru Tamura 1 and Masataka Kinjo 1*

1 Research Institute for Electronic Science, Hokkaido University

* To whom correspondence should be addressed. E-mail: kinjo{at}imd.es.hokudai.ac.jp.

Submitted on September 16, 2005
Revised on October 18, 2005
Accepted on 20 July 2006


  Abstract
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 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 (EGFP-F), on the plasma membrane 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 (SMT) confirmed that D1 was free diffusion of EGFP-F 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.

Key Words: Diffusion constant, Farnesylated EGFP, Fluorescence correlation spectroscopy, Plasma membrane, Single-molecule tracking, Total internal reflection fluorescence microscopy




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Copyright © 2006 by the Biophysical Society.