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Biophys. J. BioFAST: First Published February 26, 2007. doi:10.1529/biophysj.106.095398
© 2007 by the Biophysical Society.


A more recent version of this article appeared on May 15, 2007.
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CELL BIOPHYSICS

(Un)confined Diffusion of CD59 in the Plasma Membrane determined by High-resolution Single Molecule Microscopy

Stefan Wieser 1, Manuel Moertelmaier 1, Elke Fürtbauer 2, Hannes Stockinger 2 and Gerhard J. Schütz 1*

1 Johannes Kepler University Linz
2 Medical University of Vienna

* To whom correspondence should be addressed. E-mail: gerhard.schuetz{at}jku.at.

Submitted on August 16, 2006
Revised on October 8, 2006
Accepted on 22 January 2007


   Abstract
There has been emerging interest whether plasma membrane constituents are moving according to free Brownian motion or hop diffusion. In the latter model, lipids, lipid-anchored proteins and transmembrane proteins would be transiently confined to periodic corrals in the cell membrane, which are structured by the underlying membrane skeleton. As this model is based exclusively on results provided by one experimental strategy - high resolution single particle tracking -, we attempted in this study to confirm or amend it using a complementary technique. We developed a novel strategy which employs single molecule fluorescence microscopy to detect confinements to free diffusion of CD59 - a GPI-anchored protein - in the plasma membrane of living T24 (ECV) cells. With this method, minimum invasive labeling via fluorescent Fab fragments was sufficient to measure the lateral motion of individual protein molecules on a millisecond time scale, yielding a positional accuracy down to 22 nm. While no hop diffusion was directly observable, based on a full analytical description our results provide upper boundaries for confinement size and strength.

Key Words: confinement strength, fluorescence, hop diffusion, localization precision, positional accuracy




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Copyright © 2007 by the Biophysical Society.