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Biophys. J. BioFAST: First Published May 27, 2005. doi:10.1529/biophysj.104.055640
© 2005 by the Biophysical Society.

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CELL BIOPHYSICS

Ras diffusion is sensitive to plasma membrane viscosity

J. Shawn Goodwin 1, Kimberly R. Drake 1, Catha L. Remmert 1 and Anne K. Kenworthy 2*

1 Vanderbilt University
2 Vanderbilt University School of Medicine

* To whom correspondence should be addressed. E-mail: anne.kenworthy{at}vanderbilt.edu.

Submitted on November 3, 2004
Revised on December 16, 2004
Accepted on 17 May 2005


  Abstract
The cell surface contains a variety of barriers and obstacles that slow the lateral diffusion of glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins below the theoretical limit imposed by membrane viscosity. How the diffusion of proteins residing exclusively on the inner leaflet of the plasma membrane is regulated has been largely unexplored. We show here that the diffusion of the small GTPase Ras is sensitive to the viscosity of the plasma membrane. Using confocal fluorescence recovery after photobleaching (FRAP), we examined the diffusion of GFP-tagged HRas, NRas, and KRas in COS-7 cells loaded with or depleted of cholesterol, a well-known modulator of membrane bilayer viscosity. In cells loaded with excess cholesterol, the diffusional mobilities of GFP-HRas, GFP-NRas, and GFP-KRas were significantly reduced, paralleling the behavior of the viscosity-sensitive lipid probes DiIC16 and DiIC18. However, the effects of cholesterol depletion on protein and lipid diffusion in cell membranes were highly dependent on the depletion method used. Cholesterol depletion with M<=CD slowed Ras diffusion by a viscosity-independent mechanism, while overnight cholesterol depletion slightly increased both protein and lipid diffusion. The ability of Ras to sense membrane viscosity may represent a general feature of protein residents of the cytoplasmic face of the plasma membrane.

Key Words: DiI, FRAP, cholesterol, lateral mobility, membrane microdomains




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Copyright © 2005 by the Biophysical Society.