Sphingolipid-Cholesterol Rafts Diffuse as Small Entities in the Plasma Membrane of Mammalian Cells

doi:10.1083/jcb.148.5.997

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© The Rockefeller University Press, 0021-9525/2000//997 $5.00
The Journal of Cell Biology, Volume 148, Number 5, , 2000 997-1008

Original Article

Sphingolipid–Cholesterol Rafts Diffuse as Small Entities in the Plasma Membrane of Mammalian Cells

A. Pralle^a, P. Keller^a, E.-L. Florin^a, K. Simons^a, and J.K.H. Hörber^a

^a Cell Biology and Biophysics, European Molecular Biology Laboratory, D-69117 Heidelberg, Germany
Cell Biology and Biophysics, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.49-6221-38730649-6221-387569

horber{at}embl-heidelberg.de

To probe the dynamics and size of lipid rafts in the membraneof living cells, the local diffusion of single membrane proteinswas measured. A laser trap was used to confine the motion ofa bead bound to a raft protein to a small area (diam $≤$ 100 nm)and to measure its local diffusion by high resolution singleparticle tracking. Using protein constructs with identical ectodomainsand different membrane regions and vice versa, we demonstratethat this method provides the viscous damping of the membranedomain in the lipid bilayer. When glycosylphosphatidylinositol(GPI) -anchored and transmembrane proteins are raft-associated,their diffusion becomes independent of the type of membraneanchor and is significantly reduced compared with that of nonrafttransmembrane proteins. Cholesterol depletion accelerates thediffusion of raft-associated proteins for transmembrane raftproteins to the level of transmembrane nonraft proteins andfor GPI-anchored proteins even further. Raft-associated GPI-anchoredproteins were never observed to dissociate from the raft withinthe measurement intervals of up to 10 min. The measurementsagree with lipid rafts being cholesterol-stabilized complexesof 26 ± 13 nm in size diffusing as one entity for minutes.

Key Words: laser trap • lipid raft • protein diffusion • single particle tracking • thermal position fluctuation analysis

Abbreviations used in this paper: DIG, detergent insoluble glycolipid-enrichedcomplex; GFP, green fluorescent protein; GPI, glycosylphosphatidylinositol;HA, influenza virus hemagglutinin; LFPGT46, artificial transmembraneYFP; PLAP, placental alkaline phosphatase; SPT, single particletracking; TfR, transferrin receptor; TPF, two-photon fluorescence;YFP, yellow color variant of green fluorescent protein; YFPGLGPI,artificial GPI-anchored YFP.

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