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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
horber{at}embl-heidelberg.de
To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured. A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam
100 nm) and to measure its local diffusion by high resolution single particle tracking. Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further. Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-stabilized complexes of 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
© 2000 The Rockefeller University Press
Abbreviations used in this paper: DIG, detergent insoluble glycolipid-enriched complex; GFP, green fluorescent protein; GPI, glycosylphosphatidylinositol; HA, influenza virus hemagglutinin; LFPGT46, artificial transmembrane YFP; PLAP, placental alkaline phosphatase; SPT, single particle tracking; TfR, transferrin receptor; TPF, two-photon fluorescence; YFP, yellow color variant of green fluorescent protein; YFPGLGPI, artificial GPI-anchored YFP.
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