Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells

To probe the dynamics and size of lipid rafts in the membrane of living cel ls, the local diffusion of single membrane proteins was measured. A laser t rap was used to confine the motion of a bead bound to a raft protein to a s mall area (diam less than or equal to 100 nm) and to measure its local diff usion 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 membran e domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anc hored and transmembrane proteins are raft-associated, their diffusion becom es 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 ra ft proteins to the level of transmembrane nonraft proteins and for GPI-anch ored proteins even further. Raft-associated GPI-anchored proteins were neve r observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-sta bilized complexes of 26 +/- 13 nm in size diffusing as one entity for minut es.