Transient confinement of a glycosylphosphatidylinositol-anchored protein in the plasma membrane

Glycosylphosphatidylinositol (GPI)-anchored proteins participate in many cell surface functions; however, the molecular associations of these lipid- linked proteins within the plasma membrane are not well understood. Recent biochemical analyses of detergent insoluble membrane fractions have suggested that GPI-anchored proteins may be associated with glycosphingolipid (GSL)- enriched domains that also contain cholesterol and signaling molecules such as Src family kinases and, in some cases, caveolae. The movements of two components of the putative GSL-enriched domains, Thy-1, a GPI-anchored protein, and GMI, a GSL, were followed with single particle tracking on C3H 10T1/2 cell surfaces and categorized into four modes of lateral transport, fast diffusion, slow anomalous diffusion, diffusion confined to 325-370 nm diameter regions, and a fraction of molecules that was essentially stationary on the 6.6 s time scale. Longer observations (60 s) showed that Thy-1 and GM1 are transiently confined for 7-9 s to regions averaging 260-330 nm in diameter. Approximately 35-37% of both Thy-1 and GM1 undergo confined diffusion, whereas only 16% of fluorescein phosphatidylethanolamine, a phospholipid analog which is not expected to be found in the GSL domains, experience confined diffusion to regions averaging ~230 nm in diameter. Further, when glycosphingolipid expression was reduced ~40% with the glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoylamino-3- morpholino-1-propanol, the percentage of trajectories exhibiting confinement and the size of the confining domain for Thy-1 were reduced ~1.5-fold. In contrast, extraction of cells with Triton X-100 leaves the fraction of molecules confined and the domain sizes of Thy-1 and GM1 unchanged. Our results are consistent with the preferential association of GPI-anchored proteins with glycosphingolipid-enriched domains and suggest that the confining domains may be the in vivo equivalent of the detergent insoluble membrane fractions.