DISTRIBUTION OF A GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEIN AT THE APICAL SURFACE OF MDCK CELLS EXAMINED AT A RESOLUTION OF LESS-THAN-100 ANGSTROM USING IMAGING FLUORESCENCE RESONANCE ENERGY-TRANSFER

Membrane microdomains (''lipid rafts'') enriched in glycosylphosphatid ylinositol (GPI)-anchored proteins, glycosphingolipids, and cholestero l have been implicated in events ranging from membrane trafficking to signal transduction. Although there is biochemical evidence for such m embrane microdomains, they have not been visualized by light or electr on microscopy. To probe for microdomains enriched in GPI-anchored prot eins in intact cell membranes, we used a novel form of digital microsc opy, imaging fluorescence resonance energy transfer (FRET), which exte nds the resolution of fluorescence microscopy to the molecular level ( <100 Angstrom). We detected significant energy transfer between donor- and acceptor-labeled antibodies against the GPI-anchored protein 5' n ucleotidase (5' NT) at the apical membrane of MDCK cells. The efficien cy of energy transfer correlated strongly with the surface density of the acceptor-labeled antibody. The FRET data conformed to theoretical predictions for two-dimensional FRET between randomly distributed mole cules and were inconsistent with a model in which 5' NT is constitutiv ely clustered. Though we cannot completely exclude the possibility tha t some 5' NT is in clusters, the data imply that most 5' NT molecules are randomly distributed across the apical surface of MDCK cells. Thes e findings constrain current models for lipid rafts and the membrane o rganization of GPI-anchored proteins.