Distribution of a glycosylphosphatidylinositol-anchored protein at the apical surface of MDCK cells examined at a resolution of <100 A using imaging fluorescence resonance energy transfer.

摘要: Membrane microdomains (“lipid rafts”) enriched in glycosylphosphatidylinositol (GPI)-anchored proteins, glycosphingolipids, and cholesterol have been implicated events ranging from membrane trafficking to signal transduction. Although there is biochemical evidence for such microdomains, they not visualized by light or electron microscopy. To probe GPI- anchored proteins intact cell membranes, we used a novel form of digital microscopy, imaging fluorescence resonance energy transfer (FRET), which extends the resolution microscopy molecular level (<100 Å). We detected significant between donor- acceptor-labeled antibodies against GPI-anchored protein 5′ nucleotidase (5′ NT) at apical MDCK cells. The efficiency correlated strongly with surface density antibody. FRET data conformed theoretical predictions two-dimensional randomly distributed molecules were inconsistent model NT constitutively clustered. Though cannot completely exclude possibility that some clusters, imply most are across These findings constrain current models lipid rafts organization proteins.