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A correction to this article has been published: J. Cell Biol. 142 (3) 883
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© The Rockefeller University Press, 0021-9525/1998//69 $5.00
The Journal of Cell Biology, Volume 142, Number 1, , 1998 69-84

Articles

Distribution of a Glycosylphosphatidylinositol-anchored Protein at the Apical Surface of MDCK Cells Examined at a Resolution of <100 Å Using Imaging Fluorescence Resonance Energy Transfer



A.K. Kenworthy and M. Edidin

Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218

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

Key Words: cell membrane • cell polarity • 5' nucleotidase • fluorescence microscopy • membrane lipids

Abbreviations used in this paper: 5' NT, 5' nucleotidase; cA, reduced acceptor surface density; cD, reduced donor surface density; D:A, molar donor/acceptor ratio; E, energy transfer efficiency; Eclustered, theoretical energy transfer efficiency for clustered molecules; Emixture, theoretical energy transfer efficiency for a mixture of randomly distributed and clustered molecules; Erandom, theoretical energy transfer efficiency for randomly distributed molecules; fA, mole fraction of acceptors; fD, mole fraction of donors; fU, mole fraction of unlabeled molecules; fclustered, fraction of clustered molecules; frandom, fraction of randomly distributed molecules; Fab, monovalent antibody fragment; FRET, fluorescence resonance energy transfer; GPI, glycosylphosphatidylinositol; GSL, glycosphingolipids.

Address all correspondence to A.K. Kenworthy, Department of Biology, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218. Tel.: (410) 516-7295. Fax: (410) 516-5213. E-mail: kenwrthy{at}jhuvms.hcf.jhu.edu


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