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J. Cell Biol.,
Volume 142, Number 1, July 13, 1998 69-84
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.
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