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J. Biol. Chem., Vol. 278, Issue 8, 6384-6403, February 21, 2003
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From the Department of Physiology and Pharmacology, Texas A & M
University, Texas Veterinary Medical Center,
College Station, Texas 77843-4466
Although cholesterol is an essential component of
mammalian membranes, resolution of cholesterol organization in
membranes and organelles (i.e. lysosomes) of living cells
is hampered by the paucity of nondestructive, nonperturbing methods
providing real time structural information. Advantage was taken of the
fact that the emission maxima of a naturally occurring fluorescent sterol (dehydroergosterol) were resolvable into two structural forms,
monomeric (356 and 375 nm) and crystalline (403 and 426 nm). Model
membranes (sterol:phospholipid ratios in the physiological range,
e.g. 0.5-1.0), subcellular membrane fractions (plasma
membranes, lysosomal membranes, microsomes, and mitochondrial
membranes), and lipid rafts/caveolae (plasma membrane cholesterol-rich
microdomain purified by a nondetergent method) contained primarily
monomeric sterol and only small quantities (i.e. 1-5%) of
the crystalline form. In contrast, the majority of sterol in isolated
lysosomes was crystalline. However, addition of sterol carrier
protein-2 in vitro significantly reduced the proportion of
crystalline dehydroergosterol in the isolated lysosomes. Multiphoton
laser scanning microscopy (MPLSM) of living L-cell fibroblasts cultured
with dehydroergosterol for the first time provided real time images
showing the presence of monomeric sterol in plasma membranes, as well
as other intracellular membrane structures of living cells.
Furthermore, MPLSM confirmed that crystalline sterol colocalized in
highest amounts with LysoTracker Green, a lysosomal marker dye.
Although crystalline sterol was also detected in the cytoplasm, the
extralysosomal crystalline sterol did not colocalize with BODIPY FL
C5-ceramide, a Golgi marker, and crystals were not
associated with the cell surface membrane. These noninvasive,
nonperturbing methods demonstrated for the first time that multiple
structural forms of sterol normally occurred within membranes, membrane
microdomains (lipid rafts/caveolae), and intracellular organelles of
living cells, both in vitro and visualized in real time by
MPLSM.
Fluorescence and Multiphoton Imaging Resolve Unique Structural
Forms of Sterol in Membranes of Living Cells*
*
This work was supported in part by United States Public
Health Service Grant GM31651 from the National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Physiology
and Pharmacology, Texas A & M University, TVMC, College Station, TX 77843-4466. Tel.: 979-862-1433; Fax: 979-862-4929; E-mail: Fschroeder@cvm.tamu.edu.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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