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J Biol Chem, Vol. 275, Issue 17, 12769-12780, April 28, 2000
From the Fluorescent sterols, dehydroergosterol and
NBD-cholesterol, were used to examine high density lipoprotein-mediated
cholesterol uptake and intracellular targeting in L-cell fibroblasts.
The uptake, but not esterification or targeting to lipid droplets, of
these sterols differed >100-fold, suggesting significant differences in uptake pathways. NBD-cholesterol uptake kinetics and
lipoprotein specificity reflected high density lipoprotein-mediated
sterol uptake via the scavenger receptor B1. Fluorescence energy
transfer showed an average intermolecular distance of 26 Å between the two fluorescent sterols in L-cells. Indirect
immunofluorescence revealed that both fluorescent sterols localized to
L-cell lipid droplets, the surface of which contained adipose
differentiation-related protein. This lipid droplet-specific protein
specifically bound NBD-cholesterol with high affinity
(Kd = 2 nM) at a single site. Thus,
NBD-cholesterol and dehydroergosterol were useful fluorescent probes of
sterol uptake and intracellular sterol targeting. NBD-cholesterol more
selectively probed high density lipoprotein-mediated uptake and
rapid intracellular targeting of sterol to lipid droplets. Targeting of
sterol to lipid droplets was correlated with the presence of adipose
differentiation related protein, a lipid droplet-specific protein shown
for the first time to bind unesterified sterol with high affinity.
High Density Lipoprotein-mediated Cholesterol Uptake and
Targeting to Lipid Droplets in Intact L-cell Fibroblasts
A SINGLE- AND MULTIPHOTON FLUORESCENCE APPROACH*
,
,
Department of Pathobiology, the
§ Department of Physiology and Pharmacology, Texas A & M
University, Texas Veterinary Medical Center, College Station, Texas
77843-4466, the ¶ Department of Mechanical Engineering,
Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and the Laboratory for Fluorescence Dynamics, Department of
Physics, University of Illinois, Urbana, Illinois 61801, and the
** Department of Pharmaceutical Sciences, University of Maryland School
of Pharmacy, Baltimore, Maryland 21201-1180
*
This work was supported in part by United States Public
Health Service, National Institutes of Health Grants GM 31561 (to F. S.), 5P41RR03155 (to E. G.), and DK41463 and American
Heart Association Grant 9951222U (to G. S.). These data were
presented in part at the 43rd Annual Meeting of the
Biophysical Society (Frolov, A., Petrescu, A., Atshaves, B. P.,
So, P. T. C., Gratton, E., Serrero, G., and Schroeder, F. (1999) Biophys. J. 76, A99, poster 397).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. Tel: 409-862-1433;
Fax: 409-862-4929; E-mail: fschroeder@cvm.tamu.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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