High density lipoprotein-mediated cholesterol uptake and targeting to lipid droplets in intact L-cell fibroblasts. A single- and multiphoton fluorescence approach.
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 A 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 (K(d) = 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.
Frolov
A
A
Department of Pathobiology, Texas A & M University, Texas Veterinary Medical Center, College Station, Texas 77843-4466, USA.
Petrescu
A
A
Atshaves
B P
BP
So
P T
PT
Gratton
E
E
Serrero
G
G
Schroeder
F
F
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D051242:Caveolin 1; D022461:Caveolins; D002460:Cell Line; D002784:Cholesterol / Q000031:analogs & derivatives / Q000378:metabolism / Q000493:pharmacokinetics*; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004875:Ergosterol / Q000031:analogs & derivatives / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D000070780:Perilipin-2; D017785:Photons; D011971:Receptors, Immunologic / Q000096:biosynthesis; D018110:Receptors, Lipoprotein; D051116:Receptors, Scavenger; D011994:Recombinant Proteins / Q000378:metabolism; D051122:Scavenger Receptors, Class B; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors
C077527:7-nitrobenz-2-oxa-1,3-diazol-4-ylcholesterol; D051242:Caveolin 1; D022461:Caveolins; D005456:Fluorescent Dyes; D008075:Lipoproteins, HDL; D008565:Membrane Proteins; D000070780:Perilipin-2; C000602983:Plin2 protein, mouse; D011971:Receptors, Immunologic; D018110:Receptors, Lipoprotein; D051116:Receptors, Scavenger; D011994:Recombinant Proteins; C454739:Scarb1 protein, mouse; D051122:Scavenger Receptors, Class B; C010905:dehydroergosterol; D002784:Cholesterol; D009327:4-Chloro-7-nitrobenzofurazan; D004875:Ergosterol