High density lipoprotein-mediated cholesterol uptake and targeting to lipid droplets in intact L-cell fibroblasts: a single- and multiphoton fluorescence approach

• Published in: The Journal of biological chemistry 2000-04, Vol.275 (17), p.12769-12780
• Main Authors: Frolov, A, Petrescu, A, Atshaves, B.P, So, P.T.C, Gratton, E, Serrero, G, Schroeder, F
• Format: Article
• Language: English
• Subjects: 4-Chloro-7-nitrobenzofurazan - analogs & derivatives; 4-Chloro-7-nitrobenzofurazan - metabolism; Animals; binding proteins; Caveolin 1; Caveolins; Cell Line; cell lines; cholesterol; Cholesterol - analogs & derivatives; Cholesterol - metabolism; Cholesterol - pharmacokinetics; Chromatography, High Pressure Liquid; cytochemistry; dehydroergosterol; Dose-Response Relationship, Drug; Ergosterol - analogs & derivatives; Ergosterol - metabolism; fibroblasts; Fibroblasts - metabolism; fluorescence; fluorescent antibody technique; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes - metabolism; high density lipoprotein; humans; Kinetics; lipid bodies; lipid droplets; Lipoproteins, HDL - metabolism; Membrane Proteins - biosynthesis; Membrane Proteins - metabolism; Mice; Microscopy, Confocal - methods; Perilipin-2; Photons; receptors; Receptors, Immunologic - biosynthesis; Receptors, Lipoprotein; Receptors, Scavenger; Recombinant Proteins - metabolism; Scavenger Receptors, Class B; Spectrometry, Fluorescence - methods; sterols; Time Factors; uptake
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.17.12769

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.