Abstract
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.
Highlights
Fluorescent sterols, dehydroergosterol and NBD-cholesterol, were used to examine high density lipoproteinmediated cholesterol uptake and intracellular targeting in L-cell fibroblasts
NBD-cholesterol (NBD-chol) proved to be an alternate fluorescent cholesterol analog to visualize HDL-mediated uptake and intracellular targeting of unesterified cholesterol in L-cell fibroblasts
Based on the above findings, the fluorescence properties of DHE and NBD-chol as well as cultured L-cell fibroblasts, a cell line that grows in serum-free medium [26], were used to (i) compare the uptake and esterification of these sterols in the same cell type, (ii) determine if uptake of either fluorescent sterol was characteristic of HDL receptor mediated uptake, (iii) resolve the specificity of intracellular targeting of fluorescent sterol, and (iv) begin to determine the molecular basis for trafficking of cholesterol to lipid droplets
Summary
(Received for publication, September 24, 1999, and in revised form, January 13, 2000). NBD-cholesterol more selectively probed high density lipoprotein-mediated uptake and rapid intracellular targeting of sterol to lipid droplets. NBD-cholesterol (NBD-chol) proved to be an alternate fluorescent cholesterol analog to visualize HDL-mediated uptake and intracellular targeting of unesterified cholesterol in L-cell fibroblasts. Based on the above findings, the fluorescence properties of DHE and NBD-chol as well as cultured L-cell fibroblasts, a cell line that grows in serum-free medium [26], were used to (i) compare the uptake and esterification of these sterols in the same cell type, (ii) determine if uptake of either fluorescent sterol was characteristic of HDL receptor mediated uptake, (iii) resolve the specificity of intracellular targeting of fluorescent sterol, and (iv) begin to determine the molecular basis for trafficking of cholesterol to lipid droplets
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