High Density Lipoprotein-mediated Cholesterol Uptake and Targeting to Lipid Droplets in Intact L-cell Fibroblasts A SINGLE- AND MULTIPHOTON FLUORESCENCE APPROACH* Academic Article uri icon

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.

author list (cited authors)

  • Frolov, A., Petrescu, A., Atshaves, B. P., So, P., Gratton, E., Serrero, G., & Schroeder, F.

citation count

  • 104

complete list of authors

  • Frolov, A||Petrescu, A||Atshaves, BP||So, PT||Gratton, E||Serrero, G||Schroeder, F

publication date

  • April 2000