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Originally published In Press as doi:10.1194/jlr.M600457-JLR200 on May 7, 2007

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Journal of Lipid Research, Vol. 48, 1689-1700, August 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Interaction of high density lipoprotein particles with membranes containing cholesterol

Susana A. Sanchez1,*, Maria A. Tricerri{dagger} and Enrico Gratton*

* Laboratory for Fluorescence Dynamics, University of California-Irvine, Irvine, CA
{dagger} Instituto de Investigaciones Bioquímicas, Facultad de Ciencias Medicas, Universidad Nacional de La Plata, La Plata, Argentina

Published, JLR Papers in Press, May 7, 2007.

1 To whom correspondence should be addressed. e-mail: susanas{at}uci.edu

In this study, free cholesterol (FC) efflux mediated by human HDL was investigated using fluorescence methodologies. The accessibility of FC to HDL may depend on whether it is located in regions rich in unsaturated phospholipids or in domains containing high levels of FC and sphingomyelin, known as "lipid rafts." Laurdan generalized polarization and two-photon microscopy were used to quantify FC removal from different pools in the bilayer of giant unilamellar vesicles (GUVs). GUVs made of POPC and FC were observed after incubation with reconstituted particles containing apolipoprotein A-I and POPC [78Å diameter reconstituted high density lipoprotein (rHDL)]. Fluorescence correlation spectroscopy data show an increase in rHDL size during the incubation period. GUVs made of two "raft-like" mixtures [DOPC/DPPC/FC (1:1:1) and POPC/SPM/FC (6:1:1)] were used to model liquid-ordered/liquid-disordered phase coexistence. Through these experiments, we conclude that rHDL preferentially removes cholesterol from the more fluid phases. These data, and their extrapolation to in vivo systems, show the significant role that phase separation plays in the regulation of cholesterol homeostasis.

Supplementary key words raft mixtures • giant unilamellar vesicles • two-photon microscopy • 6-lauroyl-2-(dimethylamino) naphthalene

Abbreviations: apoA-I, apolipoprotein A-I; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; FC, free cholesterol; FCS, fluorescence correlation spectroscopy; GP, generalized polarization; GUV, giant unilamellar vesicle; LAURDAN, 6-lauroyl-2-(dimethylamino) naphthalene; lo, ld, liquid-ordered and liquid-disordered phases, respectively; MßCD, methyl-ß-cyclodextrin; rHDL, reconstituted high density lipoprotein; SPM, porcine brain sphingomyelin


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Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.