title: Specific cellular incorporation of a pyrene-labelled cholesterol: lipoprotein-mediated delivery toward ordered intracellular membranes.
abstract: In the aim of testing tools for tracing cell trafficking of exogenous cholesterol, two fluorescent derivatives of cholesterol, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and 21-methylpyrenyl-cholesterol (Pyr-met-Chol), with distinctive chemico-physical characteristics, have been compared for their cell incorporation properties, using two cell models differently handling cholesterol, with two incorporation routes. In the Caco-2 cell model, the cholesterol probes were delivered in bile salt micelles, as a model of intestinal absorption. The two probes displayed contrasting behaviors for cell uptake characteristics, cell staining, and efflux kinetics. In particular, Pyr-met-Chol cell incorporation involved SR-BI, while that of NBD-Chol appeared purely passive. In the PC-3 cell model, which overexpresses lipoprotein receptors, the cholesterol probes were delivered via the serum components, as a model of systemic delivery. We showed that Pyr-met-Chol-labelled purified LDL or HDL were able to specifically deliver Pyr-met-Chol to the PC-3 cells, while NBD-Chol incorporation was independent of lipoproteins. Observations by fluorescence microscopy evidenced that, while NBD-Chol readily stained the cytosolic lipid droplets, Pyr-met-Chol labelling led to the intense staining of intracellular structures of membranous nature, in agreement with the absence of detectable esterification of Pyr-met-Chol. A 48 h incubation of PC-3 cells with either Pyr-met-Chol-labelled LDL or HDL gave same staining patterns, mainly colocalizing with Lamp1, caveolin-1 and CD63. These data indicated convergent trafficking downwards their respective receptors, LDL-R and SR-BI, toward the cholesterol-rich internal membrane compartments, late endosomes and multivesicular bodies. Interestingly, Pyr-met-Chol staining of these structures exhibited a high excimer fluorescence emission, revealing their ordered membrane environment, and indicating that Pyr-met-Chol behaves as a fair cholesterol tracer regarding its preferential incorporation into cholesterol-rich domains. We conclude that, while NBD-Chol is a valuable marker of cholesterol esterification, Pyr-met-Chol is a reliable new lipoprotein fluorescent marker which allows to probe specific intracellular trafficking of cholesterol-rich membranes.
authors: INSERM U563/1048, CHU Purpan, 31024, Toulouse, cedex 3, France; CEA, SB2SM and UMR8221/UMR9198 CNRS, I2BC, IBiTec-Saclay, 91191, Gif-sur-Yvette, cedex, France; Université Toulouse III, UMR 1048, F-31000, Toulouse, France.
authors: Gérald
authors: G
authors: Gaibelet
authors: Université Toulouse III, UMR 1048, F-31000, Toulouse, France; Plateau technique d'Imagerie Cellulaire, INSERM U1043, F-31300, Toulouse, France.
authors: Sophie
authors: S
authors: Allart
authors: Université Toulouse III, UMR 1048, F-31000, Toulouse, France; INSERM U1048, F-31400, Toulouse, France.
authors: François
authors: F
authors: Tercé
authors: Université Toulouse III, UMR 1048, F-31000, Toulouse, France; INSERM U1048, F-31400, Toulouse, France.
authors: Vincent
authors: V
authors: Azalbert
authors: Université Toulouse III, UMR 1048, F-31000, Toulouse, France; INSERM U1048, Lipidomic Platform Metatoul, F-31400, Toulouse, France.
authors: Justine
authors: J
authors: Bertrand-Michel
authors: INSERM U563/1048, CHU Purpan, 31024, Toulouse, cedex 3, France.
authors: Safouane
authors: S
authors: Hamdi
authors: Université Toulouse III, UMR 1048, F-31000, Toulouse, France; INSERM U1048, F-31400, Toulouse, France.
authors: Xavier
authors: X
authors: Collet
authors: INSERM U563/1048, CHU Purpan, 31024, Toulouse, cedex 3, France; CEA, SB2SM and UMR8221/UMR9198 CNRS, I2BC, IBiTec-Saclay, 91191, Gif-sur-Yvette, cedex, France.
authors: Stéphane
authors: S
authors: Orlowski
mesh_terms: D001692:Biological Transport; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002784:Cholesterol / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008075:Lipoproteins, HDL / Q000737:chemistry / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000737:chemistry / Q000378:metabolism*; D011721:Pyrenes / Q000737:chemistry
chemical_list: D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D011721:Pyrenes; D002784:Cholesterol; C030984:pyrene