Specific cellular incorporation of a pyrene-labelled cholesterol: lipoprotein-mediated delivery toward ordered intracellular membranes.

Gérald Gaibelet (1, 2) , Sophie Allart (3) , François Tercé (4) , Vincent Azalbert (4) , Justine Bertrand-Michel (4) , Safouane Hamdi (4) , Xavier Collet (4) , Stéphane Orlowski (1, 2, 4)

1 I2BC - Institut de Biologie Intégrative de la Cellule
2 IBITECS - Institut de Biologie et de Technologies de Saclay
3 CPTP - Centre de Physiopathologie Toulouse Purpan
4 I2MC - Institut des Maladies Métaboliques et Cardiovasculaires

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.

Domaines: Sciences du Vivant [q-bio]

Gérald Gaibelet, Sophie Allart, François Tercé, Vincent Azalbert, Justine Bertrand-Michel, et al.. Specific cellular incorporation of a pyrene-labelled cholesterol: lipoprotein-mediated delivery toward ordered intracellular membranes.. PLoS ONE, 2015, 10 (4), pp.e0121563. ⟨10.1371/journal.pone.0121563⟩. ⟨hal-01447879⟩