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

PLoS One. 2015 Apr 15;10(4):e0121563. doi: 10.1371/journal.pone.0121563. eCollection 2015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Transport
  • Caco-2 Cells
  • Cell Membrane / chemistry
  • Cell Membrane / metabolism
  • Cholesterol / analogs & derivatives
  • Cholesterol / chemistry
  • Cholesterol / metabolism*
  • Humans
  • Lipoproteins, HDL / chemistry
  • Lipoproteins, HDL / metabolism*
  • Lipoproteins, LDL / chemistry
  • Lipoproteins, LDL / metabolism*
  • Pyrenes / chemistry

Substances

  • Lipoproteins, HDL
  • Lipoproteins, LDL
  • Pyrenes
  • Cholesterol
  • pyrene

Grants and funding

This work was supported by grants from Institut National de la Santé Et de la Recherche Médicale, from Agence Nationale de la Recherche (06-PNRA 5.34 “Absinte”), and from Vaincre La Mucoviscidose (FC0906). XC was a recipient of a “Contrat d’Interface” from Centre Hospitalo-Universitaire de Toulouse; GG was a recipient of a “Prix de Recherche 2008” from Société Française de Nutrition. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.