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Cholesterol-pyrene as a probe for cholesterol distribution on ordered and disordered membranes: Determination of spectral wavelengths.

ABSTRACT: Biological membranes contain a large variety of lipids species compartmentalized in different domains heterogeneous in size, composition and dynamics. Cholesterol induces membrane ordered domains thanks to its affinity for saturated lipids. Membrane domains had been studied with fluorescent probes either linked to phospholipids and proteins or as individual fluorophore. However, no efficient formulation of a cholesterol probe has been available so far. Herein, we described a cholesterol-pyrene probe behaviour in heterogeneous membranes. We characterised the pyrene fluorescence spectra in liquid-ordered (Lo) and liquid-disordered (Ld) membranes. Using statistical multivariate analysis, we found out the most appropriate wavelengths for membrane domains studies. 373 nm and 379 nm were the most discriminant wavelengths to follow the liquid-ordered and the liquid-disordered environments. Cholesterol clustering behaviour was quantified by the modulation of the cholesterol-pyrene excimers peak (474 nm). In liquid-ordered membranes at low temperature, cholesterol-pyrene was found as multimers and as monomers. At high temperature, the liquid-ordered status of the membrane decreases and cholesterol-pyrene tends to cluster. In liquid-disordered membranes, cholesterol-pyrene was present mostly as monomers and the small quantity of excimers increased with temperature. Cholesterol-pyrene was used to test the ceramide effect on membranes, and presented a behaviour in agreement with the cholesterol behaviour reported in the literature. Overall, the presented data show that cholesterol-pyrene is an efficient sensor to study liquid ordered and liquid disordered organisation in membranes.

SUBMITTER: Almeida C

PROVIDER: S-EPMC6086420 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Cholesterol-pyrene as a probe for cholesterol distribution on ordered and disordered membranes: Determination of spectral wavelengths.

Almeida Claudia C De Wreede Anaëlle A Lamazière Antonin A Ayala-Sanmartin Jesus J

PloS one 20180810 8

Biological membranes contain a large variety of lipids species compartmentalized in different domains heterogeneous in size, composition and dynamics. Cholesterol induces membrane ordered domains thanks to its affinity for saturated lipids. Membrane domains had been studied with fluorescent probes either linked to phospholipids and proteins or as individual fluorophore. However, no efficient formulation of a cholesterol probe has been available so far. Herein, we described a cholesterol-pyrene p ...[more]

PMID: 30096186

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Project description: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.

Dataset Information

Cholesterol-pyrene as a probe for cholesterol distribution on ordered and disordered membranes: Determination of spectral wavelengths.

Publications

Cholesterol-pyrene as a probe for cholesterol distribution on ordered and disordered membranes: Determination of spectral wavelengths.

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