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.
Highlights
Cholesterol is an essential component of mammalian cells, mainly present in the plasma membrane, and distributed in various intracellular compartments
We show here that (i) Pyr-met-Chol and NBD-Chol display clearcut differences for their cellular uptake processes, (ii) scavenger receptor class B type I (SR-BI) is involved in Pyr-met-Chol, but not NBD-Chol, incorporation in Caco-2 cells from biliary micelles and in PC-3 cells from high density lipoproteins (HDL), (iii) Pyr-met-Chol, stably associated with HDL and low density lipoprotein (LDL) in contrast to NBD-Chol, can be delivered to PC-3 cells harboring the corresponding lipoprotein receptors, and (iv) Pyr-met-Chol incorporation leads to an intracellular staining of membrane compartments characterized by their cholesterol richness and their ordered structure, as revealed by pyrene excimer fluorescence emission
NBD-Chol has been reported on various cell systems to be esterified by Acyl-CoA:cholesterol acyltransferase (ACAT), even more efficiently than cholesterol, and embedded into lipid bodies, leading to a huge fluorescence emission enhancement due to the hydrophobic environment of the NBD fluorophore in the core of these lipid bodies [14,15,16]
Summary
Cholesterol is an essential component of mammalian cells, mainly present in the plasma membrane, and distributed in various intracellular compartments. It is both involved in membrane lipid composition, with a structural role and functional modulation of many membrane proteins, and in various cellular metabolic pathways and regulations. Cholesterol is provided both by synthesis in peripheral tissues and by absorption through the enterocytes in the intestine. Cellular exchanges of cholesterol with the external medium are mediated by lipoproteins, which act as “donor” and “acceptor” particles, ensuring a global regulation through its handling in the systemic circulation. In some pathophysiological situations, the regulation of cholesterol metabolism and traffic is altered [4,5]
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