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.
Highlights
The cell membrane lipids are not randomly distributed but they form different membrane domains because of specific lipid-lipid and lipid-protein affinities
The Py-met-chol probe has been studied in model and biological membranes [35]
Membrane domains had been studied with the help of numerous fluorescent probes
Summary
The cell membrane lipids are not randomly distributed but they form different membrane domains because of specific lipid-lipid and lipid-protein affinities. Cholesterol-pyrene probe for ordered and disordered membranes functions as suggested by numerous experimental data (for reviews see [1,2,3,4,5]). The modulatory effects of cellular functions by lipid composition and membrane domain separation seem to be directly related to the physical-chemical properties of the different domains such as membrane thickness, lipid compaction and fluidity. Cholesterol is an important regulator and inducer of membrane domains separation. The cholesterol ability to induce the formation of membrane domains is related to its stronger affinity for saturated lipid acyl chains compared to that for non-saturated chains [13,14,15] and by its ability to form an hydrogen bond with the amide group of sphingolipids [5]. Cholesterol is not found exclusively in strong ordered domains but has been observed in Ld domains all over the plasma membrane [5,18,19]
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