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The diversity of the liquid ordered (Lo) phase of phosphatidylcholine/cholesterol membranes: a variable temperature multinuclear solid-state NMR and x-ray diffraction study.

ABSTRACT: To investigate the properties of a pure liquid ordered (Lo) phase in a model membrane system, a series of saturated phosphatidylcholines combined with cholesterol were examined by variable temperature multinuclear (1H, 2H, 13C, 31P) solid-state NMR spectroscopy and x-ray scattering. Compositions with cholesterol concentrations>or=40 mol %, well within the Lo phase region, are shown to exhibit changes in properties as a function of temperature and cholesterol content. The 2H-NMR data of both cholesterol and phospholipids were used to more accurately map the Lo phase boundary. It has been established that the gel-Lo phase coexistence extends to 60 mol % cholesterol and a modified phase diagram is presented. Combined 1H-, 2H-, 13C-NMR, and x-ray scattering data indicate that there are large changes within the Lo phase region, in particular, 1H-magic angle spinning NMR and wide-angle x-ray scattering were used to examine the in-plane intermolecular spacing, which approaches that of a fluid Lalpha phase at high temperature and high cholesterol concentrations. Although it is well known for cholesterol to broaden the gel-to-fluid transition temperature, we have observed, from the 13C magic angle spinning NMR data, that the glycerol region can still undergo a "melting", though this is broadened with increasing cholesterol content and changes with phospholipid chain length. Also from 2H-NMR order parameter data it was observed that the effect of temperature on chain length became smaller with increasing cholesterol content. Finally, from the cholesterol order parameter, it has been previously suggested that it is possible to determine the degree to which cholesterol associates with different phospholipids. However, we have found that by taking into account the relative temperature above the phase boundary this relationship may not be correct.

SUBMITTER: Clarke JA

PROVIDER: S-EPMC1403185 | biostudies-literature | 2006 Apr

REPOSITORIES: biostudies-literature

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The diversity of the liquid ordered (Lo) phase of phosphatidylcholine/cholesterol membranes: a variable temperature multinuclear solid-state NMR and x-ray diffraction study.

Clarke James A JA Heron Andrew J AJ Seddon John M JM Law Robert V RV

Biophysical journal 20060401 7

To investigate the properties of a pure liquid ordered (Lo) phase in a model membrane system, a series of saturated phosphatidylcholines combined with cholesterol were examined by variable temperature multinuclear (1H, 2H, 13C, 31P) solid-state NMR spectroscopy and x-ray scattering. Compositions with cholesterol concentrations>or=40 mol %, well within the Lo phase region, are shown to exhibit changes in properties as a function of temperature and cholesterol content. The 2H-NMR data of both chol ...[more]

PMID: 16537550

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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

The diversity of the liquid ordered (Lo) phase of phosphatidylcholine/cholesterol membranes: a variable temperature multinuclear solid-state NMR and x-ray diffraction study.

Publications

The diversity of the liquid ordered (Lo) phase of phosphatidylcholine/cholesterol membranes: a variable temperature multinuclear solid-state NMR and x-ray diffraction study.

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