Absence of Fluid-Ordered/Fluid-Disordered Phase Coexistence in Ceramide/POPC Mixtures Containing Cholesterol

Abstract

The effect of temperature on the lateral structure of lipid bilayers composed of porcine brain ceramide and 1-palmitoyl 2-oleoyl-phosphatidylcholine (POPC), with and without addition of cholesterol, were studied using differential scanning calorimetry, Fourier transformed infrared spectroscopy, atomic force microscopy, and confocal/two-photon excitation fluorescence microscopy (which included LAURDAN generalized polarization function images). A broad gel/fluid phase coexistence temperature regime, characterized by the presence of micrometer-sized gel-phase domains with stripe and flowerlike shapes, was observed for different POPC/ceramide mixtures (up to approximately 25 mol % ceramide). This observed phase coexistence scenario is in contrast to that reported previously for this mixture, where absence of gel/fluid phase coexistence was claimed using bulk LAURDAN generalized polarization (GP) measurements. We demonstrate that this apparent discrepancy (based on the direct comparison between the LAURDAN GP data obtained in the microscope and the fluorometer) disappears when the additive property of the LAURDAN GP function is taken into account to examine the data obtained using bulk fluorescence measurements. Addition of cholesterol to the POPC/ceramide mixtures shows a gradual transition from a gel/fluid to gel/liquid-ordered phase coexistence scenario as indicated by the different experimental techniques used in our experiments. This last result suggests the absence of fluid-ordered/fluid-disordered phase coexistence in the ternary mixtures studied in contrast to that observed at similar molar concentrations with other ceramide-base-containing lipid mixtures (such as POPC/sphingomyelin/cholesterol, which is used as a canonical raft model membrane). Additionally, we observe a critical cholesterol concentration in the ternary mixtures that generates a peculiar lateral pattern characterized by the observation of three distinct regions in the membrane.

Enzymatic generation of ceramide induces membrane restructuring: Correlated AFM and fluorescence imaging of supported bilayers

“…The importance of ceramide in modulating membrane physical properties and biological function has prompted a number of studies of ceramide incorporation in model membranes. Several recent investigations have examined the effects of ceramide incorporation on coexisting ordered and fluid domains in phase separated binary and ternary lipid mixtures that are used to model membrane rafts Fidorra et al, 2006;Silva et al, 2007;Sot et al, 2006Sot et al, , 2008. This work has demonstrated that ceramide leads to increased membrane heterogeneity and that its effects depend on the membrane composition.…”

“…For example, treating phosphatidylcholine/SM vesicles with SMase produced ceramide patches that coalesced into larger domains and then formed vesicles (Holopainen et al, 2000;Nurminen et al, 2002). Several other studies have provided evidence for ceramide-enriched domains in monolayers (Fanani et al, 2002;Hartel et al, 2005), in fluid phospholipid vesicles (Taniguchi et al, 2006) and in giant vesicles prepared from SM/ceramide or ceramide/POPC/cholesterol mixtures (Fidorra et al, 2006;Sot et al, 2006). These studies in monolayers and vesicles indicate that enzymatic generation of ceramide leads to more complex changes in membrane morphology than does direct ceramide incorporation in the same lipid membranes.…”

Hallmarks of Reversible Phase Separation in Living, Unperturbed Cell Membranes

“…Complementary GUV imaging techniques, such as fluorescence lifetime imaging microscopy (FLIM) or the use of environmental probes such as Laurdan can also be useful in identifying phases. [55][56][57] Complementary techniques using geometries other than GUVs (e.g. nuclear magnetic resonance or electron paramagnetic resonance on multilamellar vesicles) produce excellent determinations of the lipid ratios and temperatures at which all three phases coexist.…”

Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting ordered-disordered and gel-fluid domains

“…GUVs were prepared using the electroformation method developed by Angelova et al ( 19 ), using a specialized chamber supplied by L. A. Bagatolli (Odense, Denmark) that allows direct visualization under the microscope ( 53 ). Stock solutions of lipids (0.2 mg/ml total lipid containing 0.2 mol% DiI) were prepared in a chloroform-methanol (9:1, v/v) solution.…”