Ceramide-Induced Lamellar Gel Phases in Fluid Cell Lipid Extracts

Abstract

The effects of increasing amounts of palmitoylceramide (pCer) on human red blood cell lipid membranes have been studied using atomic force microscopy of supported lipid bilayers, in both imaging (bilayer thickness) and force-spectroscopy (nanomechanical resistance) modes. Membranes appeared homogeneous with pCer concentrations up to 10 mol % because of the high concentration of cholesterol (Chol) present in the membrane (∼45 mol %). However, the presence of pCer at 30 mol % gave rise to a clearly distinguishable segregated phase with a nanomechanical resistance 7-fold higher than the continuous phase. These experiments were validated using differential scanning calorimetry. Furthermore, Chol depletion of the bilayers caused lipid domain generation in the originally homogeneous samples, and Chol-depleted domain stiffness significantly increased with higher amounts of pCer. These results point to the possibility of different kinds of transient and noncompositionally constant, complex gel-like phases present in RBC lipid membranes rich in both pCer and Chol, in contrast to the widespread opinion about the displacements between pCer-enriched "gel-like" domains and liquid-ordered "raft-like" Chol-enriched phases. Changes in the biophysical properties of these complex gel-like phases governed by local modulation of pCer:Chol ratios could be a cell mechanism for fine-tuning the properties of membranes as required.

Pb(II) Induces Scramblase Activation and Ceramide-Domain Generation in Red Blood Cells

“…It has been observed in many systems, including RBC lipids 24 , 29 , that Cer can give rise to rigid domains in the RBC membranes. In order to confirm that this was the case with our samples, RBC were incubated overnight with Pb(II) and the corresponding lipid extracts dispersed in buffer and analysed by differential scanning calorimetry.…”

“…The generation of non-purely ceramidic gel domains under saturating conditions is also possible and gel phases containing both ceramide and cholesterol can appear if both lipids are saturating the membrane 27 , 28 which could be the case for RBC membranes under Pb 2+ stress. A recent study from our laboratory demonstrated that RBC lipid extracts in the presence of high concentrations of ceramide exhibit lamellar gel domains enriched in both cholesterol and ceramide 29 .…”

Young modulus of supported lipid membranes containing milk sphingomyelin in the gel, fluid or liquid-ordered phase, determined using AFM force spectroscopy

“…On the other hand, interfacial elasticity has been shown to be lowest for the Lo phase, when compared to that of liquid-condensed (~gel) or liquid-expansed (~fluid) phases of sphingomyelin monolayers [75]. In bilayers made of red blood cell lipid extract, García-Arribas et al [76] measured as low breakthrough forces as those of a fluid phase, even though the bilayers were expected to be in Lo phase due to their cholesterol content.…”

“…Meanwhile, the human erythrocyte membrane contains up to 45 mol% of cholesterol [82,83]. Even though the distribution and fluxes of cholesterol in cells are complex [84], this indicates that gel phase is probably inhibited in many biologically relevant situations, even though indirect observations pointed to some gel phase domains in the MFGM [15], not to mention the role of other compounds like ceramides [76]. The high amounts of cholesterol in plasmatic membranes, compared to those used in model vesicle or supported membranes, may account for the lower Young modulus values generally found when directly probing cells or milk fat globules.…”

“…The high amounts of cholesterol in plasmatic membranes, compared to those used in model vesicle or supported membranes, may account for the lower Young modulus values generally found when directly probing cells or milk fat globules. Phase diagrams [85] or nanomechanical data [76] point to the complex, and sometimes unexpected, behavior of polar lipid mixtures with biological composition. In the same line, milk SM vesicles are significantly less stiff that vesicles of the single sphingomyelins, probably as a result of disorder in the packing of milk SM [86].…”