Structure of Cholesterol/Ceramide Monolayer Mixtures: Implications to the Molecular Organization of Lipid Rafts

Abstract

The structure of monolayers of cholesterol/ceramide mixtures was investigated using grazing incidence x-ray diffraction, immunofluorescence, and atomic force microscopy techniques. Grazing incidence x-ray diffraction measurements showed the existence of a crystalline mixed phase of the two components within a range of compositions of cholesterol/ceramide between 100:0 and 67:33. The mixed phase coexists with the ceramide crystalline phase in the range of compositions between 50:50 and 30:70; between 30:70 and 0:100 only the highly crystalline phase of ceramide was detected. The latter was determined and modeled. Immunolabeling was performed with an antibody specific to the cholesterol monohydrate crystalline arrangement. The antibody recognizes crystalline cholesterol monolayers, but does not interact with crystalline ceramide. Immunofluorescence and atomic force microscopy data show that in uncompressed ceramide monolayers, the highly crystalline phase coexists with a disordered loosely packed phase. In contrast, no disordered phase coexists with the new crystalline mixed phase. We conclude that the new mixed phase represents a stable homogeneous arrangement of cholesterol with ceramide. As ceramide incorporates the lipid backbone common to all sphingolipids, this arrangement may be relevant to the understanding of the molecular organization of lipid rafts.

Revisiting Plant Plasma Membrane Lipids in Tobacco: A Focus on Sphingolipids

“…9C), in good agreement with the values of the interfacial area either calculated or measured by the Langmuir monolayer technique, as reviewed (Deleu et al, 2014). By contrast, phospholipids occupy 95 to 110 Å 2 per molecule in an expanded state and 45 to 55 Å 2 per molecule in a condensed state (Deleu et al, 2001;Eeman et al, 2005), and sterols display an interfacial area of 38 to 48 Å 2 per molecule (Eeman et al, 2005;Scheffer et al, 2005). Predictions based on the geometrical properties of glycosphingolipid molecules indicate that the separation of a glycosphingolipid-rich phase in a phospholipid bilayer would imply a minimization of the interfacial free energy required to accommodate the amphipathic glycosphingolipid in the bilayer.…”

Crystalline Lipid Domains: Characterization by X‐Ray Diffraction and their Relation to Biology

“…We chose to show here, however, cases where significant dissimilarities are found, in order to illustrate their possible significance in terms of the molecular interactions that generate them. Figure 6 shows the d-spacing of monolayers and bilayers composed of SM/Chol, [10d, 19] DPPC/Chol, [9f, g] and Cer/Chol [22] (Cer/Chol bilayer data are taken from Ref. [53]).…”

“…Figure 8 shows Bragg peaks of ceramide and Cer/Chol mixtures with 35-40 mol % ceramide from monolayers, [22] single bilayers, [53] and nonoriented multilayers, that is, multilamellar vesicles (MLVs).…”

Antibody Labeling of Cholesterol/Ceramide Ordered Domains in Cell Membranes

“…This phase is a solid solution of ceramide within the cholesterol structure; each molecule of ceramide displaces two molecules of cholesterol, with subsequent deformation of the cholesterol monolayer lattice. [21] Notwithstanding the similarity in the structural arrangement of the two phases, antibody Cer-Chol 405F interacts selectively with the cholesterol/C 16 -ceramide-mixed monolayer phase, compared to pure cholesterol or ceramide A C H T U N G T R E N N U N G monolayers. [20] When the ceramide content is above 40 %, the A C H T U N G T R E N N U N G mixture undergoes phase separation into a phase that is A C H T U N G T R E N N U N G composed exclusively of ceramide that coexists with the 60:40 mol % cholesterol/C 16 -ceramide phase.…”