The interfacial elastic packing interactions of different galactosylceramides (GalCers), sphingomyelins (SMs), and phosphatidylcholines (PC) were compared by determining their elastic area compressibility moduli (Cs-1) as a function of lateral packing pressure (pi) in a Langmuir-type film balance. To assess the relative contributions of the lipid headgroups as well as those of the ceramide and diacylglycerol hydrocarbon regions, we synthesized various GalCer and SM species with identical, homogeneous acyl residues and compared their behavior to that of PCs possessing similar hydrocarbon structures. For PCs, this meant that the sn-1 acyl chain was long and saturated (e.g., palmitate) and the sn-2 chain composition was varied to match that of GalCer or SM. When at equivalent pi and in either the chain-disordered (liquid-expanded) or chain-ordered (liquid-condensed) state, GalCer films were less elastic than either SM or PC films. When lipid headgroups were identical (SM and PC), Cs-1 values (at equivalent pi) for chain-disordered SMs, but not chain-ordered SMs, were 25-30% higher than those of PCs. Typical values for fluid phase (liquid-expanded) GalCer at 30 mN/m and 24 degrees C were 158 (+/- 7) mN/m, whereas those of SM were 135 (+/- 7) mN/m and those of PC were 123 (+/- 2) mN/m. Pressure-induced transitions to chain-ordered states (liquid-condensed) resulted in significant increases (two- to fourfold) in the "in-plane" compressibility for all three lipid types. Typical Cs-1 values for chain-ordered GalCers at 30 mN/m and 24 degrees C were between 610 and 650 mN/m, whereas those of SM and of PC were very similar and were between 265 and 300 mN/m. Under fluid phase conditions, the pi-Cs-1 behavior for each lipid type was insensitive to whether the acyl chain was saturated or monounsaturated. Measurement of the Cs-1 values also provided an effective way to evaluate the two-dimensional phase transition region of SMs, GalCers, and PCs. Modest heterogeneity in the acyl composition led to transitional broadening. Our findings provide useful information regarding the in-plane elasticity of lipids that are difficult to investigate by alternative methods, i.e., micropipette aspiration technique. The results also provide insight into the stability of sphingolipid-enriched, membrane microdomains that are thought to play a role in the sorting and trafficking of proteins containing glycosylphosphatidylinositol anchors with cells.

The molecular basis of bilayer tubule formation in hydrated galactosylceramide (GalCer) dispersions has been investigated by synthesizing different chain-pure GalCers and examining their aqueous mesomorphic phase structure by freeze fracture and negative-stain electron microscopy. Thermotropic characterization of the GalCer species by differential scanning calorimetry provided supplementary information that verified the phase state under which morphological observations were carried out. Under aqueous conditions and at room temperature, N-24:1 delta 15(cis) GalSph, the predominant monounsaturated, nonhydroxy acyl species of bovine brain GalCer (NFA-GalCer), formed cylindrical mesomorphic self-assemblies consisting almost exclusively of "nanotubes," i.e., lipid bilayer tubules of relatively uniform length and diameter (length, 250-400 nm; diameter, 25-30 nm). In contrast, N-24:0 GalSph, the major saturated, nonhydroxy acyl species of bovine brain GalCer, displayed no tendency to form these relatively small "nanotubes." Rather, N-24:0 GalSph formed larger, variable-length ribbon-like structures (length, 5,000-10,000 nm) that often appeared to undulate and, occasionally, appeared to be helically twisted. Interestingly, bovine brain GalCer, which contains high levels of the N-24:1 delta 15(cis) and N-24:0 species as well as 2-hydroxy acyl chains, formed multilamellar liposomes of variable size and showed little tendency to form cylindrical structures. This result suggested that changes to the polar interface/headgroup region imparted by the 2-hydroxy acyl species strongly influenced bilayer tubule and cylinder formation in GalCer. To define this influence more clearly, other sphingoid-based and glycerol-based lipids were investigated. Morphological characterization of N-24:1 delta 15(cis) sphingosylphosphorylcholine (24:1 SM) revealed no evidence of bilayer cylinder or tubule formation. Similar results were obtained with aqueous dispersions of 1-palmitoyl-2-nervonoyl phosphatidylcholine (16:0, 24:1 PC). Hence, the bulkier, more hydrated, zwitterionic phosphocholine headgroup inhibited the formation of bilayer nanotubes and cylinders under physiological saline conditions.

The interfacial interactions occurring between cholesterol and either galactosylceramides (GalCers) or sphingomyelins (SMs) with identical acyl chains have been investigated using Langmuir film balance techniques. Included among the synthesized GalCers and SMs were species containing palmitoyl (16:0), stearoyl (18:0), oleoyl [18:1 ∆9(c) ], nervonoyl [24:1 ∆15(c) ], or linoleoyl [18:2 ∆9,12(c) ] acyl residues. The cholesterol-induced condensations in the average molecular areas of the monolayers were determined by classic mean molecular area vs composition plots as well as by expressing the changes in terms of sphingolipid cross-sectional area reduction over the surface pressure range from 1 to 40 mN/m (at 1 mN/m intervals). The results show that, at surface pressures approximating bilayer conditions (30 mN/m), acyl heterogeneity in naturally occurring SMs (bovine or egg SM) enhanced the area condensation induced by cholesterol compared to their predominant molecular species (e.g. 18:0 SM in bovine SM; 16:0 SM in egg SM). Nonetheless, cholesterol always had a greater condensing effect on SM compared to GalCer when these sphingolipids were acyl chain matched and in similar phase states (prior to mixing with cholesterol). Also, the cholesterol-induced area changes for a given sphingolipid type (e.g. SM or GalCer) were similar whether the acyl chains were saturated, cis-∆9-monounsaturated, or cis-∆9,12-diunsaturated if the sphingolipids were in similar phase states (prior to mixing with cholesterol) and compared at equivalent surface pressures. These results indicate that, under conditions where hydrocarbon structure is matched, the sphingolipid head group plays a dominant role in determining the extent to which cholesterol reduces sphingolipid cross-sectional area. Despite the larger cholesterol-induced area condensations observed in SMs compared to those in GalCers, the molecular-packing densities showed that equimolar GalCercholesterol films were generally packed as tight as or slightly tighter than those of the SMcholesterol films. The results are discussed in terms of a molecular model for sphingolipidcholesterol interactions. Our findings also not only raise questions as to whether cholesterolinduced condensation data provide a reliable measure of the affinity, i.e. interaction strength, between cholesterol and different lipids but also provide insight regarding the stability of sterol/ sphingolipid-rich microdomains thought to exist in caveolae and other cell membrane regions. † This investigation was supported by USPHS Grant GM45928 (R.E.B.) and the Hormel Foundation. The automated Langmuir film balance used in this study receives major support from USPHS Grants HL49180 and HL17371 (H. L. Brockman, P. I.).