Coexistence of immiscible mixtures of palmitoylsphingomyelin and palmitoylceramide in monolayers and bilayers

Abstract

A combination of lipid monolayer- and bilayer-based model systems has been applied to explore in detail the interactions between and organization of palmitoylsphingomyelin (pSM) and the related lipid palmitoylceramide (pCer). Langmuir balance measurements of the binary mixture reveal favorable interactions between the lipid molecules. A thermodynamically stable point is observed in the range approximately 30-40 mol % pCer. The pSM monolayer undergoes hyperpolarization and condensation with small concentrations of pCer, narrowing the liquid-expanded (LE) to liquid-condensed (LC) pSM main phase transition by inducing intermolecular interactions and chain ordering. Beyond this point, the phase diagram no longer reveals the presence of the pSM-enriched phase. Differential scanning calorimetry (DSC) of multilamellar vesicles reveals a widening of the pSM main gel-fluid phase transition (41 degrees C) upon pCer incorporation, with formation of a further endotherm at higher temperatures that can be deconvoluted into two components. DSC data reflect the presence of pCer-enriched domains coexisting, in different proportions, with a pSM-enriched phase. The pSM-enriched phase is no longer detected in DSC thermograms containing >30 mol % pCer. Direct domain visualization has been carried out by fluorescence techniques on both lipid model systems. Epifluorescence microscopy of mixed monolayers at low pCer content shows concentration-dependent, morphologically different pCer-enriched LC domain formation over a pSM-enriched LE phase, in which pCer content close to 5 and 30 mol % can be determined for the LE and LC phases, respectively. In addition, fluorescence confocal microscopy of giant vesicles further confirms the formation of segregated pCer-enriched lipid domains. Vesicles cannot form at >40 mol % pCer content. Altogether, the presence of at least two immiscible phase-segregated pSM-pCer mixtures of different compositions is proposed at high pSM content. A condensed phase (with domains segregated from the liquid-expanded phase) showing enhanced thermodynamic stability occurs near a compositional ratio of 2:1 (pSM/pCer). These observations become significant on the basis of the ceramide-induced microdomain aggregation and platform formation upon sphingomyelinase enzymatic activity on cellular membranes.

Figure 1

Compression isotherms of pSM-pCer mixed monolayers. (A) Surface pressure versus molecular area of representative isotherms for pure pSM (thin solid line), pure pCer (thick solid line), and pSM-pCer mixtures at 5 mol % (thin short-dashed line), 10 mol % (thin dotted line), 25 mol % (thin dot-dashed line), 33 mol % (thin double-dot-dashed line), 40 mol % (thin long-dashed line), 50 mol % (thick short-dashed line), and 75 mol % (thick dotted line) of pCer. The arrow indicates the LE-LC phase transition pressure for pure pSM. (B) Compressibility modulus (κ) versus molecular area for the mixed monolayers indicated in A. The inset shows the dependence of the pure pCer compression isotherm and compressibility modulus on molecular area (note the expanded molecular area scale). Arrowhead indicates LC-LC rearrangement.

Figure 2

Condensation of pSM induced by the presence of pCer. (A) Compression isotherms of pure pSM (solid line) and pure pCer (dashed line) and the extrapolated molecular area for pSM in a condensed phase at low surface pressures (straight gray line). (B–D) Variation of mean molecular area of the pSM-pCer mixture with pCer mole fractions at 5, 10, and 35 mN/m, respectively. Solid lines represent the molecular area for an ideal pSM-pCer mixture. Dashed gray lines represent the molecular area for the ideal pSM (in LC phase)-pCer mixture.

Figure 3

Surface pressure versus pCer mole fraction phase diagram for pSM-pCer monolayers. LE-LC (solid triangles), LC-LC (open triangles), and LC-to-collapsed phase (CP) (solid circles) transition pressures. Error bars represent the mean ± SE, and the lines are provided as guides to the eye only. The shaded zone indicates the range of composition in which the mixed monolayer undergoes a behavior change.

Figure 4

Surface potential/unit molecular surface density (ΔV/n) in pSM-pCer mixed monolayers. (A) ΔV/n versus molecular area for pure pSM (1), pure pCer (7), and pSM-pCer mixtures at 5 mol % (2), 10 mol % (3), 25 mol % (4), 50 mol % (5), and 75 mol % (6) pCer. (B and C) Variation of ΔV/n of pSM-pCer mixtures with pCer mole fraction at 10 and 35 mN/m, respectively. Solid lines represent the molecular area for an ideal pSM-pCer mixture.

Figure 5

Excess mixing free energy for the pSM-pCer mixture. ΔG excess was calculated as the difference in the work of compression (from the area below the compression isotherm curve) between the experimental and theoretical isotherms for ideally mixed pSM-pCer monolayers. Error bars represent the mean ± SE of duplicated experiments.

Figure 6

Epifluorescence micrographs of pSM-pCer monolayers. The figure shows images of pure pSM (A) and mixed pSM-pCer monolayers containing 5 mol % (B), 10 mol % (C), and 25 mol % (D) pCer at 10 mN/m. The monolayers were doped with 0.5 mol % of the fluorescent probe DiI-C₁₈. All images are 331 × 263 μm in size. (E and F) Surface dependency of the LC (dark, probe-depleted) area extent and the pCer mole fraction present in the LE phase (bright, probe-enriched), respectively. The data were calculated from images of mixed monolayers containing 5 mol % (solid circles), 10 mol % (open circles), and 20 mol % pCer (gray circles). Error bars represent the mean ± SE values for E and the propagation error for the calculated values in F.

Figure 7

DSC of pSM-pCer vesicles. Representative thermograms for pSM vesicles with increasing proportions of pCer (in mol %). Dotted lines correspond to deconvoluted endotherms.

Figure 8

Confocal microscopy of DiIC₁₈-stained pSM GUVs in the presence of increasing proportions of pCer at room temperature. Bright and dark areas represent probe-enriched (pSM-enriched) and probe-depleted (pCer-enriched) phases, respectively. Scale bars, 10 μm.