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Comparative Study
. 2003 Oct;85(4):2406-16.
doi: 10.1016/S0006-3495(03)74664-5.

Sphingomyelin/phosphatidylcholine/cholesterol phase diagram: boundaries and composition of lipid rafts

Rodrigo F M de Almeida  1 , Aleksandre Fedorov, Manuel Prieto
Affiliations
Comparative Study

Sphingomyelin/phosphatidylcholine/cholesterol phase diagram: boundaries and composition of lipid rafts

Rodrigo F M de Almeida et al. Biophys J. 2003 Oct.

Abstract

The ternary system palmitoylsphingomyelin (PSM)/palmitoyloleoylphosphatidylcholine (POPC)/cholesterol is used to model lipid rafts. The phase behavior of the three binary systems PSM/POPC, PSM/cholesterol, and POPC/cholesterol is first experimentally determined. Phase coexistence boundaries are then determined for ternary mixtures at room temperature (23 degrees C) and the ternary phase diagram at that temperature is obtained. From the diagram at 23 degrees C and the binary phase diagrams, a reasonable expectation is drawn for the ternary phase diagram at 37 degrees C. Several photophysical methodologies are employed that do not involve detergent extraction, in addition to literature data (e.g., differential scanning calorimetry) and thermodynamic rules. For the ternary phase diagrams, some tie-lines are calculated, including the one that contains the PSM/POPC/ cholesterol 1:1:1 mixture, which is often used in model raft studies. The diagrams here described are used to rationalize literature results, some of them apparently discrepant, and to discuss lipid rafts within the framework of liquid-ordered/liquid-disordered phase coexistence.

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Figures

FIGURE 1
FIGURE 1
Determination of solid ordered (so)/liquid disordered (ld) phase coexistence boundaries. Steady-state anisotropy of DPH (〈r〉DPH) as a function of temperature for MLV composed of 100% PSM (circles), 75 mol % PSM/25 mol % POPC (diamonds), and 100% POPC (squares).
FIGURE 2
FIGURE 2
Phase diagram for PSM/POPC MLV. Experimental data are shown as circles. The dotted line is hypothetical. The point for pure POPC is from Curatolo et al. (1985). It is considered that there are one ld phase and two so phases (so1 rich in POPC and so2 rich in PSM).
FIGURE 3
FIGURE 3
Determination of ld/liquid ordered (lo) phase coexistence boundaries for the PSM/cholesterol (chol) lipid mixtures at 50°C determined from the 〈r〉DPH (A). The three straight lines distinguish the three regimes: only ld phase for low chol mol fraction (xchol), ld/lo coexistence for intermediate xchol, and only lo phase for high xchol. The amplitudes of the fluorescence decay components of DPH (short component, diamonds: (0.4 ± 0.1) ns; intermediate component, squares: (3.4 ± 1.2) ns; long component, triangles: (9.6 ± 1.4) ns) are also shown (B) together with the phase boundaries determined from the anisotropy data. DPH mean fluorescence lifetime versus chol mol fraction (C) is fairly constant.
FIGURE 4
FIGURE 4
PSM/chol phase diagram. The points shown were determined from 〈r〉DPH (circles), DPH quenching by 5-NS (squares), and literature (diamonds, see text for details). Between the dotted lines is the regime of strong 〈r〉DPH variation for pure PSM.
FIGURE 5
FIGURE 5
POPC/chol phase diagram. The experimental points were determined from 〈r〉DPH and t-PnA lifetime.
FIGURE 6
FIGURE 6
Determination of so/lo phase coexistence boundaries. The lipid mixtures contain 15 mol % of POPC and varying proportions of PSM and chol, at 23°C. The method is based on the quenching of DPH fluorescence by 5-NS observed as a decrease in the lifetime of the fluorophore. The dotted lines separate the high quenching efficiency (one phase (so for low xchol, lo for high xchol)) from the low quenching efficiency (so/lo coexistence and segregation of probe and quencher, for intermediate xchol).
FIGURE 7
FIGURE 7
PSM/POPC/chol phase diagram at 23°C (A) and at 37°C (B). The circles are experimental points. The red (quasi) tie-line on the tie-triangle describes the lo/ld composition at the right of which there is also so phase. The blue tie-lines are the interval for the possible tie-lines that contain the 1:1:1 composition. The purple point marks the 1:1:1 composition and the green point marks the 2:1:1 composition. The phase boundaries for the diagram at 37°C are dashed lines to highlight the fact that no experimental determinations were performed for ternary mixtures at that temperature, due to technical limitations as described in the text. The dashed horizontal line for xchol = 0.66 represents the cholesterol solubility limit on the lipid bilayer. The region above that line is of no interest for the present study because we are only interested in lamellar phases and it does not affect any of the conclusions. The value was chosen as the limit determined for several PC (Huang et al., 1999) at room temperature, but it should be noted that the value can be different for a lipid mixture, i.e., the line may not be horizontal, there can be temperature variations, and it was not determined for SM.
FIGURE 7
FIGURE 7
PSM/POPC/chol phase diagram at 23°C (A) and at 37°C (B). The circles are experimental points. The red (quasi) tie-line on the tie-triangle describes the lo/ld composition at the right of which there is also so phase. The blue tie-lines are the interval for the possible tie-lines that contain the 1:1:1 composition. The purple point marks the 1:1:1 composition and the green point marks the 2:1:1 composition. The phase boundaries for the diagram at 37°C are dashed lines to highlight the fact that no experimental determinations were performed for ternary mixtures at that temperature, due to technical limitations as described in the text. The dashed horizontal line for xchol = 0.66 represents the cholesterol solubility limit on the lipid bilayer. The region above that line is of no interest for the present study because we are only interested in lamellar phases and it does not affect any of the conclusions. The value was chosen as the limit determined for several PC (Huang et al., 1999) at room temperature, but it should be noted that the value can be different for a lipid mixture, i.e., the line may not be horizontal, there can be temperature variations, and it was not determined for SM.
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