Effect of the structure of lipids favoring disordered domain formation on the stability of cholesterol-containing ordered domains (lipid rafts): identification of multiple raft-stabilization mechanisms

Abstract

Despite the importance of lipid rafts, commonly defined as liquid-ordered domains rich in cholesterol and in lipids with high gel-to-fluid melting temperatures (T(m)), the rules for raft formation in membranes are not completely understood. Here, a fluorescence-quenching strategy was used to define how lipids with low T(m), which tend to form disordered fluid domains at physiological temperatures, can stabilize ordered domain formation by cholesterol and high-T(m) lipids (either sphingomyelin or dipalmitoylphosphatidylcholine). In bilayers containing mixtures of low-T(m) phosphatidylcholines, cholesterol, and high-T(m) lipid, the thermal stability of ordered domains decreased with the acyl-chain structure of low-T(m) lipids in the following order: diarachadonyl > diphytanoyl > 1-palmitoyl 2-docosahexenoyl = 1,2 dioleoyl = dimyristoleoyl = 1-palmitoyl, 2-oleoyl (PO). This shows that low-T(m) lipids with two acyl chains having very poor tight-packing propensities can stabilize ordered domain formation by high-T(m) lipids and cholesterol. The effect of headgroup structure was also studied. We found that even in the absence of high-T(m) lipids, mixtures of cholesterol with PO phosphatidylethanolamine (POPE) and PO phosphatidylserine (POPS) or with brain PE and brain PS showed a (borderline) tendency to form ordered domains. Because these lipids are abundant in the inner (cytofacial) leaflet of mammalian membranes, this raises the possibility that PE and PS could participate in inner-leaflet raft formation or stabilization. In bilayers containing ternary mixtures of PO lipids, cholesterol, and high-T(m) lipids, the thermal stability of ordered domains decreased with the polar headgroup structure of PO lipids in the order PE > PS > phosphatidylcholine (PC). Analogous experiments using diphytanoyl acyl chain lipids in place of PO acyl chain lipids showed that the stabilization of ordered lipid domains by acyl chain and headgroup structure was not additive. This implies that it is likely that there are two largely mutually exclusive mechanisms by which low-T(m) lipids can stabilize ordered domain formation by high-T(m) lipids and cholesterol: 1), by having structures resulting in immiscibility of low-T(m) and high-T(m) lipids, and 2), by having structures allowing them to pack tightly within ordered domains to a significant degree.

FIGURE 1

Effect of cholesterol and TEMPO concentration upon quenching of DPH fluorescence. (A) Effect of cholesterol concentration upon the temperature dependence of TEMPO-induced quenching. Samples contained a 1:1 (mol/mol) mixture of DPPC and DOPC plus 0 mol % (+), 15 mol % (▵), 25 mol % (▿), 33 mol % (□), or 40 mol % (○) cholesterol. F samples contained 2 mM TEMPO, whereas Fo samples did not contain TEMPO. Unless otherwise noted, in this and all subsequent experiments, samples contained SUV with 50 μM total lipid plus 0.5 mol % DPH, and were dispersed in PBS at pH 7.4. In Figs. 1–7, average F/Fo is shown for at least duplicate samples, and ordered domain melting temperatures (defined by the point of maximum slope when a sigmoidal temperature-dependence was observed) did not vary by >±1.5°C. (B) Effect of TEMPO concentration. Samples contained DOPC (□), 3:1 (mol/mol) DOPC/cholesterol (+), DPPC (○), or 3:1 (mol/mol) DPPC/cholesterol (▵). At each successive point, 1-μl aliquots of 339 mM TEMPO dissolved in ethanol was added. Fluorescence (arbitrary units) reached final values immediately after each addition of TEMPO. Experiments were carried out at room temperature.

FIGURE 2

Melting curves for ordered domains in vesicles containing phospholipids mixed with cholesterol. Each sample contained 50 μM total lipid dispersed in PBS at pH 7.4. The ratio of DPH fluorescence in the presence and absence of TEMPO (F/Fo) was measured with increasing temperature. (A) Melting curves of binary mixtures of lipids with the PC headgroup and variable acyl chains. Phospholipids were SM (▿), DPPC (▵), POPC (⋄), SOPC (□), DOPC (•), DMoPC (▾), DPhPC (+), DArPC (+), or PDoPC (○). (B) Melting curves for mixtures of lipids with 40 mol % cholesterol. Symbols are the same as in A, with the addition of 1:1 (mol/mol) POPE/POPS (⋄). (C) Melting curves of mixtures of lipids with varied headgroups. Phospholipids were 1:1 (mol/mol) POPE/POPS (▿), 1:1 (mol/mol) porcine brain PE/brain PS (▵), DPhPC (+), and 1:1 (mol/mol) DPhPE/DPhPS (□).

FIGURE 3

Melting curves for ordered domains in vesicles containing 1:1 (mol/mol) mixtures of high-T_m lipid/low-T_m PC plus cholesterol. Each sample contained 50 μM total lipid dispersed in PBS at pH 7.4. The ratio of DPH fluorescence in the presence and absence of TEMPO (F/Fo) was measured with increasing temperature. (A) Melting curves of lipid mixtures containing 25 mol % cholesterol and DPPC as the high-T_m lipid, and DOPC (▿), POPC (+), DPhPC (▵), DMoPC (⋄), DArPC (□), or PDoPC (○) as the low-T_m lipid. (B) Melting curves of lipid mixtures containing 25 mol % cholesterol and SM as the high-T_m lipid, and low-T_m lipids as in A, except when low-T_m lipid was SOPC (•). (C) Melting curves in mixtures containing 40 mol % cholesterol, DPPC as the high-T_m lipid, and low-T_m lipids as in B.

FIGURE 4

FRET assay of ordered-domain stability in mixtures of 25 mol % cholesterol with 1:1 (mol/mol) DPPC/low-T_m PC. Each sample contained 100 μM total lipid dispersed in PBS at pH 7.4. Unless otherwise noted, sample composition was as in Fig. 3, but also containing FRET donor (2 mol % LW peptide), and, when desired, FRET acceptor (1 mol % LcTMADPH). Mixtures with the low-T_m lipids DArPC (□), DPhPC (▿), or POPC (○) are shown. The open triangle represents 1:1 (mol/mol) mixture of vesicles containing DPPC, 25 mol % cholesterol, and 1 mol % LcTMADPH with vesicles containing POPC, 25 mol % cholesterol, and 2 mol % LW peptide.

FIGURE 5

Melting curves of ordered domains in mixtures of cholesterol (25 mol %), high-T_m lipid (37.5 mol %), and various low-T_m PO lipids (37.5 mol %). Each sample contained 50 μM SUV dispersed in PBS at pH 7.4. The ratio of DPH fluorescence in the presence and absence of TEMPO (F/Fo) was measured with increasing temperature. (A) Melting curves of lipid mixtures containing DPPC as the high-T_m lipid. (B) Melting curves of lipid mixtures containing SM as the high-T_m lipid. Mixtures contained POPC (▵), POPE (▿), or 1:1 (mol/mol) POPE/POPS (□) as low-T_m lipid. Mixtures containing 1-palmitoyl-2-oleoyl-phosphatidic acid and 1-palmitoyl-2-oleoyl-phosphatidylglycerol exhibited quenching profiles similar to that for mixtures containing POPE/POPS (data not shown).

FIGURE 6

Melting curves of ordered domains in mixtures of cholesterol (25 mol %), high-T_m lipid (37.5 mol %), and various low-T_m lipids (37.5 mol %). Each sample contained 50 μM SUV dispersed in PBS at pH 7.4. The ratio of DPH fluorescence in the presence and absence of TEMPO (F/Fo) was measured with increasing temperature. (A) Melting curves of lipid mixtures with DPPC as the high-T_m lipid and PO lipids as the low-T_m component. Mixtures contained POPC (▴), POPE (∇), or POPS (+) as the low-T_m lipid. (B) Melting curves of lipid mixtures with DPPC as the high-T_m lipid and DPh acyl-chained lipids as the low-T_m component. Mixtures contained DPhPC (▴), DPhPE (▿), or DPhPS (+) as the low-T_m lipid.

FIGURE 7

Schematic illustration of the effect of interactions between loosely (low-T_m) and tightly (high-T_m) packing lipids upon the stability of the ordered phases. Surface view of a portion of a lipid bilayer is shown. Heavy line represents boundary between disordered and ordered phases. The locations of individual lipids that prefer to form ordered phase (open circles) or those that prefer to form disordered phase (shaded circles) is also shown. The darker the shading of circles representing the disordered-phase-preferring lipids, the poorer their ability to pack tightly. Notice that the ordered phase is predominantly composed of lipids that prefer to form ordered phase but also contains some disordered-phase-preferring lipid, whereas the disordered phase is predominantly composed of lipids that prefer to form disordered phase but also contains some ordered-phase-preferring lipid. Ordered-phase stability is greater when disordered-phase-preferring lipid has a moderate ability to pack tightly (center) than when disordered-phase-preferring lipid has a poor ability to pack tightly (left). Ordered-phase stability is also greater when disordered-phase-preferring lipids pack so poorly that they do not dissolve well in the ordered phase (right).