Miscibility of ternary mixtures of phospholipids and cholesterol in monolayers, and application to bilayer systems

We investigate miscibility transitions of two different ternary lipid mixtures, DOPC/DPPC/Chol and POPC/PSM/Chol. In vesicles, both of these mixtures of an unsaturated lipid, a saturated lipid, and cholesterol form micron-scale domains of immiscible liquid phases for only a limited range of compositions. In contrast, in monolayers, both of these mixtures produce two distinct regions of immiscible liquid phases that span all compositions studied, the alpha-region at low cholesterol and the beta-region at high cholesterol. In other words, we find only limited overlap in miscibility phase behavior of monolayers and bilayers for the lipids studied. For vesicles at 25 degrees C, the miscibility phase boundary spans portions of both the monolayer alpha-region and beta-region. Within the monolayer beta-region, domains persist to high pressures, yet within the alpha-region, miscibility phase transition pressures always fall below 15 mN/m, far below the bilayer equivalent pressure of 32 mN/m. Approximately equivalent phase behavior is observed for monolayers of DOPC/DPPC/Chol and for monolayers of POPC/PSM/Chol. As expected, pressure-area isotherms of our ternary lipid mixtures yield smaller molecular area and compressibility for monolayers containing more saturated acyl chains and cholesterol. All monolayer experiments were conducted under argon. We show that exposure of unsaturated lipids to air causes monolayer surface pressures to decrease rapidly and miscibility transition pressures to increase rapidly.     

A calorimetry and deuterium NMR study of mixed model membranes of 1-palmitoyl-2-oleylphosphatidylcholine and saturated phosphatidylcholines

Binary phase diagrams have been constructed from differential scanning calorimetry (DSC) data for the systems 1-palmitoyl-2-oleylphosphatidylcholine (POPC)/dimyristoylphosphatidylcholine (DMPC), POPC/dipalmitoylphosphatidylcholine (DPPC) and POPC/distearoylphosphatidylcholine (DSPC). Mixtures of POPC with DMPC exhibit complete miscibility in the gel and liquid crystalline states. Mixtures of POPC with DPPC or with DSPC exhibit gel phase immiscibility over the composition range 0-75% DPPC (or DSPC). These results, when taken together with previous studies of mixtures of phosphatidylcholines, are consistent with the hypothesis that PCs whose order-disorder transition temperatures (Tm values) differ by less than 33 deg. C exhibit gel state miscibility. Those whose Tm values differ by more than 33 deg. C exhibit gel state immiscibility. 2H-NMR spectroscopy has been used to further study mixed model membranes composed of POPC and DPPC, in which either lipid has been labeled with deuterium in the 2-, 10- or 16-position of the palmitoyl chain(s) or in the N-methyls of the choline head group. POPC/DPPC mixtures in the liquid crystalline state are intermediate in order between pure POPC and DPPC at the same temperature. The POPC palmitoyl chain is always more disordered than the palmitoyl chains of DPPC in liquid crystalline POPC/DPPC mixtures. This is attributed to the fact that a POPC palmitoyl chain is constrained by direct bonding to have at least one oleyl chain among its nearest neighbors, while a DPPC palmitoyl chain must have at least one neighboring palmitoyl chain. When liquid crystalline POPC, DPPC and POPC/DPPC mixtures are compared at a reduced temperature (relative to the acyl chain order-disorder transition), POPC/DPPC mixtures are more disordered than predicted from the behavior of the pure components, in agreement with enthalpy data derived from DSC studies. Within the temperature range of the broad phase transition of 1:1 POPC/DPPC, a superposition of gel and liquid crystalline spectra is observed for 1:1 POPC/[2H]DPPC, while 1:1[2H]POPC/DPPC exhibits only a liquid crystalline spectrum. Thus, at temperatures within the phase transition region, the liquid crystalline phase is POPC-rich and the gel phase is DPPC-rich. Comparison of the liquid crystalline quadrupole splittings within the thermal phase transition range suggests that mixing of the residual liquid crystalline POPC and DPPC is highly non-ideal.     

Calcitonin-derived carrier peptide plays a major role in the membrane localization of a peptide-cargo complex

Bilayers made of dioleoylphosphatidylcholine (DOPC)/dipalmitoylphosphatidylcholine (DPPC) mixture containing or not cholesterol (Chl) were used to investigate the interaction of a carrier peptide with membranes. Atomic force microscopy revealed that the C-terminal 9-32 fragment of human calcitonin (hCT (9-32)), free or coupled to enhanced green fluorescent protein (hCT-eGFP) cargo forms aggregates in the DOPC fluid phase in absence of Chl and in the DPPC enriched liquid-ordered phase when Chl is present. The data show that hCT (9-32) plays a determinant role in the membrane localization of the peptide-cargo complex. They suggest that carpet-like mechanism for membrane destabilization may be involved in the carrier function of hCT (9-32).     

The fluorescent cholesterol analog dehydroergosterol induces liquid-ordered domains in model membranes

The fluorescent sterol dehydroergosterol (DHE) is often used as a marker for cholesterol in cellular studies. We show by vesicle fluctuation analysis that DHE has a lower ability than cholesterol to stiffen lipid bilayers suggesting less efficient packing with phospholipid acyl chains. Despite this difference, we found by fluorescence and atomic force microscopy, that DHE induces liquid-ordered/-disordered coexistent domains in giant unilamellar vesicles (GUVs) and supported bilayers made of dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylcholine (DOPC) and DHE or cholesterol. DHE-induced phases have a height difference of 0.9-1 nm similar as known for cholesterol-containing domains. DHE not only promotes formation of liquid-liquid immiscibility but also shows strong partition preference for the liquid-ordered phase further supporting its suitability as cholesterol probe.     

Differential dynamic and structural behavior of lipid-cholesterol domains in model membranes

Changes in the cholesterol (Chol) content of biological membranes are known to alter the physicochemical properties of the lipid lamella and consequently the function of membrane-associated enzymes. To characterize these changes, we used steady-state and time resolved fluorescence spectroscopy and two photon-excitation microscopy techniques. The membrane systems were chosen according to the techniques that were used: large unilamellar vesicles (LUVs) for cuvette and giant unilamellar vesicles (GUVs) for microscopy measurements; they were prepared from dipalmitoyl phosphatidylcholine (DPPC) and dioctadecyl phosphatidylcholine (DOPC) in mixtures that are well known to form lipid domains. Two fluorescent probes, which insert into different regions of the bilayer, were selected: 1,6-diphenyl-1,3,5-hexatriene (DPH) was located at the deep hydrophobic core of the acyl chain regions and 2-dimethylamino-6-lauroylnaphthalene (Laurdan) at the hydrophilic-hydrophobic membrane interface. Our spectroscopy results show that (i) the changes induced by cholesterol in the deep hydrophobic phospholipid acyl chain domain are different from the ones observed in the superficial region of the hydrophilic-hydrophobic interface, and these changes depend on the state of the lamella and (ii) the incorporation of cholesterol into the lamella induces an increase in the orientation dynamics in the deep region of the phospholipid acyl chains with a corresponding decrease in the orientation at the region close to the polar lipid headgroups. The microscopy data from DOPC/DPPC/Chol GUVs using Laurdan generalized polarization (Laurdan GP) suggest that a high cholesterol content in the bilayer weakens the stability of the water hydrogen bond network and hence the stability of the liquid-ordered phase (Lo).     

Lowering line tension with high cholesterol content induces a transition from macroscopic to nanoscopic phase domains in model biomembranes

Chemically simplified lipid mixtures are used here as models of the cell plasma membrane exoplasmic leaflet. In such models, phase separation and morphology transitions controlled by line tension in the liquid-disordered (Ld)?+?liquid-ordered (Lo) coexistence regime have been described [1]. Here, we study two four-component lipid mixtures at different cholesterol fractions: brain sphingomyelin (BSM) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol (Chol). On giant unilamellar vesicles (GUVs) display a nanoscopic-to-macroscopic transition of Ld?+?Lo phase domains as POPC is replaced by DOPC, and this transition also depends on the cholesterol fraction. Line tension decreases with increasing cholesterol mole fractions in both lipid mixtures. For the ternary BSM/DOPC/Chol mixture, the published phase diagram [19] requires a modification to show that when cholesterol mole fraction is >~0.33, coexisting phase domains become nanoscopic.     

A region-matched hydrophobic interaction between melittin and dimyristoylphosphatidylcholine in a ternary mixture of phosphatidylcholines

Interaction of melittin with phosphatidylcholine molecules in pure vesicles, binary mixtures and a ternary mixture of dimyristoylphosphatidylcholine IDMPC), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC) was investigated by differential scanning calorimetry. Melittin binds preferentially with DMPC, and results in segregation of DMPC in binary mixtures of DMPC/DPPC and DMPC/DSPC and in a ternary mixture of DMPC/DPPC/DSPC. The results indicate that the hydrophobic part of peptide interacts preferentially with the phospholipid which has the same size of hydrophobic region or fatty acyl chains.     

Lipid interactions of an actinoporin pore-forming oligomer

The actinoporins are cytolytic toxins produced by sea anemones. Upon encountering a membrane, preferably containing sphingomyelin, they oligomerize and insert their N-terminal helix into the membrane, forming a pore. Whether sphingomyelin is specifically recognized by the protein or simply induces phase coexistence in the membrane has been debated. Here, we perform multi-microsecond molecular dynamics simulations of an octamer of fragaceatoxin C, a member of the actinoporin family, in lipid bilayers containing either pure 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or a 1:1 mixture of DOPC and palmitoyl sphingomyelin (PSM). The complex is highly stable in both environments, with only slight fraying of the inserted helices near their N-termini. Analyzing the structural parameters of the mixed membrane in the course of the simulation, we see signs of a phase transition for PSM in the inner leaflet of the bilayer. In both leaflets, cross-interactions between lipids of different type decrease over time. Surprisingly, the aromatic loop thought to be responsible for sphingomyelin recognition interacts more with DOPC than PSM by the end of the simulation. These results support the notion that the key membrane property that actinoporins recognize is lipid phase coexistence.     

Temperature-pressure phase diagram of a heterogeneous anionic model biomembrane system: results from a combined calorimetry, spectroscopy and microscopy study

By using Fourier transform infrared (FT-IR) spectroscopy in combination with differential scanning calorimetry (DSC) coupled with pressure perturbation calorimetry (PPC), ultrasound velocimetry, Laurdan fluorescence spectroscopy, fluorescence microscopy and atomic force microscopy (AFM), the temperature and pressure dependent phase behavior of the five-component anionic model raft lipid mixture DOPC/DOPG/DPPC/DPPG/cholesterol (20:5:45:5:25 mol%) was investigated. A temperature range from 5 to 65 °C and a pressure range up to 16 kbar were covered to establish the temperature-pressure phase diagram of this heterogeneous model biomembrane system. Incorporation of 10-20 mol% PG still leads to liquid-ordered (l(o))-liquid-disordered (l(d)) phase coexistence regions over a wide range of temperatures and pressures. Compared to the corresponding neutral model raft mixture (DOPC/DPPC/Chol 25:50:25 mol%), the p,T-phase diagram is - as expected and in accordance with the Gibbs phase rule - more complex, the phase sequence as a function of temperature and pressure is largely similar, however. This anionic heterogeneous model membrane system will serve as a more realistic model biomembrane system to study protein interactions with anionic lipid bilayers displaying liquid-disordered/liquid-ordered domain coexistence over a wide range of the temperature-pressure plane, thus allowing also studies of biologically relevant systems encountered under extreme environmental conditions.     

Order parameters and areas in fluid-phase oriented lipid membranes using wide angle X-ray scattering

We used wide angle x-ray scattering (WAXS) from stacks of oriented lipid bilayers to measure chain orientational order parameters and lipid areas in model membranes consisting of mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/cholesterol in fluid phases. The addition of 40% cholesterol to either DOPC or DPPC changes the WAXS pattern due to an increase in acyl chain orientational order, which is one of the main properties distinguishing the cholesterol-rich liquid-ordered (Lo) phase from the liquid-disordered (Ld) phase. In contrast, powder x-ray data from multilamellar vesicles does not yield information about orientational order, and the scattering from the Lo and Ld phases looks similar. An analytical model to describe the relationship between the chain orientational distribution and WAXS data was used to obtain an average orientational order parameter, S_x-ray. When 40% cholesterol is added to either DOPC or DPPC, S_x-ray more than doubles, consistent with previous NMR order parameter measurements. By combining information about the average chain orientation with the chain-chain correlation spacing, we extended a commonly used method for calculating areas for gel-phase lipids to fluid-phase lipids and obtained agreement to within 5% of literature values.     

Lipid dynamics and domain formation in model membranes composed of ternary mixtures of unsaturated and saturated phosphatidylcholines and cholesterol

In recent years, the implication of sphingomyelin in lipid raft formation has intensified the long sustained interest in this membrane lipid. Accumulating evidences show that cholesterol preferentially interacts with sphingomyelin, conferring specific physicochemical properties to the bilayer membrane. The molecular packing created by cholesterol and sphingomyelin, which presumably is one of the driving forces for lipid raft formation, is known in general to differ from that of cholesterol and phosphatidylcholine membranes. However, in many studies, saturated phosphatidylcholines are still considered as a model for sphingolipids. Here, we investigate the effect of cholesterol on mixtures of dioleoyl-phosphatidylcholine (DOPC) and dipalmitoyl-phosphatidylcholine (DPPC) or distearoyl-phosphatidylcholine (DSPC) and compare it to that on mixtures of DOPC and sphingomyelin analyzed in previous studies. Giant unilamellar vesicles prepared from ternary mixtures of various lipid compositions were imaged by confocal fluorescence microscopy and, within a certain range of sterol content, domain formation was observed. The assignment of distinct lipid phases and the molecular mobility in the membrane bilayer was investigated by fluorescence correlation spectroscopy. Cholesterol was shown to affect lipid dynamics in a similar way for DPPC and DSPC when the two phospholipids were combined with cholesterol in binary mixtures. However, the corresponding ternary mixtures exhibited different spatial lipid organization and dynamics. Finally, evidences of a weaker interaction of cholesterol with saturated phosphatidylcholines than with sphingomyelin (with matched chain length) are discussed.     

Interaction of high density lipoprotein particles with membranes containing cholesterol

In this study, free cholesterol (FC) efflux mediated by human HDL was investigated using fluorescence methodologies. The accessibility of FC to HDL may depend on whether it is located in regions rich in unsaturated phospholipids or in domains containing high levels of FC and sphingomyelin, known as "lipid rafts." Laurdan generalized polarization and two-photon microscopy were used to quantify FC removal from different pools in the bilayer of giant unilamellar vesicles (GUVs). GUVs made of POPC and FC were observed after incubation with reconstituted particles containing apolipoprotein A-I and POPC [78A diameter reconstituted high density lipoprotein (rHDL)]. Fluorescence correlation spectroscopy data show an increase in rHDL size during the incubation period. GUVs made of two "raft-like" mixtures [DOPC/DPPC/FC (1:1:1) and POPC/SPM/FC (6:1:1)] were used to model liquid-ordered/liquid-disordered phase coexistence. Through these experiments, we conclude that rHDL preferentially removes cholesterol from the more fluid phases. These data, and their extrapolation to in vivo systems, show the significant role that phase separation plays in the regulation of cholesterol homeostasis.     

Characterization of the ternary mixture of sphingomyelin, POPC, and cholesterol: support for an inhomogeneous lipid distribution at high temperatures

A ternary lipid mixture of palmitoyl-oleoyl-phosphatidylcholine (POPC), palmitoyl-erythro-sphingosylphosphorylcholine (PSM), and cholesterol at a mixing ratio of 37.5:37.5:25 mol/mol/mol was characterized using fluorescence microscopy, ²H NMR, and electron paramagnetic resonance spectroscopy. The synthetic PSM provides an excellent molecule for studying the molecular properties of raft phases. It shows a narrow phase transition at a temperature of 311 K and is commercially available with a perdeuterated sn-2 chain. Fluorescence microscopy shows that large inhomogeneities in the mixed membranes are observed in the coexistence region of liquid-ordered and liquid-disordered lipid phases. Above 310 K, no optically detectable phase separation was shown. Upon decrease in temperature, a redistribution of the cholesterol into large liquid-ordered PSM/cholesterol domains and depletion of cholesterol from liquid-disordered POPC domains was observed by ²H NMR and electron paramagnetic resonance experiments. However, there is no complete segregation of the cholesterol into the liquid-ordered phase and also POPC-rich domains contain the sterol in the phase coexistence region. We further compared order parameters and packing properties of deuterated PSM or POPC in the raft mixture at 313 K, i.e., in the liquid crystalline phase state. PSM shows significantly larger ²H NMR order parameters in the raft phase than POPC. This can be explained by an inhomogeneous interaction of cholesterol between the lipid species and the mutual influence of the phospholipids on each other. These observations point toward an inhomogeneous distribution of the lipids also in the liquid crystalline phase at 313 K. From the prerequisite that order parameters are identical in a completely homogeneously mixed membrane, we can determine a minimal microdomain size of 45-70 nm in PSM/POPC/cholesterol mixtures above the main phase transition of all lipids.     

The polar nature of 7-ketocholesterol determines its location within membrane domains and the kinetics of membrane microsolubilization by apolipoprotein A-I

7-Ketocholesterol is an oxidized derivative of cholesterol with numerous physiological effects. In model membranes, 7-ketocholesterol and cholesterol were compared by physical measures of bilayer order and polarity, formation of detergent resistant domains (DRM), phase separation, and membrane microsolubilization by apolipoprotein A-I. In binary mixtures of a saturated phosphatidylcholine (PC), dipalmitoyl-PC (DPPC), and cholesterol or 7-ketocholesterol, the sterols modulate bilayer order and polarity and induce DRM formation to a similar extent. Cholesterol induces formation of ordered lipid domains (rafts) in tertiary mixtures with dioleoyl-PC (DOPC) and DPPC, or DOPC and sphingomyelin (SM). In tertiary mixtures, cholesterol increased lipid order and reduces bilayer polarity more than 7-ketocholesterol. This effect was more pronounced when the mixtures were in a miscible liquid-disordered (L(d)) phase. Substitution of 7-ketocholesterol for cholesterol dramatically reduced the extent of DRM formation in DOPC/DPPC and DOPC/SM bilayers and ordered lipid phase separation in mixtures of a spin-labeled PC with DPPC and with SM. Compared to cholesterol, 7-ketocholesterol decreased the rate for the microsolubilization of dimyristoyl-PC multilamellar vesicles by apolipoprotein A-I. The membrane effects of 7-ketocholesterol were dependent on the phospholipid matrix. In L(d) phase phospholipids, a model for 7-ketocholesterol indicates that the proximity of the 7-keto and 3beta-OH groups puts both polar moieties at the lipid-water interface to tilt the sterol nucleus to the plane of the bilayer. 7-Ketocholesterol was less effective in forming ordered lipid domains, in decreasing the level of bilayer hydration, and in forming phase boundary bilayer defects. Compared to cholesterol, 7-ketocholesterol can differentially modulate membrane properties involved in protein-membrane association and function.     

Structure and dynamics of sphingomyelin bilayer: insight gained through systematic comparison to phosphatidylcholine

Sphingomyelin, one of the main lipid components of biological membranes, is actively involved in various cellular processes such as protein trafficking and signal transduction. In particular, specific lateral domains enriched in sphingomyelin and cholesterol have been proposed to play an important functional role in biomembranes, although their precise characteristics have remained unclear. A thorough understanding of the functional role of membranes requires detailed knowledge of their individual lipid components. Here, we employ molecular dynamics simulations to conduct a systematic comparison of a palmitoylsphingomyelin (PSM, 16:0-SM) bilayer with a membrane that comprises dipalmitoylphosphatidylcholine (DPPC) above the main phase transition temperature. We clarify atomic-scale properties that are specific to sphingomyelin due to its sphingosine moiety, and further discuss their implications for SM-rich membranes. We find that PSM bilayers, and in particular the dynamics of PSM systems, are distinctly different from those of a DPPC bilayer. When compared with DPPC, the strong hydrogen bonding properties characteristic to PSM are observed to lead to considerable structural changes in the polar headgroup and interface regions. The strong ordering of PSM acyl chains and specific ordering effects in the vicinity of a PSM-water interface reflect this issue clearly. The sphingosine moiety and related hydrogen bonding further play a crucial role in the dynamics of PSM bilayers, as most dynamic properties, such as lateral and rotational diffusion, are strongly suppressed. This is most evident in the rotational motion characterized by spin-lattice relaxation times and the decay of hydrogen bond autocorrelation functions that are expected to be important in complexation of SM with other lipids in many-component bilayers. A thorough understanding of SM bilayers would greatly benefit from nuclear magnetic resonance experiments for acyl chain ordering and dynamics, allowing full comparison of these simulations to experiments.     

N-Nervonoylsphingomyelin (C24:1) Prevents Lateral Heterogeneity in Cholesterol-Containing Membranes

This study was conducted to explore how the nature of the acyl chains of sphingomyelin (SM) influence its lateral distribution in the ternary lipid mixture SM/cholesterol/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), focusing on the importance of the hydrophobic part of the SM molecule for domain formation. Atomic force microscopy (AFM) measurements showed that the presence of a double bond in the 24:1 SM molecule in mixtures with cholesterol (CHO) or in pure bilayers led to a decrease in the molecular packing. Confocal microscopy and AFM showed, at the meso- and nanoscales respectively, that unlike 16:0 and 24:0 SM, 24:1 SM does not induce phase segregation in ternary lipid mixtures with DOPC and CHO. This ternary lipid mixture had a nanomechanical stability intermediate between those displayed by liquid-ordered (Lo) and liquid-disordered (Ld) phases, as reported by AFM force spectroscopy measurements, demonstrating that 24:1 SM is able to accommodate both DOPC and CHO, forming a single phase. Confocal experiments on giant unilamellar vesicles made of human, sheep, and rabbit erythrocyte ghosts rich in 24:1 SM and CHO, showed no lateral domain segregation. This study provides insights into how the specific molecular structure of SM affects the lateral behavior and the physical properties of both model and natural membranes. Specifically, the data suggest that unsaturated SM may help to keep membrane lipids in a homogeneous mixture rather than in separate domains.     

N-Nervonoylsphingomyelin (C24:1) Prevents Lateral Heterogeneity in Cholesterol-Containing Membranes

This study was conducted to explore how the nature of the acyl chains of sphingomyelin (SM) influence its lateral distribution in the ternary lipid mixture SM/cholesterol/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), focusing on the importance of the hydrophobic part of the SM molecule for domain formation. Atomic force microscopy (AFM) measurements showed that the presence of a double bond in the 24:1 SM molecule in mixtures with cholesterol (CHO) or in pure bilayers led to a decrease in the molecular packing. Confocal microscopy and AFM showed, at the meso- and nanoscales respectively, that unlike 16:0 and 24:0 SM, 24:1 SM does not induce phase segregation in ternary lipid mixtures with DOPC and CHO. This ternary lipid mixture had a nanomechanical stability intermediate between those displayed by liquid-ordered (Lo) and liquid-disordered (Ld) phases, as reported by AFM force spectroscopy measurements, demonstrating that 24:1 SM is able to accommodate both DOPC and CHO, forming a single phase. Confocal experiments on giant unilamellar vesicles made of human, sheep, and rabbit erythrocyte ghosts rich in 24:1 SM and CHO, showed no lateral domain segregation. This study provides insights into how the specific molecular structure of SM affects the lateral behavior and the physical properties of both model and natural membranes. Specifically, the data suggest that unsaturated SM may help to keep membrane lipids in a homogeneous mixture rather than in separate domains.     

The partition of cholesterol between ordered and fluid bilayers of phosphatidylcholine: a synchrotron X-ray diffraction study

The structure and composition of coexisting bilayer phases separated in binary mixtures of dipalmitoylphosphatidylcholine and cholesterol and ternary mixtures of equimolar proportions of dipalmitoyl- and dioleoylphosphatidycholines containing different proportions of cholesterol have been characterized by synchrotron X-ray diffraction methods. The liquid-ordered phase is distinguished from gel and fluid phases by a disordering of the hydrocarbon chains intermediate between the two phases as judged from the wide-angle X-ray scattering profiles. Electron density distribution calculated in coexisting bilayer phases shows that liquid-ordered phase is enriched in dipalmitoylphosphatidylcholine and cholesterol and a higher electron density in the methylene chain region of the bilayer ascribed to the location of the sterol ring of cholesterol. The ratio of the two constituents in the liquid-ordered phase is not constant because the stoichiometry is temperature-dependent as seen by respective changes in bilayer thickness over the range 20 degrees to 36 degrees C where coexisting phases are observed. Three coexisting phases were deconvolved in the ternary mixture at 20 degrees C. From an analysis of the ternary mixtures containing mole fractions of cholesterol from 0.09 to 0.15 it was found that the liquid-crystal and gel phases each contained about 10% of the cholesterol molecules and the liquid-ordered phase was comprised of 30% cholesterol molecules.     

Complexes in ternary cholesterol-phospholipid mixtures

Recent work by Veatch and Keller has described micron-scale liquid-liquid immiscibility in giant unilamellar vesicles composed of ternary mixtures of cholesterol, dipalmitoylphosphatidylcholine (DPPC), and dioleoylphosphatidylcholine (DOPC). Significantly, they do not observe micron-scale immiscibility in any of the three corresponding binary mixtures under the same conditions. It is shown here that this unexpected result can be accounted for by the formation of a complex between cholesterol and DPPC. The complex is miscible with DPPC and cholesterol, and immiscible with DOPC. A simple, idealized thermodynamic treatment of this model leads to theoretical ternary phase diagrams that are similar to the experimental diagram reported by Veatch and Keller. The model also accounts for significant qualitative features of the deuterium NMR spectra of these mixtures in bilayers.     

Epifluorescence microscopic studies of monolayers containing mixtures of dioleoyl- and dipalmitoylphosphatidylcholines.

Monolayers of dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and some mixtures of these lipids were investigated using an epifluorescence microscopic surface balance. Monolayers were visualized at 23 +/- 1 degree C through the fluorescence of 1 mol% of two different fluorescent probes, 1-palmitoyl-2-(12-[(7-nitro-2-1,3-benzoxadizole-4- yl)amino]dodecanoyl)phosphatidylcholine (NBD-PC), which partitions into the liquid expanded (LE) or disordered lipid phase and 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO-C18), which preferentially associates with the liquid condensed (LC) phase or lipid with ordered chains. LC domains were observed in pure DPPC monolayers at relatively low surface pressures (pi), and these domains grew with increasing surface pressure. Only liquid expanded phase was observed in pure DOPC monolayers up to the point of monolayer collapse. In monolayers containing 29:70:1, 49:50:1, and 69:30:1 (mol/mol/mol) of DPPC:DOPC:probe the domains of LC phase were smaller than those seen in DPPC monolayers at equivalent surface pressures. Quantitative analysis of the visual fields shown by the mixed monolayers showed a distribution of sizes of condensed domains at any given pi. At pi = 30 mN m-1, liquid-expanded, or fluid, regions occupied more than 70% of the total monolayer area in all three mixtures studied, whereas DPPC monolayers were more than 75% condensed or solid at that pressure. For monolayers of DPPC:DOPC:NBD-PC 49:50:1 and 69:30:1 the average domain size and the percentage of the total area covered with LC, or rigid, areas increased to a maximum at pi around 35 mN m-1 followed by a decrease at higher pi.(ABSTRACT TRUNCATED AT 250 WORDS)