Differential ability of cholesterol-enriched and gel phase domains to resist benzyl alcohol-induced fluidization in multilamellar lipid vesicles

Benzyl alcohol (BA) has a well-known fluidizing effect on both artificial and cellular membranes. BA is also likely to modulate the activities of certain membrane proteins by decreasing the membrane order. This phenomenon is presumably related to the ability of BA to interrupt interactions between membrane proteins and the surrounding lipids by fluidizing the lipid bilayer. The components of biological membranes are laterally diversified into transient assemblies of varying content and order, and many proteins are suggested to be activated or inactivated by their localization in or out of membrane domains displaying different physical phases. We studied the ability of BA to fluidize artificial bilayer membranes representing liquid-disordered, cholesterol-enriched and gel phases. Multilamellar vesicles were studied by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and trans-parinaric acid, which display different phase partitioning. Domains of different degree of order and thermal stability showed varying abilities to resist fluidization by BA. In bilayers composed of mixtures of an unsaturated phosphatidylcholine, a saturated high melting temperature lipid (sphingomyelin or phosphatidylcholine) and cholesterol, BA fluidized and lowered the melting temperature of the ordered and gel phase domains. In general, cholesterol-enriched domains were more resistant to BA than pure gel phase domains. In contrast, bilayers containing high melting temperature gel phase domains containing a ceramide or a galactosylceramide proved to be the most effective in resisting fluidization. The results of our study suggest that the ability of BA to affect the fluidity and lateral organization of the membranes was dependent on the characteristic features of the membrane compositions studied and related to the intermolecular cohesion in the domains.     

Nonequilibrium behavior in supported lipid membranes containing cholesterol

We investigate lateral organization of lipid domains in vesicles versus supported membranes and monolayers. The lipid mixtures used are predominantly DOPC/DPPC/Chol and DOPC/BSM/Chol, which have been previously shown to produce coexisting liquid phases in vesicles and monolayers. In a monolayer at an air-water interface, these lipids have miscibility transition pressures of approximately 12-15 mN/m, which can rise to 32 mN/m if the monolayer is exposed to air. Lipid monolayers can be transferred by Langmuir-Sch?fer deposition onto either silanized glass or existing Langmuir-Blodgett supported monolayers. Micron-scale domains are present in the transferred lipids only if they were present in the original monolayer before deposition. This result is valid for transfers at 32 mN/m and also at lower pressures. Domains transferred to glass supports differ from liquid domains in vesicles because they are static, do not align in registration across leaflets, and do not reappear after temperature is cycled. Similar static domains are found for vesicles ruptured onto glass surfaces. Although supported membranes on glass capture some aspects of vesicles in equilibrium (e.g., gel-liquid transition temperatures and diffusion rates of individual lipids), the collective behavior of lipids in large liquid domains is poorly reproduced.     

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.     

Non-raft forming sphingomyelin-cholesterol mixtures

Sphingomyelin from biological membranes forms segregated domains with cholesterol in fluid bilayers. However, a synthetic form of sphingomyelin with an oleoyl chain linked to sphingosine is not incorporated into cholesterol-rich domains. We have studied the properties of mixtures of oleoyl-sphingomyelin and cholesterol as well as mixtures of oleoyl-sphingomyelin with 1-stearoyl-2-oleoyl-phosphatidylcholine by DSC and NMR. Cholesterol has a high miscibility with oleoyl-sphingomyelin and it does not separate in crystalline form until the mol fraction of cholesterol reaches a value above 0.6. A large fraction of the cholesterol crystals that are formed are in the monohydrate form. Furthermore, these crystals rehydrate relatively rapidly compared with pure cholesterol crystals in the absence of phospholipid. The environment of the carbonyl group of the phospholipid indicates that it is similar to other forms of sphingomyelin with saturated acyl chains. Also similar to other forms of sphingomyelin, the quaternary ammonium group of oleoyl-sphingomyelin is more rigid than that of phosphatidylcholines, as indicated by the strong resonance observed with cross-polarization/magic angle spinning. Additionally, oleoyl-sphingomyelin produces a larger alteration than egg sphingomyelin of the phase transition of 1-stearoyl-2-oleoyl-phosphatidylcholine. These studies indicate that oleoyl-sphingomyelin, unlike saturated forms of sphingomyelin, does not form segregated domains with cholesterol because of its greater miscibility with phosphatidylcholine.     

Seeing spots: complex phase behavior in simple membranes

Liquid domains in model lipid bilayers are frequently studied as models of raft domains in cell plasma membranes. Micron-scale liquid domains are easily produced in vesicles composed of ternary mixtures of a high melting temperature lipid, a low melting temperature lipid, and cholesterol. Here, we describe the rich phase behavior observed in binary and ternary systems. We then discuss experimental challenges inherent in mapping phase diagrams of even simple lipid systems. For example, miscibility behavior varies with lipid type, lipid ratio, lipid oxidation, and level of impurity. Liquid domains are often circular, but can become noncircular when membranes are near critical points. Finally, we reflect on applications of phase diagrams in model systems to rafts in cell membranes.     

HDLs induce raft domain vanishing in heterogeneous giant vesicles

Cholesterol efflux from the plasma membrane to HDLs is essential for cell cholesterol homeostasis. Recently, cholesterol-enriched ordered membrane domains, i.e. lipid rafts have been proposed to play an important role in this process. Here we introduce a new method to investigate the role of HDL interactions with the raft lipid phase and to directly visualize the effects of HDL-induced cholesterol efflux on rafts in model membranes. Addition of HDLs to giant lipid vesicles containing raft-type domains promoted decrease in size and disappearance of such domains as visualized by fluorescence microscopy. This was interpreted as resulting from cholesterol efflux from the vesicles to the HDLs. The raft vanishing rate was directly related to the HDL concentration. Evidence for a direct interaction of HDLs with the membrane was obtained by observing mutual adhesion of vesicles. It is suggested that the present method can be used to study the selective role of the bilayer lipid phase (raft and non-raft) in cholesterol efflux and membrane-HDL interaction and their underlying mechanisms. Such mechanisms may contribute to cholesterol efflux in vivo.     

Phosphatidylcholine and sphingomyelin containing an elaidoyl fatty acid can form cholesterol-rich lateral domains in bilayer membranes

Elaidic acid is a trans-fatty acid found in many food products and implicated for having potentially health hazardous effects in humans. Elaidic acid is readily incorporated into membrane lipids in vivo and therefore affects processes regulating membrane physical properties. In this study the membrane properties of sphingomyelin and phosphatidylcholine containing elaidic acid (N-E-SM and PEPC) were determined in bilayer membranes with special emphasis on their interaction with cholesterol and participation in ordered domain formation. In agreement with previous studies the melting temperatures were found to be about 20 °C lower for the elaidoyl than for the corresponding saturated lipids. The trans-unsaturation increased the polarity at the membrane-water interface as reported by Laurdan fluorescence. Fluorescence quenching experiments using cholestatrienol as a probe showed that both N-E-SM and PEPC were incorporated in lateral membrane domains with sterol and saturated lipids. At low temperatures the elaidoyl lipids were even able to form sterol-rich domains without any saturated lipids present in the bilayer. We conclude from this study that the ability of N-E-SM and PEPC to form ordered domains together with cholesterol and saturated phospho- and sphingolipids in model membranes indicates that they might have an influence on raft formation in biological membranes.     

Liquid-liquid phase transition temperatures increase when lipid bilayers are supported on glass

Membranes made from certain ternary mixtures of lipids can display coexisting liquid phases. In giant unilamellar vesicles, these phases appear as liquid domains which diffuse and coalesce after the vesicle is cooled below its miscibility transition temperature (T_m). Converting vesicles to supported lipid bilayers alters the mobility of the lipids and domains in the bilayer. At the same time, the miscibility transition temperature of the lipid mixture is altered. Here we compare T_m in vesicles and in supported bilayers formed by rupturing the same vesicles onto glass. We determine transition temperatures using fluorescence microscopy, and identify an increase in T_m when it is measured in identical membranes in solution and on a glass surface. We systematically alter the lipid composition of our membranes in order to observe the correlation between membrane composition and variation in T_m.     

Sterol chemical configuration and conformation influence the thermotropic phase behaviour of dipalmitoylphosphatidylcholine mixtures containing 5β-cholestan-3β- and -3α-ol

We report here our differential scanning calorimetry measurements investigating the thermotropic phase behaviour of binary dipalmitoylphosphatidylcholine (DPPC)/sterol mixtures containing two saturated sterols with different ring configurations (5β-H and either 3α-OH or 3β-OH). These measurements differ in the proportions of sharp and broad components in the heating endotherms, representing the melting of the sterol-poor and sterol-rich lipid micro-domains of the DPPC bilayer, respectively. Our results suggest that the 5,10-cis ring configuration of both saturated sterols and the ring A conformations have the greatest influence on DPPC bilayer properties, most likely by inducing small increases in the mean area/molecule as compared to cholesterol. However, the C3-OH orientation also influences sterol miscibility, likely due to variations in the strength and number of interfacial H-bonds with changes in molecular area, which in turn probably reflect the depth of the sterol in the DPPC bilayer. This influence of C3-OH orientation is significantly greater than was observed in our earlier study of cholesterol/- and epicholesterol/DPPC mixtures. Overall, our results show that both saturated and unsaturated 3α-ols are less miscible than the corresponding 3β-ols, but that the presence of a Δ⁵ double bond can improve the sterol miscibility in the DPPC bilayer at high sterol concentrations.     

Study of the miscibility of cholesteryl oleate in a matrix of ceramide, cholesterol and fatty acid

Cholesteryl esters (CE) are not generally abundant but are ubiquitous in living organisms and have markedly different properties from cholesterol because of their acyl chain. The miscibility/immiscibility of CE with biological lipid structures is a key property for their functions. In this work we study the solubility of cholesteryl oleate (ChO) in a model of the stratum corneum lipid matrix composed of ceramide C16, cholesterol and palmitic acid in excess water. Experiments were done in conditions of fully ionized (pH = 9.0) and fully neutralized fatty acid (pH = 4.0), and differential scanning calorimetry of the ternary mixtures with added ChO at pH = 9.0 clearly displayed a main transition with the same maximum temperature, peak shape, and enthalpy, suggesting that ChO was excluded from the remaining lipids. This technique is not conclusive at pH = 4.0 because the transitions of the lipid matrix and ChO overlap. The insolubility of ChO at both pH values is supported by X-ray diffraction. Adding the ceramide:cholesterol:fatty acid lipid mixture to ChO did not change the X-ray pattern of the mixture nor that of the ChO. To supplement the above physical techniques, we applied ¹³C MAS NMR spectroscopy with C-13 carbonyl-labeled ChO. A single ¹³C carbonyl peak from the ChO at 171.5 ppm was observed, indicating exposure to only one environment. The chemical shift was identical to pure ChO below and above the temperature of isotropic liquid formation. Taken together, our results lead to the conclusion that the solubility of ChO is negligible in the ceramide:cholesterol:fatty acid lipid mixture.     

Thermal stability of outer membrane protein porin from Paracoccus denitrificans: FT-IR as a spectroscopic tool to study lipid-protein interaction

Lipid protein interactions play a key role in the stability and function of various membrane proteins. Earlier we have reported the extreme thermal stability of porin from Paracoccus denitrificans reconstituted into liposomes. Here, we used Fourier transform infrared spectroscopy for a label free analysis of the global secondary structural changes and local changes in the tyrosine microenvironment. Our results show that a mixed lipid system (non-uniform bilayer) optimizes the thermal stability of porin as compared to the porin in pure lipids (uniform bilayer) or detergent micelles. This is in line with the fact that the bacterial outer membrane is a dynamic system made up of lipids of varying chain lengths, head groups and the barrel wall height contacting the membrane is uneven.     

Differential lipid packing abilities and dynamics in giant unilamellar vesicles composed of short-chain saturated glycerol-phospholipids, sphingomyelin and cholesterol

The ability of membrane components to arrange themselves heterogeneously within the bilayer induces the formation of microdomains. Much work has been devoted to mimicking domain-assembly in artificial bilayers and characterizing their physico-chemical properties. Ternary lipid mixtures composed of unsaturated phospholipids, sphingomyelin and cholesterol give rise to large, round domains. Here, we replaced the unsaturated phospholipid in the ternary mixture with sphingomyelin and cholesterol by saturated glycero-phospholipids of different chain length and characterized the critical role of cholesterol in sorting these lipids by confocal imaging and fluorescence correlation spectroscopy (FCS). More cholesterol is needed to obtain phase segregation in ternary mixtures, in which the unsaturated phospholipid is replaced by a saturated one. Finally, lipid dynamics in distinct phases is very low and astonishingly similar, thereby suggesting the poor ability of cholesterol in sorting short-chain saturated glycero-phospholipids and sphingomyelin.     

Vesicles with charged domains

This work summarizes results obtained on membranes composed of the ternary mixture dioleoylphosphatidylglycerol (DOPG), egg sphingomyelin (eSM) and cholesterol (Chol). The membrane phase state as a function of composition is characterized from data collected with fluorescence microscopy on giant unilamellar vesicles. The results suggest that the presence of the charged DOPG significantly decreases the composition region of coexistence of liquid ordered and liquid disordered phases as compared to that in the ternary mixture of dioleoylphosphatidycholine, sphingomyelin and cholesterol. The addition of calcium chloride to DOPG:eSM:Chol vesicles, and to a lesser extent the addition of sodium chloride, leads to the stabilization of the two-phase coexistence region, which is expressed in an increase in the miscibility temperature. On the other hand, addition of the chelating agent EDTA has the opposite effect, suggesting that impurities of divalent cations in preparations of giant vesicles contribute to the stabilization of charged domains. We also explore the behavior of these membranes in the presence of extruded unilamellar vesicles made of the positively charged lipid dioleoyltrimethylammoniumpropane (DOTAP). The latter can induce domain formation in DOPG:eSM:Chol vesicles with initial composition in the one-phase region.     

Characterization of membrane properties of inositol phosphorylceramide

Inositol phosphorylceramides (IPCs) are a class of anionic sphingolipids with a single inositol-phosphate head group coupled to ceramide. IPCs and more complex glycosylated IPCs have been identified in fungi, plants and protozoa but not in mammals. IPCs have also been identified in detergent resistant membranes in several organisms. Here we report on the membrane properties of the saturated N-palmitoyl-IPC (P-IPC) in one component bilayers as well as in complex bilayers together with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and cholesterol. The membrane properties of P-IPC were shown to be affected by calcium. According to anisotropy changes reported by DPH, the gel-to-liquid transition temperature (T_m) of P-IPC was 48 °C. Addition of 5 mM CaCl₂ during vesicle preparation markedly increased the T_m (65 °C). According to fluorescence quenching experiments in complex lipid mixtures, P-IPC formed sterol containing domains in an otherwise fluid environment. The P-IPC containing domains melted at a lower temperature and appeared to contain less sterol as compared to domains containing N-palmitoyl-sphingomyelin. Calcium further reduced the sterol content of the ordered domains and also increased the thermal stability of the domains. Calcium also induced vesicle aggregation of unilamellar vesicles containing P-IPC, as was observed by 4D confocal microscopy and dynamic light scattering. We believe that IPCs and the calcium induced effects could be important in numerous membrane associated cellular processes such as membrane fusion and in membrane raft linked processes.     

Domain formation and stability in complex lipid bilayers as reported by cholestatrienol

In this study, we used cholestatrienol (CTL) as a fluorescent reporter molecule to study sterol-rich L(o) domains in complex lipid bilayers. CTL is a fluorescent cholesterol analog that mimics the behavior of cholesterol well. The ability of 12SLPC to quench the fluorescence of cholestatrienol gives a measure of the amount of sterol included in L(o) domains in mixed lipid membranes. The stability of sterol-rich domains formed in complex lipid mixtures containing saturated sphingomyelins, phosphatidylcholines, or galactosylceramide as potential domain-forming lipids were studied. The amount of sterol associated with sterol-rich domains seemed to always increase with increasing temperature. The quenching efficiency was highly dependent on the domain-forming lipid present in complex lipid mixtures. Sphingomyelins formed stable sterol-enriched domains and were able to shield CTL from quenching better than the other lipids included in this study. The saturated phosphatidylcholines also formed sterol-rich domains, but the quenching efficiency in membranes with these was higher than with sphingomyelins and the domains melted at lower temperatures. PGalCer was not able to form sterol-enriched domains. However, we found that PGalCer stabilized sterol-rich domains formed in PSM-containing bilayers. Using a fluorescent ceramide analog, we also demonstrated that N-palmitoyl-ceramide displaced the sterol from sphingolipid-rich domains in mixed bilayer membranes.     

Theory of Raft Formation by the Cross-Linking of Saturated or Unsaturated Lipids in Model Lipid Bilayers

We consider the effect of cross-linking a small fraction of lipids, either saturated or unsaturated, in a mixture of saturated and unsaturated lipids and cholesterol. The change in phase behavior is examined utilizing a recent phenomenological model of the ternary system, which is extended to include a fourth component representing the cross-linked lipids. These lipids are taken to be identical to monomeric ones except for their reduced entropy of mixing. We find that even a relatively small amount of cross-linked lipids, less than 5 mol %, is sufficient to significantly expand the range of compositions within which there is coexistence between liquid-ordered and liquid-disordered phases. Equivalently, the cross-linking of lipids increases the liquid-liquid miscibility transition temperature, and therefore could bring about phase separation at a temperature at which, before cross-linking, there was only a single liquid phase.     

Miscibility of acyl-chain defined phosphatidylcholines with N-palmitoyl sphingomyelin in bilayer membranes

In this study we have used differential scanning calorimetry (DSC) to study the miscibility of different saturated phosphatidylcholines (PCs) with d-erythro-N-palmitoyl-sphingomyelin (16:0-SM). Information about the miscibility was obtained by observing the thermotropic phase behavior of binary mixtures of saturated PCs and 16:0-SM. The results obtained showed that PC miscibility in 16:0-SM was markedly affected by the PC acyl-chain composition. According to phase diagrams prepared from DSC data and the mid-transition temperatures of the main phase transition, the PC which formed the most ideal mixture with 16:0-SM was di-14:0-PC. However, the cooperativity of the main transition in PC/16:0-SM bilayers increased until the average acyl-chain length in the PC reached 15 carbons. Based on the criteria of the most ideal miscibility or the highest cooperativity of the main transition, we conclude that di-14:0-PC, 15:0/15:0-PC, and 14:0/16:0-PC interacted most favorably with 16:0-SM in bilayer membranes. Di-16:0-PC, to which 16:0-SM is often compared in biophysical studies, showed much less ideal miscibility.     

Melting behaviour of D-sucrose, D-glucose and D-fructose

The melting behaviour of d-sucrose, d-glucose and d-fructose was studied. The melting peaks were determined with DSC and the start of decomposition was studied with TG at different rates of heating. In addition, melting points were determined with a melting point apparatus. The samples were identified as d-sucrose, alpha-d-glucopyranose and beta-d-fructopyranose by powder diffraction measurements. There were differences in melting between the different samples of the same sugar and the rate of heating had a remarkable effect on the melting behaviour. For example, T(o), DeltaH(f) and T(i) (initial temperature of decomposition) at a 1 degrees Cmin(-1) rate of heating were 184.5 degrees C, 126.6Jg(-1) and 171.3 degrees C for d-sucrose, 146.5 degrees C, 185.4Jg(-1) and 152.0 degrees C for d-glucose and 112.7 degrees C, 154.1Jg(-1) and 113.9 degrees C for d-fructose. The same parameters at 10 degrees Cmin(-1) rate of heating were 188.9 degrees C, 134.4Jg(-1) and 189.2 degrees C for d-sucrose, 155.2 degrees C, 194.3Jg(-1) and 170.3 degrees C for d-glucose and 125.7 degrees C, 176.7Jg(-1) and 136.8 degrees C d-fructose. At slow rates of heating, there were substantial differences between the different samples of the same sugar. The melting point determination is a sensitive method for the characterization of crystal quality but it cannot be used alone for the identification of sugar samples in all cases. Therefore, the melting point method should be validated for different sugars.     

Thermodynamics of lipid interactions in complex bilayers

The mutual interactions between lipids in bilayers are reviewed, including mixtures of phospholipids, and mixtures of phospholipids and cholesterol (Chol). Binary mixtures and ternary mixtures are considered, with special emphasis on membranes containing Chol, an ordered phospholipid, and a disordered phospholipid. Typically the ordered phospholipid is a sphingomyelin (SM) or a long-chain saturated phosphatidylcholine (PC), both of which have high phase transitions temperatures; the disordered phospholipid is 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) or dioleoylphosphatidylcholine (DOPC). The unlike nearest-neighbor interaction free energies (ω_AB) between lipids (including Chol), obtained by an variety of unrelated methods, are typically in the range of 0-400 cal/mol in absolute value. Most are positive, meaning that the interaction is unfavorable, but some are negative, meaning it is favorable. It is of special interest that favorable interactions occur mainly between ordered phospholipids and Chol. The interpretation of domain formation in complex mixtures of Chol and phospholipids in terms of phase separation or condensed complexes is discussed in the light of the values of lipid mutual interactions.