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.     

Microviscosity parameters and protein mobility in biological membranes.

A fluorescence polarization technique with 1,6-diphenyl 1,3,5-hexatriene as a probe were employed to determine the microviscosity, n, in liposomes and biological membranes of different cholesterol to phospholipid mol ratio. From the temperature profile of n the flow activation energy, deltaE, and the unit flow volume, V, were derived. The increase of cholesterol/phospholipid ratio in liposomes is followed by a marked increase in n and a decrease in both deltaE and V. Liposomes of the same phospholipid composition as human erythrocyte membranes display in the extreme cases of cholesterol/phospholipid ratios 0 and 1.4 the values of n(25 degrees C) = 1.8 and 9.1 P, and deltaE = 15.0 and 6.5 kcal/mol, respectively. For most membranes studied the fluorescence polarization characteristics and the corresponding n values are similar to those obtained with these liposomes when the cholesterol/phospholipid level of the liposomes and the membranes were the same. However, unlike in liposomes deltaE of all membranes is in the narrow range of 6.5-8.5 kcal/mol, regardless of its cholesterol/phospholipid level. It is plausible that this is a general characteristic of biological membranes which originates from the vertical movement of membrane proteins to an equilibrium position which maintains constant deltaE and V values. This type of movement should affect the interrelation between lipid fluidity and protein mobility. Lipid microviscosity and the degree of rotational mobility of concanavalin A receptor sites in cell membranes were therefore determined. The examined cells were normal and malignant fibroblasts, as an example of cells that form solid tumours in vivo, and normal and malignant lymphocytes, as an example of cells that form ascites tumours in vivo. In both cell systems, opposite correlations between the lipid fluidity and the mobility of concanavalin A receptors were observed. In the fibroblasts the malignant cells possess a lower lipid fluidity but a higher receptor mobility, whereas in the lymphocytes the malignant cells possess a higher lipid fluidity but a lower receptor mobility. Thus, in these cell systems the degree of rotational mobility of concanavalin A receptors increases upon decreasing the lipid fluidity and decreases upon increasing the fluidity of the lipid core. This dynamic feature is in line with the above proposal according to which the concanavalin A receptor sites become more exposed to the aqueous surrounding upon increasing the microviscosity of the lipid layer and vice versa.     

Does cholesterol suppress the antimicrobial peptide induced disruption of lipid raft containing membranes?

The activity of antimicrobial peptides has been shown to depend on the composition of the target cell membrane. The bacterial selectivity of most antimicrobial peptides has been attributed to the presence of abundant acidic phospholipids and the absence of cholesterol in bacterial membranes. The high amount of cholesterol present in eukaryotic cell membranes is thought to prevent peptide-induced membrane disruption by increasing the cohesion and stiffness of the lipid bilayer membrane. While the role of cholesterol on an antimicrobial peptide-induced membrane disrupting activity has been reported for simple, homogeneous lipid bilayer systems, it is not well understood for complex, heterogeneous lipid bilayers exhibiting phase separation (or "lipid rafts"). In this study, we show that cholesterol does not inhibit the disruption of raft-containing 1,2-dioleoyl-sn-glycero-3-phosphocholine:1,2-dipalmitoyol-sn-glycero-3-phosphocholine model membranes by four different cationic antimicrobial peptides, MSI-78, MSI-594, MSI-367 and MSI-843 which permeabilize membranes. Conversely, the presence of cholesterol effectively inhibits the disruption of non-raft containing 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dipalmitoyol-sn-glycero-3-phosphocholine lipid bilayers, even for antimicrobial peptides that do not show a clear preference between the ordered gel and disordered liquid-crystalline phases. Our results show that the peptide selectivity is not only dependent on the lipid phase but also on the presence of phase separation in heterogeneous lipid systems.     

Ordering effects of cholesterol and its analogues

Without any exaggeration, cholesterol is one of the most important lipid species in eukaryotic cells. Its effects on cellular membranes and functions range from purely mechanistic to complex metabolic ones, besides which it is also a precursor of the sex hormones (steroids) and several vitamins. In this review, we discuss the biophysical effects of cholesterol on the lipid bilayer, in particular the ordering and condensing effects, concentrating on the molecular level or inter-atomic interactions perspective, starting from two-component systems and proceeding to many-component ones e.g., modeling lipid rafts. Particular attention is paid to the roles of the methyl groups in the cholesterol ring system, and their possible biological function. Although our main research methodology is computer modeling, in this review we make extensive comparisons between experiments and different modeling approaches.     

Ordering effects of cholesterol and its analogues

Without any exaggeration, cholesterol is one of the most important lipid species in eukaryotic cells. Its effects on cellular membranes and functions range from purely mechanistic to complex metabolic ones, besides which it is also a precursor of the sex hormones (steroids) and several vitamins. In this review, we discuss the biophysical effects of cholesterol on the lipid bilayer, in particular the ordering and condensing effects, concentrating on the molecular level or inter-atomic interactions perspective, starting from two-component systems and proceeding to many-component ones e.g., modeling lipid rafts. Particular attention is paid to the roles of the methyl groups in the cholesterol ring system, and their possible biological function. Although our main research methodology is computer modeling, in this review we make extensive comparisons between experiments and different modeling approaches.     

Physical properties of the lipid bilayer membrane made of calf lens lipids: EPR spin labeling studies

The physical properties of a membrane derived from the total lipids of a calf lens were investigated using EPR spin labeling and were compared with the properties of membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in all three membranes. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are practically identical in lens lipid and POPC/Chol membranes, but differ drastically from profiles in pure POPC membranes. In both lens lipid and POPC/Chol membranes, the lipids are strongly immobilized at all depths, which is in contrast to the high fluidity of the POPC membrane. Hydrophobicity and oxygen transport parameter profiles in lens lipid and POPC/Chol membranes have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. At this position, hydrophobicity increases from the level of methanol to the level of hexane, and the oxygen transport parameter increases by a factor of 2-3. These profiles in POPC membranes are bell-shaped. It is concluded that the high level of cholesterol in lens lipids makes the membrane stable, immobile, and impermeable to both polar and nonpolar molecules.     

Physical properties of the lipid bilayer membrane made of calf lens lipids: EPR spin labeling studies

The physical properties of a membrane derived from the total lipids of a calf lens were investigated using EPR spin labeling and were compared with the properties of membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in all three membranes. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are practically identical in lens lipid and POPC/Chol membranes, but differ drastically from profiles in pure POPC membranes. In both lens lipid and POPC/Chol membranes, the lipids are strongly immobilized at all depths, which is in contrast to the high fluidity of the POPC membrane. Hydrophobicity and oxygen transport parameter profiles in lens lipid and POPC/Chol membranes have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. At this position, hydrophobicity increases from the level of methanol to the level of hexane, and the oxygen transport parameter increases by a factor of 2-3. These profiles in POPC membranes are bell-shaped. It is concluded that the high level of cholesterol in lens lipids makes the membrane stable, immobile, and impermeable to both polar and nonpolar molecules.     

Effect of cholesterol on lateral diffusion of fluorescent lipid probes in native hippocampal membranes

Cholesterol is an abundant lipid of mammalian membranes and plays a crucial role in membrane organization, dynamics, function and sorting. The role of cholesterol in membrane organization has been a subject of intense investigation that has largely been carried out in model membrane systems. An extension of these studies in natural membranes, more importantly in neuronal membranes, is important to establish a relationship between disease states and changes in membrane physical properties resulting from an alteration in lipid composition. We have monitored the lateral diffusion of lipid probes, DiIC(18)(3) and FAST DiI which are similar in their intrinsic fluorescence properties but differ in their structure, in native and cholesterol-depleted hippocampal membranes using the fluorescence recovery after photobleaching (FRAP) approach. Our results show that the mobility of these probes is in general higher in hippocampal membranes depleted of cholesterol. Interestingly, the increase in mobility of these probes does not linearly correlate with the extent of cholesterol depletion. These results assume significance in the light of recent reports on the requirement of cholesterol to support the function of the G-protein coupled serotonin(1A) receptor present endogenously in hippocampal membranes.     

Aspirin inhibits formation of cholesterol rafts in fluid lipid membranes

Aspirin and other non-steroidal anti-inflammatory drugs have a high affinity for phospholipid membranes, altering their structure and biophysical properties. Aspirin has been shown to partition into the lipid head groups, thereby increasing membrane fluidity. Cholesterol is another well known mediator of membrane fluidity, in turn increasing membrane stiffness. As well, cholesterol is believed to distribute unevenly within lipid membranes leading to the formation of lipid rafts or plaques. In many studies, aspirin has increased positive outcomes for patients with high cholesterol. We are interested if these effects may be, at least partially, the result of a non-specific interaction between aspirin and cholesterol in lipid membranes.We have studied the effect of aspirin on the organization of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) membranes containing cholesterol. Through Langmuir–Blodgett experiments we show that aspirin increases the area per lipid and decreases compressibility at 32.5 mol% cholesterol, leading to a significant increase of fluidity of the membranes. Differential scanning calorimetry provides evidence for the formation of meta-stable structures in the presence of aspirin. The molecular organization of lipids, cholesterol and aspirin was studied using neutron diffraction. While the formation of rafts has been reported in binary DPPC/cholesterol membranes, aspirin was found to locally disrupt membrane organization and lead to the frustration of raft formation. Our results suggest that aspirin is able to directly oppose the formation of cholesterol structures through non-specific interactions with lipid membranes.     

Effects of cholesterol on the divalent cation-mediated interactions of vesicles containing amino and choline phospholipids

We have used assays of lipid probe mixing, contents mixing and contents leakage to monitor the divalent cation-mediated interactions between lipid vesicles containing phosphatidylserine (PS) as a minority component together with mixtures of phosphatidylethanolamine (PE), phosphatidylcholine (PC) or sphingomyelin, and cholesterol in varying proportions. The initial rates of calcium- and magnesium-induced lipid probe quenching between vesicles, which reflect primarily the rates of vesicle aggregation, are strongly reduced as progressively higher proportions of PC or sphingomyelin are incorporated into PE/PS vesicles. The initial rates of divalent cation-induced contents mixing and contents leakage for PE/PS vesicles are also strongly reduced when choline phospholipids are incorporated into the vesicles in even low molar proportions. Sphingomyelin has a more potent inhibitory effect on these processes than does PC at an equal level in the vesicle membranes. The inclusion of cholesterol in these vesicles, at levels up to 1:2 moles sterol/mole phospholipid, has little effect on the rates of calcium- or magnesium-induced vesicle aggregation. However, cholesterol significantly enhances the initial rates of vesicle contents mixing and contents leakage in the presence of divalent cations when the vesicles contain choline as well as amino phospholipids. This effect is substantial only when the level of cholesterol exceeds the level of choline phospholipids in the vesicles. These results may have significance for the fusion of certain cellular membranes in mammalian cells, whose cytoplasmic faces have lipid compositions very similar to those of the vesicles examined in this study.     

Role of cholesterol in lipid raft formation: lessons from lipid model systems

Biochemical and cell-biological experiments have identified cholesterol as an important component of lipid 'rafts' and related structures (e.g., caveolae) in mammalian cell membranes, and membrane cholesterol levels as a key factor in determining raft stability and organization. Studies using cholesterol-containing bilayers as model systems have provided important insights into the roles that cholesterol plays in determining lipid raft behavior. This review will discuss recent progress in understanding two aspects of lipid-cholesterol interactions that are particularly relevant to understanding the formation and properties of lipid rafts. First, we will consider evidence that cholesterol interacts differentially with different membrane lipids, associating particularly strongly with saturated, high-melting phospho- and sphingolipids and particularly weakly with highly unsaturated lipid species. Second, we will review recent progress in reconstituting and directly observing segregated raft-like (liquid-ordered) domains in model membranes that mimic the lipid compositions of natural membranes incorporating raft domains.     

Cholesterol modulates maculosin's orientation in model systems of biological membranes. Relevance towards putative molecular recognition

Fluorescence techniques were used to study (1) the extent of insertion of the bioactive cyclic dipeptide cyclo(l-tyrosyl-l-prolyl), maculosin, in model systems of membranes of 1, 2-palmitoyl-sn-glycero-3-phosphatidyl choline (DPPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl choline (POPC), (2) its in-depth location in those lipidic membranes, and (3) the influence of cholesterol on the dipeptides's location and orientation. Partition into lipidic bilayers is extensive, mainly for liquid crystalline phase membranes (K(p)=1.3x10(4)). Maculosin locates at the lipid head groups level regardless of the membrane system. Nevertheless, its orientation is lipid phase dependent. When maculosin was inserted in liquid crystalline phase bilayers, its phenolic ring was perpendicular to the membrane surface, whereas it changed orientation when inserted in gel phase membranes. Cholesterol was able to reverse the lipid phase influence on maculosin's orientation.     

Effect of cholesterol on the physical state and function of lymphocyte membranes

Effect of gradual increase of cholesterol content in T-lymphocyte membranes on the structure and physical state of plasmic membrane lipids and activities of the membrane-bound enzymes was investigated. The increase in cholesterol content was shown to result in a two-phase change of luminescence parameters of the fluorescent probes dimethylaminochalcone and pyrene, which indicates heterogeneity of cholesterol in the membranes. With the growth of steroid content in the cell membranes, at first, we observed a sharp decrease in the lipid bilayer fluidity and inhibition of Na+, K+-ATPase activity, which at the molar ratio cholesterol/phospholipids 0.6 in thymocyte membranes, remains at the same level. With higher cholesterol concentrations ATPase activity did not change. The effect of cholesterol on ATPase activity was in a good agreement with the effect of membrane lipids on fluidity. It is suggested that two pools of cholesterol molecules exist in the membranes, differing in their effects of bilayer fluidity and functional activity of the membranes.     

Role of cholesterol in lipid raft formation: lessons from lipid model systems

Biochemical and cell-biological experiments have identified cholesterol as an important component of lipid ‘rafts’ and related structures (e.g., caveolae) in mammalian cell membranes, and membrane cholesterol levels as a key factor in determining raft stability and organization. Studies using cholesterol-containing bilayers as model systems have provided important insights into the roles that cholesterol plays in determining lipid raft behavior. This review will discuss recent progress in understanding two aspects of lipid-cholesterol interactions that are particularly relevant to understanding the formation and properties of lipid rafts. First, we will consider evidence that cholesterol interacts differentially with different membrane lipids, associating particularly strongly with saturated, high-melting phospho- and sphingolipids and particularly weakly with highly unsaturated lipid species. Second, we will review recent progress in reconstituting and directly observing segregated raft-like (liquid-ordered) domains in model membranes that mimic the lipid compositions of natural membranes incorporating raft domains.     

Effects of cholesterol on the structural transitions induced by diacylglycerol in phosphatidylcholine and phosphatidylethanolamine bilayer systems

The transient membrane lipid diacylglycerol (DG) is known to modify and destabilize phospholipid bilayers and can lead to the formation of nonbilayer structures. Since cholesterol forms a major fraction of many plasma membranes, we have investigated how it modifies the structural effects of DG on bilayers of egg phosphatidylcholine (PC) and egg phosphatidylethanolamine (PE). We view these systems as modelling the behaviour of local, DG-containing sites in membranes. Using X-ray diffraction, we have characterized the lamellar (L alpha) and inverse hexagonal (HII) structures that these ternary lipid mixtures form in excess aqueous solution. As the DG level increases, the lipid progresses from a single L alpha structure to a mixture of L alpha and HII, and then to a pure HII structure. This allows determination of the DG levels at which the HII transition begins, which we interpret as those levels that destabilize bilayers. In both PC and PE bilayers, the presence of 30 mol% cholesterol reduces the amounts of DG required to destabilize the bilayer structure. The destabilization can be translated into the number of neighbouring lipid molecules that a DG molecule perturbs, and of bilayer areas that it affects. The data show that the presence of cholesterol greatly enhances the perturbing effects of DG. We examine the possible role of DG in enzyme activation and membrane fusion.     

Inhibition of cholesterol efflux by 7-ketocholesterol: comparison between cells, plasma membrane vesicles, and liposomes as cholesterol donors.

Cholesterol removal from lipid-loaded macrophages is an important, potentially antiatherogenic process, and we have previously shown that an oxysterol, 7-ketocholesterol (7K), can impair efflux to lipid-free apoprotein A-1 (apoA-1). This publication investigates whether incorporation of 7K into membranes could account for this impairment of cholesterol efflux. Cholesterol efflux was studied from lipoprotein-loaded THP-1 cells, from plasma membrane vesicles obtained from these cells, and from artificial, protein-free liposomes. Impairment of cholesterol efflux by 7K was observed for all cholesterol donor systems whether measured as decline in cholesterol removal rates or as the percentage mass of total cellular cholesterol exported. 7-Ketocholesterol itself was not removed by apoA-1 from any of the cholesterol donor systems. Increasing membrane cholesterol content increased the rate of cholesterol removal by apoA-1 (as seen with plasma membrane vesicles), the quantity of cholesterol removed at equilibrium (liposomes), or both (whole cells). Although the minimum inhibitory 7K concentrations varied between the cholesterol donor systems, 7K inhibited cholesterol efflux in all systems. It was concluded that 7K induces alteration in membranes which decreased the efficiency of cholesterol efflux and the quantity of removed cholesterol induced by apoA-1. As cell membrane proteins are not essential for cholesterol efflux in these systems, the impairment of such by 7K suggests that its effect on membrane lipid composition and its structure are key regulatory elements in this efflux process.     

Influence of the membrane environment on cholesterol transfer

Cholesterol, an essential component in biological membranes, is highly unevenly distributed within the cell, with most localized in the plasma membrane while only a small fraction is found in the endoplasmic reticulum, where it is synthesized. Cellular membranes differ in lipid composition and protein content, and these differences can exist across their leaflets too. This thermodynamic landscape that cellular membranes impose on cholesterol is expected to modulate its transport. To uncover the role the membrane environment has on cholesterol inter- and intra-membrane movement, we used time-resolved small angle neutron scattering to study the passive movement of cholesterol between and within membranes with varying degrees of saturation content. We found that cholesterol moves systematically slower as the degree of saturation in the membranes increases, from a palmitoyl oleyl phosphotidylcholine membrane, which is unsaturated, to a dipalmitoylphosphatidylcholine (DPPC) membrane, which is fully saturated. Additionally, we found that the energetic barrier to move cholesterol in these phosphatidylcholine membranes is independent of their relative lipid composition and remains constant for both flip-flop and exchange at ∼100 kJ/mol. Further, by replacing DPPC with the saturated lipid palmitoylsphingomyelin, an abundant saturated lipid of the outer leaflet of the plasma membrane, we found the rates decreased by a factor of two. This finding is in stark contrast with recent molecular dynamic simulations that predict a dramatic slow-down of seven orders of magnitude for cholesterol flipping in membranes with a similar phosphocholine and SM lipid composition.     

Preparation of Artificial Plasma Membrane Mimicking Vesicles with Lipid Asymmetry

Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the most important features of eukaryotic cellular membranes. However, commonly used model membrane vesicles cannot provide control of lipid distribution between inner and outer leaflets. We recently developed methods to prepare asymmetric model membrane vesicles, but facile incorporation of a highly controlled level of cholesterol was not possible. In this study, using hydroxypropyl-α-cyclodextrin based lipid exchange, a simple method was devised to prepare large unilamellar model membrane vesicles that closely resemble mammalian plasma membranes in terms of their lipid composition and asymmetry (sphingomyelin (SM) and/or phosphatidylcholine (PC) outside/phosphatidylethanolamine (PE) and phosphatidylserine (PS) inside), and in which cholesterol content can be readily varied between 0 and 50 mol%. We call these model membranes “artificial plasma membrane mimicking” (“PMm”) vesicles. Asymmetry was confirmed by both chemical labeling and measurement of the amount of externally-exposed anionic lipid. These vesicles should be superior and more realistic model membranes for studies of lipid-lipid and lipid-protein interaction in a lipid environment that resembles that of mammalian plasma membranes.     

Kinetics of cholesterol extraction from lipid membranes by methyl-beta-cyclodextrin--a surface plasmon resonance approach

The kinetics of cholesterol extraction from cellular membranes is complex and not yet completely understood. In this paper we have developed an experimental approach to directly monitor the extraction of cholesterol from lipid membranes by using surface plasmon resonance and model lipid systems. Methyl-beta-cyclodextrin was used to selectively remove cholesterol from large unilamellar vesicles of various compositions. The amount of extracted cholesterol is highly dependent on the composition of lipid membrane, i.e. the presence of sphingomyelin drastically reduced and slowed down cholesterol extraction by methyl-beta-cyclodextrin. This was confirmed also in the erythrocyte ghosts system, where more cholesterol was extracted after erythrocytes were treated with sphingomyelinase. We further show that the kinetics of the extraction is mono-exponential for mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol. The kinetics is complex for ternary lipid mixtures composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine, bovine brain sphingomyelin and cholesterol. Our results indicate that the complex kinetics observed in experiments with cells may be the consequence of lateral segregation of lipids in cell plasma membrane.     

Investigation of stratum corneum lipid model membranes with free fatty acid composition by neutron diffraction

The outermost epidermal layer, the stratum corneum (SC), is the main skin barrier. Studies of SC model systems enable characterization of the influence of individual lipids on the organization of the SC lipid matrix, which is the main pathway of water through the skin. This work presents a neutron diffraction study of the SC model membranes based on short-chain ceramide 6 with nearly realistic composition of free fatty acids (FFA) at physiological temperature of the SC. The influence of FFA and the effect of cholesterol–cholesterol sulfate substitution on the structure and hydration of the SC model membranes are described. The structure of the SC membrane with FFA is close to the structure of the earlier studied SC membrane based on short-chain palmitic acid (PA) and does not vary significantly under changes of the ratio of the main membrane components. FFA accelerates membrane swelling at the same low level of hydration of both PA- and FFA-containing membranes. The substitution of cholesterol sulfate by cholesterol in the membrane composition decreases membrane swelling and leads to phase separation in the model system.