Fluid-phase chain unsaturation controlling domain microstructure and phase in ternary lipid bilayers containing GalCer and cholesterol

We report the microstructure and phase behavior of three ternary mixtures each containing a long-chain saturated glycosphingolipid, galactosylceramide (GalCer), and cholesterol at room temperature. The unsaturation level of the fluid-phase component was varied by lipid choice, i.e., saturated 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), singly unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or doubly unsaturated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). GalCer was used because of its biological significance, for example, as a ligand in the sexual transmission of HIV and stimulator of natural killer T-cells. Supported lipid bilayers of the ternary mixtures were imaged by atomic force microscopy and GalCer-rich domains were characterized by area/perimeter ratios (A/P). GalCer domain phase transitions from solid (S) to liquid (L) phase were verified by domain behavior in giant unilamellar vesicles, which displayed two-dimensional microstructure similar to that of supported lipid bilayers. As cholesterol concentration was increased, we observed approximately 2.5, approximately 10, and approximately 20-fold decreases in GalCer domain A/P for bilayers in L-S phase coexistence containing DOPC, POPC, and DLPC, respectively. The transition to L-L phase coexistence occurred at approximately 10 mol % cholesterol for bilayers containing DOPC or POPC and was accompanied by maintenance of a constant A/P. L-L phase coexistence did not occur for bilayers containing DLPC. We systematically relate our results to the impact of chain unsaturation on the interaction of the fluid-phase lipid and cholesterol. Physiologically, these observations may give insight into the interplay of fatty acid chain unsaturation, sterol concentration, and lipid hydrophobic mismatch in membrane phenomena.     

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.     

Transmembrane helices can induce domain formation in crowded model membranes

We studied compositionally heterogeneous multi-component model membranes comprised of saturated lipids, unsaturated lipids, cholesterol, and α-helical TM protein models using coarse-grained molecular dynamics simulations. Reducing the mismatch between the length of the saturated and unsaturated lipid tails reduced the driving force for segregation into liquid-ordered (l(o)) and liquid-disordered (l(d)) lipid domains. Cholesterol depletion had a similar effect, and binary lipid mixtures without cholesterol did not undergo large-scale phase separation under the simulation conditions. The phase-separating ternary dipalmitoyl-phosphatidylcholine (DPPC)/dilinoleoyl-PC (DLiPC)/cholesterol bilayer was found to segregate into l(o) and l(d) domains also in the presence of a high concentration of ΤΜ helices. The l(d) domain was highly crowded with TM helices (protein-to-lipid ratio ~1:5), slowing down lateral diffusion by a factor of 5-10 as compared to the dilute case, with anomalous (sub)-diffusion on the μs time scale. The membrane with the less strongly unsaturated palmitoyl-linoleoyl-PC instead of DLiPC, which in the absence of TM α-helices less strongly deviated from ideal mixing, could be brought closer to a miscibility critical point by introducing a high concentration of TM helices. Finally, the 7-TM protein bacteriorhodopsin was found to partition into the l(d) domains irrespective of hydrophobic matching. These results show that it is possible to directly study the lateral reorganization of lipids and proteins in compositionally heterogeneous and crowded model biomembranes with coarse-grained molecular dynamics simulations, a step toward simulations of realistic, compositionally complex cellular membranes. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

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.     

Combining fluorescence lifetime and polarization microscopy to discriminate phase separated domains in giant unilamellar vesicles

Using fluorescence lifetime microscopy we study the structure of lipid domains in giant unilamellar vesicles made from sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine, and cholesterol. Lifetimes and orientation of a derivative of the fluorescent probe DPH embedded in the membrane were measured for binary and ternary lipid mixtures incorporating up to 42 mol % of cholesterol. The results show that adding cholesterol always increases the lifetime of the probe studied. In addition, the analysis of the probe orientation indicates that cholesterol has little influence on the ordering of the sphingomyelin alkyl chains whereas it has a noticeable effect on the structure of the 1,2-dioleoyl-sn-glycero-3-phosphocholine chains. The measurements made on the orientation and lifetime of the probe show the structure of the membrane in its liquid ordered and liquid disordered domains.     

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.     

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.     

Cholesterol Partition and Condensing Effect in Phase-Separated Ternary Mixture Lipid Multilayers

The cholesterol partitioning and condensing effect in the liquid-ordered (L_o) and liquid-disordered (L_d) phases were systematically investigated for ternary mixture lipid multilayers consisting of 1:1 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphocholine with varying concentrations of cholesterol. X-ray lamellar diffraction was used to deduce the electron density profiles of each phase. The cholesterol concentration in each phase was quantified by fitting of the electron density profiles with a newly invented basic lipid profile scaling method that minimizes the number of fitting parameters. The obtained cholesterol concentration in each phase versus total cholesterol concentration in the sample increases linearly for both phases. The condensing effect of cholesterol in ternary lipid mixtures was evaluated in terms of phosphate-to-phosphate distances, which together with the estimated cholesterol concentration in each phase was converted into an average area per molecule. In addition, the cholesterol position was determined to a precision of (±0.7Å) and an increase of disorder in the lipid packing in the L_o phase was observed for total cholesterol concentration of 20∼30%.     

A correlation between lipid domain shape and binary phospholipid mixture composition in free standing bilayers: A two-photon fluorescence microscopy study

Giant unilamellar vesicles (GUVs) composed of different phospholipid binary mixtures were studied at different temperatures, by a method combining the sectioning capability of the two-photon excitation fluorescence microscope and the partition and spectral properties of 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan) and Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (N-Rh-DPPE). We analyzed and compared fluorescence images of GUVs composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DLPC/DPPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DLPC/DSPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-diarachidoyl-sn-glycero-3-phosphocholine (DLPC/DAPC), 1, 2-dimyristoyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DMPC/DSPC) (1:1 mol/mol in all cases), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine/1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPE/DMPC) (7:3 mol/mol) at temperatures corresponding to the fluid phase and the fluid-solid phase coexistence. In addition, we studied the solid-solid temperature regime for the DMPC/DSPC and DMPE/DMPC mixtures. From the Laurdan intensity images the generalized polarization function (GP) was calculated at different temperatures to characterize the phase state of the lipid domains. We found a homogeneous fluorescence distribution in the GUV images at temperatures corresponding to the fluid region for all of the lipid mixtures. At temperatures corresponding to phase coexistence we observed concurrent fluid and solid domains in the GUVs independent of the lipid mixture. In all cases the lipid solid domains expanded and migrated around the vesicle surface as we decreased the temperature. The migration of the solid domains decreased dramatically at temperatures close to the solid-fluid-->solid phase transition. For the DLPC-containing mixtures, the solid domains showed line, quasicircular, and dendritic shapes as the difference in the hydrophobic chain length between the components of the binary mixture increases. In addition, for the saturated PC-containing mixtures, we found a linear relationship between the GP values for the fluid and solid domains and the difference between the hydrophobic chain length of the binary mixture components. Specifically, at the phase coexistence temperature region the difference in the GP values, associated with the fluid and solid domains, increases as the difference in the chain length of the binary mixture component increases. This last finding suggests that in the solid-phase domains, the local concentration of the low melting temperature phospholipid component increases as the hydrophobic mismatch decreases. At the phase coexistence temperature regime and based on the Laurdan GP data, we observe that when the hydrophobic mismatch is 8 (DLPC/DAPC), the concentration of the low melting temperature phospholipid component in the solid domains is negligible. This last observation extends to the saturated PE/PC mixtures at the phase coexistence temperature range. For the DMPC/DSPC we found that the nonfluorescent solid regions gradually disappear in the solid temperature regime of the phase diagram, suggesting lipid miscibility. This last result is in contrast with that found for DMPE/DMPC mixtures, where the solid domains remain on the GUV surface at temperatures corresponding to that of the solid region. In all cases the solid domains span the inner and outer leaflets of the membrane, suggesting a strong coupling between the inner and outer monolayers of the lipid membrane. This last finding extends previous observations of GUVs composed of DPPE/DPPC and DLPC/DPPC mixtures (, Biophys. J. 78:290-305).     

Lipid Organization in Mixed Lipid Membranes Driven by Intrinsic Curvature Difference

Laurdan fluorescence, novel spectral fitting, and dynamic light scattering were combined to determine lateral lipid organization in mixed lipid membranes of the oxidized lipid, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC), and each of the three bilayer lipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). Second harmonic spectra were computed to determine the number of elementary emissions present. All mixtures indicated two emissions. Accordingly, spectra were fit to two log-normal distributions. Changes with PGPC mole fraction, X_PGPC, of the area of the shorter wavelength line and of dynamic light scattering-derived aggregate sizes show that: DPPC and PGPC form component-separated mixed vesicles for X_PGPC ≤ 0.2 and coexisting vesicles and micelles for X_PGPC > 0.2 in gel and liquid-ordered phases and for all X_PGPC in the liquid-disordered phase; POPC and PGPC form randomly mixed vesicles for X_PGPC ≤ 0.2 and component-separated mixed vesicles for X_PGPC > 0.2. DOPC and PGPC separate into vesicles and micelles. Component segregation is due to unstable inhomogeneous membrane curvature stemming from lipid-specific intrinsic curvature differences between mixing molecules. PGPC is inverse cone-shaped because its truncated tail with a terminal polar group points into the interface. It is similar to and mixes with POPC, also an inverse cone because of mobility of its unsaturated tail. PGPC is least similar to DOPC because mobilities of both unsaturated tails confer a cone shape to DOPC, and PGPC separates form DOPC. DPPC and PGPC do not mix in the liquid-disordered phase because mobility of both tails in this phase renders DPPC a cone. DPPC is a cylinder in the gel phase and of moderate similarity to PGPC and mixes moderately with PGPC.     

Morphometric Image Analysis of Giant Vesicles: A New Tool for Quantitative Thermodynamics Studies of Phase Separation in Lipid Membranes

We have developed a strategy to determine lengths and orientations of tie lines in the coexistence region of liquid-ordered and liquid-disordered phases of cholesterol containing ternary lipid mixtures. The method combines confocal-fluorescence-microscopy image stacks of giant unilamellar vesicles (GUVs), a dedicated 3D-image analysis, and a quantitative analysis based in equilibrium thermodynamic considerations. This approach was tested in GUVs composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-palmitoyl-sn-glycero-3-phosphocholine/cholesterol. In general, our results show a reasonable agreement with previously reported data obtained by other methods. For example, our computed tie lines were found to be nonhorizontal, indicating a difference in cholesterol content in the coexisting phases. This new, to our knowledge, analytical strategy offers a way to further exploit fluorescence-microscopy experiments in GUVs, particularly retrieving quantitative data for the construction of three lipid-component-phase diagrams containing cholesterol.     

Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol

We use fluorescence microscopy to directly observe liquid phases in giant unilamellar vesicles. We find that a long list of ternary mixtures of high melting temperature (saturated) lipids, low melting temperature (usually unsaturated) lipids, and cholesterol produce liquid domains. For one model mixture in particular, DPPC/DOPC/Chol, we have mapped phase boundaries for the full ternary system. For this mixture we observe two coexisting liquid phases over a wide range of lipid composition and temperature, with one phase rich in the unsaturated lipid and the other rich in the saturated lipid and cholesterol. We find a simple relationship between chain melting temperature and miscibility transition temperature that holds for both phosphatidylcholine and sphingomyelin lipids. We experimentally cross miscibility boundaries both by changing temperature and by the depletion of cholesterol with beta-cyclodextrin. Liquid domains in vesicles exhibit interesting behavior: they collide and coalesce, can finger into stripes, and can bulge out of the vesicle. To date, we have not observed macroscopic separation of liquid phases in only binary lipid mixtures.     

Domain nucleation rates and interfacial line tensions in supported bilayers of ternary mixtures containing galactosylceramide

Domains within the plane of the plasma membrane, referred to as membrane rafts, have been a topic of considerable interest in the field of membrane biophysics. Although model membrane systems have been used extensively to study lipid phase behavior as it relates to the existence of rafts, very little work has focused on either the initial stage of lipid domain nucleation, or the relevant physical parameters such as temperature and interfacial line tension which control nucleation. In this work, we utilize a method in which the kinetic process of lipid domain nucleation is imaged by atomic force microscopy and modeled using classical theory of nucleation to map interfacial line tension in ternary lipid mixtures. These mixtures consist of a fluid phase lipid component (1,2-dilauroyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, or 1,2-dioleoyl-sn-glycero-3-phosphocholine), a solid phase component (galactosylceramide), and cholesterol. Interfacial line tension measurements of galactosylceramide-rich domains track with our previously measured area/perimeter ratios and height mismatches measured here. Line tension also follows known trends in cholesterol interactions and partitioning, as we observed previously with area/perimeter ratios. Our line tension measurements are discussed in combination with recent line tension measurements to address line tension regulation by cholesterol and the dynamic nature of membrane rafts.     

Lipid lateral diffusion in bilayers with phosphatidylcholine, sphingomyelin and cholesterol. An NMR study of dynamics and lateral phase separation

Pulsed field gradient (pfg)-NMR measurements of the lipid lateral diffusion coefficients in several macroscopically aligned bilayer systems were summarized from previous and new studies. The aim was to carry out a comparison of the translational dynamics for bilayers with various mixtures of l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and chicken egg yolk sphingomyelin (eSM), with or without cholesterol. New useful information was obtained on the dynamics in these lipid bilayers that has not been previously appreciated. Thus, we were able to propose that the driving force behind the phase separation into l(d)and l(o)phases evolves from the increasing difficulty to incorpotate DOPC into a highly ordered phase. Our results suggest that DOPC has a preference to be located in a disordered phase, while DPPC and eSM prefer the ordered phase. Quite unexpectedly, CHOL seems to partition into both phases to roughly the same extent, indicating that CHOL has no particular preference for any of the l(d)or l(o) phases, and there are no specific interactions between CHOL and saturated lipids.     

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.     

Phase Equilibria in DOPC/DPPC-d62/Cholesterol Mixtures

There is broad interest in the question of fluid-fluid phase coexistence in membranes, in particular, whether evidence for liquid-disordered (l_d)-liquid-ordered (l_o) two-phase regions or membrane “rafts” can be found in natural membranes. In model membrane systems, such phase behavior is observed, and we have used deuterium nuclear magnetic resonance spectroscopy to map the phase boundaries of ternary mixtures containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), chain-perdeuterated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC-d₆₂), and cholesterol. For both this ternary model system and the binary DPPC-d₆₂/cholesterol sytem, we present clear evidence for l_d-l_o two-phase coexistence. We have selected sample compositions to focus on this region of fluid-fluid phase coexistence and to determine its temperature and composition ranges. The deuterium nuclear magnetic resonance spectra for compositions near the l_d-l_o phase boundary at high cholesterol concentrations show evidence of exchange broadening or critical fluctuations in composition, similar to that reported by Vist and Davis. There appears to be a line of critical compositions ranging from 48°C for a DOPC/DPPC-d₆₂/cholesterol composition of 0:75:25, to ∼−8°C for the composition 57:14:29. At temperatures below this two-phase region, there is a region of three-phase coexistence (l_d-l_o-gel). These results are collected and presented in terms of a partial ternary phase diagram that is consistent with previously reported results of Vist and Davis.     

Direct measurement of phase coexistence in DPPC/cholesterol vesicles using Raman spectroscopy

The phase behavior of bilayers of binary mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol has been studied using Raman spectroscopy. It is observed that the shape of the cholesterol vibrational spectrum in lipid-cholesterol binary mixtures does not vary significantly with either the cholesterol concentration or the temperature. This permits determination of the lipid vibrational signatures of the liquid-disordered (l(d)), solid-ordered (s(o)) and liquid-ordered (l(o)) phases. Within the phase coexistence region, the measured spectra are described very well by a linear combination of the different spectral components, which permits a quantitative analysis of the phase diagram. In contrast to earlier findings, our experiments provide no indication of a phase boundary at low cholesterol concentration. The upper boundary of the phase coexistence region is found at approximately 27 and approximately 22 mol% for l(d)-l(o) and s(o)-l(o) coexistence region, respectively. Within these phase coexistence regions, the partitioning of cholesterol between the cholesterol-poor and the cholesterol-rich phases is in close agreement with the lever rule.     

Phase Separation in Lipid Membranes

Cell membranes show complex behavior, in part because of the large number of different components that interact with each other in different ways. One aspect of this complex behavior is lateral organization of components on a range of spatial scales. We found that lipid-only mixtures can model the range of size scales, from approximately 2 nm up to microns. Furthermore, the size of compositional heterogeneities can be controlled entirely by lipid composition for mixtures such as 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 or sphingomyelin (SM)/DOPC/POPC/cholesterol. In one region of special interest, because of its connection to cell membrane rafts, nanometer-scale domains of liquid-disordered phase and liquid-ordered phase coexist over a wide range of compositions.     

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.