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.     

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.     

Use of cyclodextrin for AFM monitoring of model raft formation

The lipid rafts membrane microdomains, enriched in sphingolipids and cholesterol, are implicated in numerous functions of biological membranes. Using atomic force microscopy, we have examined the effects of cholesterol-loaded methyl-beta-cyclodextrin (MbetaCD-Chl) addition to liquid disordered (l(d))-gel phase separated dioleoylphosphatidylcholine (DOPC)/sphingomyelin (SM) and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC)/SM supported bilayers. We observed that incubation with MbetaCD-Chl led to the disappearance of domains with the formation of a homogeneously flat bilayer, most likely in the liquid-ordered (l(o)) state. However, intermediate stages differed with the passage through the coexistence of l(o)-l(d) phases for DOPC/SM samples and of l(o)-gel phases for POPC/SM bilayers. Thus, gel phase SM domains surrounded by a l(o) matrix rich in cholesterol and POPC could be observed just before reaching the uniform l(o) state. This suggests that raft formation in biological membranes could occur not only via liquid-liquid but also via gel-liquid immiscibility. The data also demonstrate that MbetaCD-Chl as well as the unloaded cyclodextrin MbetaCD make holes and preferentially extract SM in supported bilayers. This strongly suggests that interpretation of MbetaCD and MbetaCD-Chl effects on cell membranes only in terms of cholesterol movements have to be treated with caution.     

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.     

Influence of 7α-hydroxycholesterol on sphingomyelin and sphingomyelin/phosphatidylcholine films - The Langmuir monolayer study complemented with theoretical calculations

Under pathological conditions, cholesterol oxidation products (oxysterols) appear in enhanced concentration in blood and cerebrospinal fluid, which leads to cytotoxic effect, especially in central nervous system. However, the mode of action of oxysterols on the membrane level has not been fully resolved. In this paper we have investigated the interaction between 7α- hydroxycholesterol, 7α-OH (one of the most abundant oxysterol in human body) and two major membrane lipids: sphingomyelin, SM (basic component of lipid rafts and nerve membrane) and 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, POPC (main phospholipid of mammalian cell membranes). 7α-OH/SM mixtures may mimic pathologically changed lipid raft (ordered phase, L_O) while the SM/POPC system can model its surrounding (liquid-disordered phase, L_α). For our study, the Langmuir monolayer technique (based on registration of the surface pressure/area, π/A isotherms), complemented with surface visualization technique (Brewster angle microscopy, BAM) and theoretical calculations, have been employed. The observed affinity of 7α-OH to SM, which appears to be stronger than in cholesterol/SM system, indicates that cholesterol might be partially replaced in lipid rafts by its oxidized derivative. Its incorporation significantly increases rigidity of the system in relation to normal (cholesterol-containing) raft, which can disturb its proper functioning. On the other hand, the poor effect of this oxysterol on the raft's environment was observed.     

Atomic force microscopy and force spectroscopy study of Langmuir-Blodgett films formed by heteroacid phospholipids of biological interest

Langmuir-Blodgett (LB) films of two heteroacid phospholipids of biological interest 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), as well as a mixed monolayer with chi(POPC)=0.4, were transferred onto mica in order to investigate by a combination of atomic force microscopy (AFM) and force spectroscopy (FS) their height, and particularly, their nanomechanical properties. AFM images of such monolayers extracted at 30 mN m(-1) revealed a smooth and defect-free topography except for the POPE monolayer. Since scratching such soft monolayers in contact mode was proved unsuccessful, their molecular height was measured by means of the width of the jump present in the respective force-extension curves. While for pure POPC a small jump occurs near zero force, for the mixed monolayer with chi(POPC)=0.4 the jump occurs at approximately 800 pN. Widths of approximately 2 nm could be established for POPC and chi(POPC)=0.4, but not for POPE monolayer at this extracting pressure. Such different mechanical stability allowed us to directly measure the threshold area/lipid range value needed to induce mechanical stability to the monolayers. AFM imaging and FS were next applied to get further structural and mechanical insight into the POPE phase transition (LC-LC') occurring at pressures >36.5 mN m(-1). This phase transition was intimately related to a sudden decrease in the area/molecule value, resulting in a jump in the force curve occurring at high force ( approximately 1.72 nN). FS reveals to be the unique experimental technique able to unveil structural and nanomechanical properties for such soft phospholipid monolayers. The biological implications of the nanomechanical properties of the systems under investigation are discussed considering that the annular phospholipids region of some transmembrane proteins is enriched in POPE.     

Domain formation in models of the renal brush border membrane outer leaflet.

The plasma membrane outer leaflet plays a key role in determining the existence of rafts and detergent-resistant membrane domains. Monolayers with lipid composition mimicking that of the outer leaflet of renal brush border membranes (BBM) have been deposited on mica and studied by atomic force microscopy. Sphingomyelin (SM) and palmitoyloleoyl phosphatidylcholine (POPC) mixtures, at molar ratios varying from 2:1 to 4:1, were phase-separated into liquid condensed (LC) SM-enriched phase and liquid expanded (LE) POPC-enriched phase. The LC phase accounted for 33 and 58% of the monolayers surface for 2:1 and 4:1 mixtures, respectively. Addition of 20-50 mol % cholesterol (Chl) to the SM/POPC (3:1) mixtures induced marked changes in the topology of monolayers. Whereas Chl promoted the connection between SM domains at 20 mol %, increasing Chl concentration progressively reduced the size of domains and the height differences between the phases. Lateral heterogeneity was, however, still present at 33 mol % Chl. The results indicate that the lipid composition of the outer leaflet is most likely responsible for the BBM thermotropic transition properties. They also strongly suggest that the common maneuver that consists of depleting membrane cholesterol to suppress rafts does not abolish the lateral heterogeneity of BBM membranes.     

The size of lipid rafts: an atomic force microscopy study of ganglioside GM1 domains in sphingomyelin/DOPC/cholesterol membranes

Atomic force microscopy has been used to study the distribution of ganglioside GM1 in model membranes composed of ternary lipid mixtures that mimic the composition of lipid rafts. The results demonstrate that addition of 1% GM1 to 1:1:1 sphingomyelin/dioleoylphosphatidylcholine/cholesterol monolayers leads to the formation of small ganglioside-rich microdomains (40-100 nm in size) that are localized preferentially in the more ordered sphingomyelin/cholesterol-rich phase. With 5% GM1 some GM1 microdomains are also detected in the dioleoylphosphatidylcholine-rich phase. A similar preferential localization of GM1 in the ordered phase is observed for bilayers with the same ternary lipid mixture in the upper leaflet. The small GM1-rich domains observed in these experiments are similar to the sizes for lipid rafts in natural membranes but considerably smaller than the ordered bilayer domains that have been shown to be enriched in GM1 in recent fluorescence microscopy studies of lipid bilayers. The combined data from a number of studies of model membranes indicate that lateral organization occurs on a variety of length scales and mimics many of the properties of natural membranes.     

Nanomechanical recognition of sphingomyelin-rich membrane domains by atomic force microscopy

Sphingomyelin (SM) is a reservoir of signaling lipids and forms specific lipid domains in biomembranes together with cholesterol. In this study, atomic force microscopy (AFM) and force measurement were applied to investigate the interaction of SM-binding protein toxin, lysenin, with N-palmitoyl-D-erythro-sphingosylphosphorylcholine (palmitoyl sphingomyelin, PSM) bilayer spread over a mica substrate, in an aqueous buffer solution. Lysenin molecules were grafted on a silicon nitride tip for AFM by siloxane-thiol-amide coupling. The bilayers were prepared by the Langmuir-Blodgett (LB)/Langmuir-Schaefer (LS) method. By repeating cycles of tip approach/retraction motion, single-molecular adhesion motions were observed on the force curve, characterized as "fishing curves". The addition of cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) did not alter the peak force but increased the peak extension. Mixtures of PSM/DOPC/cholesterol exhibited 2-dimensional two-phase domain separation. The characteristic fishing curves were observed exclusively in one of the phases, indicating the selective interaction of the lysenin tip to PSM-rich membrane domains. Our results indicate that the AFM tips conjugated with lysenin are useful to detect the surface distribution of SM-rich membrane domains as well as the nanomechanical properties of the domains.     

Interaction of ceramides with phosphatidylcholine, sphingomyelin and sphingomyelin/cholesterol bilayers

Ceramides (Cers) may exert their biological activity through changes in membrane structure and organization. To understand this mechanism, the effect of Cer on the biophysical properties of phosphatidylcholine, sphingomyelin (SM) and SM/cholesterol bilayers was determined using fluorescence probe techniques. The Cers were bovine brain Cer and synthetic Cers that contained a single acyl chain species. The phospholipids were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glyero-3-phosphocholine (DPPC) and bovine brain, egg yolk and bovine erythrocyte SM. The addition of Cer to POPC and DPPC bilayers that were in the liquid-crystalline phase resulted in a linear increase in acyl chain order and decrease in membrane polarity. The addition of Cer to DPPC and SM bilayers also resulted in a linear increase in the gel to liquid-crystalline phase transition temperature (T(M)). The magnitude of the change was dependent upon Cer lipid composition and was much higher in SM bilayers than DPPC bilayers. The addition of 33 mol% cholesterol essentially eliminated the thermal transition of SM and SM/Cer bilayers. However, there is still a linear increase in acyl chain order induced by the addition of Cer. The results are interpreted as the formation of DPPC/Cer and SM/Cer lipid complexes. SM/Cer lipid complexes have higher T(M)s than the corresponding SM because the addition of Cer reduces the repulsion between the bulky headgroup and allows closer packing of the acyl chains. The biophysical properties of a SM/Cer-rich bilayer are dependent upon the amount of cholesterol present. In a cholesterol-poor membrane, a sphingomyelinase could catalyze the isothermal conversion of a liquid-crystalline SM bilayer to a gel phase SM/Cer complex at physiological temperature.     

Direct Visualization of the Lateral Structure of Porcine Brain Cerebrosides/POPC Mixtures in Presence and Absence of Cholesterol

We studied the thermal behavior of membranes composed of mixtures of natural cerebrosides (from porcine brain) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with and without cholesterol, using differential scanning calorimetry, Fourier transform infrared spectroscopy, and confocal/multiphoton fluorescence microscopy. The POPC/cerebroside mixture display solid ordered/liquid disordered phase coexistence in a broad range of compositions and temperatures in agreement with previous results reported for POPC/(bovine brain)cerebrosides. The observed phase coexistence scenario consists of elongated, micrometer-sized cerebroside-rich solid ordered domains that span the bilayer, embedded in a POPC-rich liquid disordered phase. The data obtained from differential scanning calorimetry and Fourier transform infrared spectroscopy was in line with that obtained in the microscopy experiments for the binary mixture, except at very high cerebroside molar fractions (0.8-0.9) were some differences are observed. Cholesterol incorporation exerts strong changes on the lateral organization of POPC/porcine brain cerebroside membranes. At intermediate cholesterol concentrations (10-25 mol %) the solid ordered/liquid disordered phase coexistence scenario gradually transform to a solid ordered/liquid ordered one. Above 25 mol % of cholesterol two distinct regions with liquid ordered phase character are visualized in the membrane until a single liquid ordered phase forms at 40 mol % cholesterol. The observed cholesterol effect largely differs from that reported for POPC/porcine brain ceramide, reflecting the impact of the sphingolipids polar headgroup on the membrane lateral organization.     

Using micropatterned lipid bilayer arrays to measure the effect of membrane composition on merocyanine 540 binding

The lipophilic dye merocyanine 540 (MC540) was used to model small molecule-membrane interactions using micropatterned lipid bilayer arrays (MLBAs) prepared using a 3D Continuous Flow Microspotter (CFM). Fluorescence microscopy was used to monitor MC540 binding to fifteen different bilayer compositions simultaneously. MC540 fluorescence was two times greater for bilayers composed of liquid-crystalline (l.c.) phase lipids (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)) compared to bilayers in the gel phase (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)). The effect cholesterol (CHO) had on MC540 binding to the membrane was found to be dependent on the lipid component; cholesterol decreased MC540 binding in DMPC, DPPC and DSPC bilayers while having little to no effect on the remaining l.c. phase lipids. MC540 fluorescence was also lowered when 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS) was incorporated into DOPC bilayers. The increase in the surface charge density appears to decrease the occurrence of highly fluorescent monomers and increase the formation of weakly fluorescent dimers via electrostatic repulsion. This paper demonstrates that MLBAs are a useful tool for preparing high density reproducible bilayer arrays to study small molecule-membrane interactions in a high-throughput manner.     

Cholesterol in condensed and fluid phosphatidylcholine monolayers studied by epifluorescence microscopy

Epifluorescence microscopy was used to investigate the effect of cholesterol on monolayers of dipalmitoylphosphatidylcholine (DPPC) and 1 -palmitoyl-2-oleoyl phosphatidylcholine (POPC) at 21 +/- 2 degrees C using 1 mol% 1-palmitoyl-2-[12-[(7-nitro-2-1, 3-benzoxadizole-4-yl)amino]dodecanoyl]phosphatidylcholine (NBD-PC) as a fluorophore. Up to 30 mol% cholesterol in DPPC monolayers decreased the amounts of probe-excluded liquid-condensed (LC) phase at all surface pressures (pi), but did not effect the monolayers of POPC, which remained in the liquid-expanded (LE) phase at all pi. At low pi (2-5 mN/m), 10 mol% or more cholesterol in DPPC induced a lateral phase separation into dark probe-excluded and light probe-rich regions. In POPC monolayers, phase separation was observed at low pi when > or =40 mol% or more cholesterol was present. The lateral phase separation observed with increased cholesterol concentrations in these lipid monolayers may be a result of the segregation of cholesterol-rich domains in ordered fluid phases that preferentially exclude the fluorescent probe. With increasing pi, monolayers could be transformed from a heterogeneous dark and light appearance into a homogeneous fluorescent phase, in a manner that was dependent on pi and cholesterol content. The packing density of the acyl chains may be a determinant in the interaction of cholesterol with phosphatidylcholine (PC), because the transformations in monolayer surface texture were observed in phospholipid (PL)/sterol mixtures having similar molecular areas. At high pi (41 mN/m), elongated crystal-like structures were observed in monolayers containing 80-100 mol% cholesterol, and these structures grew in size when the monolayers were compressed after collapse. This observation could be associated with the segregation and crystallization of cholesterol after monolayer collapse.     

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.     

Characterization of the binary mixed monolayers of α-tocopherol with phospholipids at the air-water interface

The mixed Langmuir monolayers composed of model constituents of biological membranes, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), were investigated to provide information on the intermolecular interactions between these membrane components and the physiologically active vitamin E–α-tocopherol (TF), as well as on the phase behavior of these mixed systems. Additionally, topography of these monolayers transferred onto the mica support was investigated by the inverted metallurgical microscope. Morphological characteristics were directly observed by Brewster angle microscopy (BAM). From the surface pressure–area isotherms and the analysis of physicochemical parameters (compressibility and mean molecular area at the maximum compressibility) it was found that depending on the acyl chains saturation degree, TF has different effect on the phospholipids. In the case of DPPC, the addition of TF to the phospholipid film causes destabilization of the ordered hydrocarbon chains, while in the POPC/DOPC–TF systems, the attractive interactions are responsible for the monolayer increased stability. Thus, the results of these studies confirm the hypothesis that α-tocopherol may play a role in the stabilization of biological membranes.     

Distinguishing individual lipid headgroup mobility and phase transitions in raft-forming lipid mixtures with 31P MAS NMR

A model membrane system composed of egg sphingomyelin (SM), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol was studied with static and magic angle spinning (31)P NMR spectroscopy. This model membrane system is of significant biological relevance since it is known to form lipid rafts. (31)P NMR under magic angle spinning conditions resolves the SM and DOPC headgroup resonances allowing for extraction of the (31)P NMR parameters for the individual lipid components. The isotropic chemical shift, chemical shift anisotropy, and asymmetry parameter can be extracted from the spinning side band manifold of the individual components that form liquid-ordered and liquid-disordered domains. The magnitude of the (31)P chemical shift anisotropy and the line width is used to determine headgroup mobility and monitor the gel-to-gel and gel-to-liquid crystalline phase transitions of SM as a function of temperature in these mixtures. Spin-spin relaxation measurements are in agreement with the line width results, reflecting mobility differences and some heterogeneities. It will be shown that the presence of DOPC and/or cholesterol greatly impacts the headgroup mobility of SM both above and below the liquid crystalline phase transition temperature, whereas DOPC displays only minor variations in these lipid mixtures.     

Nanoscale electrostatic domains in cholesterol-laden lipid membranes create a target for amyloid binding

Amyloid fibrils are associated with multiple neurodegenerative disorders, such as Alzheimer's disease. Although biological membranes are involved in fibril plaque formation, the role of lipid membrane composition in fibril formation and toxicity is not well understood. We investigated the effect of cholesterol on the interaction of model lipid membranes with amyloid-β peptide (Aβ). With atomic force microscopy we demonstrated that binding of Aβ (1-42) to DOPC bilayer, enriched with 20% cholesterol, resulted in an intriguing formation of small nonuniform islands loaded with Aβ. We attribute this effect to the presence of nanoscale electrostatic domains induced by cholesterol in DOPC bilayers. Using frequency-modulated Kelvin probe force microscopy we were able to resolve these nanoscale electrostatic domains in DOPC monolayers. These findings directly affect our understanding of how the presence of cholesterol may induce targeted binding of amyloid deposits to biomembranes. We postulate that this nonhomogeneous electrostatic effect of cholesterol has a fundamental nature and may be present in other lipid membranes and monolayers.     

Behavior of GM3 ganglioside in lipid monolayers mimicking rafts or fluid phase in membranes

We studied the interaction of GM3 ganglioside with sphingomyelin (SM) and palmitoyl-oleoyl-phosphatidylcholine (POPC) in Langmuir monolayers mimicking, respectively, raft and fluid phase of a cellular membrane, by surface pressure measurements and fluorescence microscopy. No difference was observed in the behavior of SM-GM3 and POPC-GM3 monolayers. In both cases, a GM3 threshold concentration has been underlined between 20 and 40 mol%. Below this threshold, SM-GM3 and POPC-GM3 monolayers behave ideally, suggesting that GM3 and host lipid would form separated domains. On the contrary, above the threshold, a condensation of monolayers is observed. This could be due to a partial solubilisation of GM3 in host lipid, leading to a change in orientation of GM3 molecules at the air-water interface.