Docosahexaenoic acid regulates the formation of lipid rafts: A unified view from experiment and simulation

Docosahexaenoic acid (DHA, 22:6) is an n−3 polyunsaturated fatty acid (n−3 PUFA) that influences immunological, metabolic, and neurological responses through complex mechanisms. One structural mechanism by which DHA exerts its biological effects is through its ability to modify the physical organization of plasma membrane signaling assemblies known as sphingomyelin/cholesterol (SM/chol)-enriched lipid rafts. Here we studied how DHA acyl chains esterified in the sn-2 position of phosphatidylcholine (PC) regulate the formation of raft and non-raft domains in mixtures with SM and chol on differing size scales. Coarse grained molecular dynamics simulations showed that 1-palmitoyl-2-docosahexaenoylphosphatylcholine (PDPC) enhances segregation into domains more than the monounsaturated control, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC). Solid state ²H NMR and neutron scattering experiments provided direct experimental evidence that substituting PDPC for POPC increases the size of raft-like domains on the nanoscale. Confocal imaging of giant unilamellar vesicles with a non-raft fluorescent probe revealed that POPC had no influence on phase separation in the presence of SM/chol whereas PDPC drove strong domain segregation. Finally, monolayer compression studies suggest that PDPC increases lipid-lipid immiscibility in the presence of SM/chol compared to POPC. Collectively, the data across model systems provide compelling support for the emerging model that DHA acyl chains of PC lipids tune the size of lipid rafts, which has potential implications for signaling networks that rely on the compartmentalization of proteins within and outside of rafts.     

Assessing the nature of lipid raft membranes

The paradigm of biological membranes has recently gone through a major update. Instead of being fluid and homogeneous, recent studies suggest that membranes are characterized by transient domains with varying fluidity. In particular, a number of experimental studies have revealed the existence of highly ordered lateral domains rich in sphingomyelin and cholesterol (CHOL). These domains, called functional lipid rafts, have been suggested to take part in a variety of dynamic cellular processes such as membrane trafficking, signal transduction, and regulation of the activity of membrane proteins. However, despite the proposed importance of these domains, their properties, and even the precise nature of the lipid phases, have remained open issues mainly because the associated short time and length scales have posed a major challenge to experiments. In this work, we employ extensive atom-scale simulations to elucidate the properties of ternary raft mixtures with CHOL, palmitoylsphingomyelin (PSM), and palmitoyloleoylphosphatidylcholine. We simulate two bilayers of 1,024 lipids for 100 ns in the liquid-ordered phase and one system of the same size in the liquid-disordered phase. The studies provide evidence that the presence of PSM and CHOL in raft-like membranes leads to strongly packed and rigid bilayers. We also find that the simulated raft bilayers are characterized by nanoscale lateral heterogeneity, though the slow lateral diffusion renders the interpretation of the observed lateral heterogeneity more difficult. The findings reveal aspects of the role of favored (specific) lipid-lipid interactions within rafts and clarify the prominent role of CHOL in altering the properties of the membrane locally in its neighborhood. Also, we show that the presence of PSM and CHOL in rafts leads to intriguing lateral pressure profiles that are distinctly different from corresponding profiles in nonraft-like membranes. The results propose that the functioning of certain classes of membrane proteins is regulated by changes in the lateral pressure profile, which can be altered by a change in lipid content.     

Elasticity, strength, and water permeability of bilayers that contain raft microdomain-forming lipids

Bilayers composed of phosphatidylcholine (PC), sphingomyelin (SM), and cholesterol (CHOL) are commonly used as systems to model the raft-lipid domain structure believed to compartmentalize particular cell membrane proteins. In this work, micropipette aspiration of giant unilamellar vesicles was used to test the elasticities, water permeabilities, and rupture tensions of single-component PC, binary 1:1 PC/CHOL, and 1:1 SM/CHOL, and ternary 1:1:1 PC/SM/CHOL bilayers, one set of measurements with dioleoyl PC (DOPC; C18:1/C18:1 PC) and the other with stearoyloleoyl PC (SOPC; C18:0/C18:1 PC). Defining the elastic moduli (K_A), the initial slopes of the increase in tension (σ) versus stretch in lipid surface area (α_e) were determined for all systems at low (15°C) and high (32-33°C) temperatures. The moduli for the single-component PC and binary phospholipid/CHOL bilayers followed a descending hierarchy of stretch resistance with SM/CHOL > SOPC/CHOL > DOPC/CHOL > PC. Although much more resistant to stretch than the single-component PC bilayers, the elastic response of vesicle bilayers made from the ternary phospholipid/CHOL mixtures showed an abrupt softening (discontinuity in slope), when immediately subjected to a steady ramp of tension at the low temperature (15°C). However, the discontinuities in elastic stretch resistance at low temperature vanished when the bilayers were held at ∼1 mN/m prestress for long times before a tension ramp and when tested at the higher temperature 32-33°C. The elastic moduli of single-component PC and DOPC/CHOL bilayers changed very little with temperature, whereas the moduli of the binary SOPC/CHOL and SM/CHOL bilayers diminished markedly with increase in temperature, as did the ternary SOPC/SM/CHOL system. For all systems, increasing temperature increased the water permeability but decreased rupture tension. Concomitantly, the measurements of permeability exhibited a prominent correlation with the rupture tension across all the systems. Together, these micromechanical tests of binary and ternary phospholipid/CHOL bilayers demonstrate that PC hydrocarbon chain unsaturation and temperature are major determinants of the mechanical and permeation properties of membranes composed of raft microdomain-forming lipids.     

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

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

Effect of a 2-hydroxylated fatty acid on Cholesterol-rich membrane domains

Abstract

2-Hydroxyoleic acid (2OHOA) is a synthetic fatty acid with antihypertensive properties that is able to alter structural membranes properties. The main purpose of this study was to analyze the effect of 2OHOA on the membrane architecture in cholesterol (Cho)-rich domains. For this purpose, model membranes mimicking the composition of lipid rafts and PC- or PE-Cho-rich domains were examined in the absence and presence of 2OHOA by synchrotron X-ray diffraction, atomic force microscopy (AFM) and microcalorimetry (DSC) techniques. Our results demonstrate that 2OHOA phase separates from lipid raft domains and affects the lateral organization of lipids in the membrane. In model raft membranes, 2OHOA interacted with the sphingomyelin (SM) gel phase increasing the thickness of the water layer, which should lead to increased bilayer fluidity. The hydrogen binding competition between 2OHOA and Cho could favour the enrichment of 2OHOA in SM domains separated from the SM-Cho domains, resulting in an enhanced phase separation into SM-2OHOA-rich liquid-disordered (non-raft) and SM-Cho-rich liquid-ordered (raft) domains. The segregation into 2OHOA-rich/Cho-poor and 2OHOA-poor/Cho-rich domains was also observed in PC bilayers.     

Material Properties of Lipid Microdomains: Force-Volume Imaging Study of the Effect of Cholesterol on Lipid Microdomain Rigidity

The effect of cholesterol (CHOL) on the material properties of supported lipid bilayers composed of lipid mixtures that mimic the composition of lipid microdomains was studied by force-volume (FV) imaging under near-physiological conditions. These studies were carried out with lipid mixtures of dioleoylphosphatidylcholine, dioleoylphosphatidylserine, and sphingomyelin. FV imaging enabled simultaneous topology and force measurements of sphingomyelin-rich domains (higher domain (HD)) and phospholipid-rich domains (lower domain (LD)), which allowed quantitative measurement of the force needed to puncture the lipid bilayer with or without CHOL. The force required to penetrate the various domains of the bilayer was probed using high- and low-ionic-strength buffers as a function of increasing amounts of CHOL in the bilayer. The progressive addition of CHOL also led to a decreasing height difference between HD and LD. FV imaging further demonstrated a lack of adhesion between the atomic force microscope tip and the HD or LD at loads below the breakthrough force. These results can lead to a better understanding of the role that CHOL plays in the mechanical properties of cellular membranes in modulating membrane rigidity, which has important implications for cellular mechanotransduction.     

Lipid Raft Composition Modulates Sphingomyelinase Activity and Ceramide-Induced Membrane Physical Alterations

Lipid rafts and ceramide (Cer)-platforms are membrane domains that play an important role in several biological processes. Cer-platforms are commonly formed in the plasma membrane by the action of sphingomyelinase (SMase) upon hydrolysis of sphingomyelin (SM) within lipid rafts. The interplay among SMase activity, initial membrane properties (i.e., phase behavior and lipid lateral organization) and lipid composition, and the amount of product (Cer) generated, and how it modulates membrane properties were studied using fluorescence methodologies in model membranes. The activity of SMase was evaluated by following the hydrolysis of radioactive SM. It was observed that 1), the enzyme activity and extent of hydrolysis are strongly dependent on membrane physical properties but not on substrate content, and are higher in raft-like mixtures, i.e., mixtures with liquid-disordered/liquid-ordered phase separation; and 2), Cer-induced alterations are also dependent on membrane composition, specifically the cholesterol (Chol) content. In the lowest-Chol range, Cer segregates together with SM into small (∼8.5 nm) Cer/SM-gel domains. With increasing Chol, the ability of Cer to recruit SM and form gel domains strongly decreases. In the high-Chol range, a Chol-enriched/SM-depleted liquid-ordered phase predominates. Together, these data suggest that in biological membranes, Chol in particular and raft domains in general play an important role in modulating SMase activity and regulating membrane physical properties by restraining Cer-induced alterations.     

Domain formation in model membranes studied by pulsed-field gradient-NMR: the role of lipid polyunsaturation

The effects of increased unsaturation in the sn-2 fatty acyl chain of phosphatidylcholines (PCs) on the lipid lateral diffusion have been investigated by pulsed-field gradient NMR. Macroscopically oriented bilayers containing a monosaturated PC, egg sphingomyelin, and cholesterol (CHOL) have been studied at temperatures between 0 degrees C and 60 degrees C, and the number of double bonds in the PC was one, two, four, or six. For PC bilayers, with and without the incorporation of egg sphingomyelin and CHOL, the lateral diffusion increased with increasing number of double bonds, as a consequence of the increased headgroup area caused by the unsaturation. Addition of CHOL caused a decrease in lipid diffusion due to the condensing effect of CHOL on the headgroup area. Phase separation into large domains of liquid-disordered and liquid-ordered phases were observed in the ternary systems with PCs containing four and six double bonds, as evidenced by the occurrence of two lipid diffusion coefficients. PC bilayers with one or two double bonds appear homogeneous on the length scales probed by the experiment, but the temperature dependence of the diffusion suggests that small domains may be present also in these ternary systems.     

Lipid rafts reconstituted in model membranes

One key tenet of the raft hypothesis is that the formation of glycosphingolipid- and cholesterol-rich lipid domains can be driven solely by characteristic lipid-lipid interactions, suggesting that rafts ought to form in model membranes composed of appropriate lipids. In fact, domains with raft-like properties were found to coexist with fluid lipid regions in both planar supported lipid layers and in giant unilamellar vesicles (GUVs) formed from 1) equimolar mixtures of phospholipid-cholesterol-sphingomyelin or 2) natural lipids extracted from brush border membranes that are rich in sphingomyelin and cholesterol. Employing headgroup-labeled fluorescent phospholipid analogs in planar supported lipid layers, domains typically several microns in diameter were observed by fluorescence microscopy at room temperature (24 degrees C) whereas non-raft mixtures (PC-cholesterol) appeared homogeneous. Both raft and non-raft domains were fluid-like, although diffusion was slower in raft domains, and the probe could exchange between the two phases. Consistent with the raft hypothesis, GM1, a glycosphingolipid (GSL), was highly enriched in the more ordered domains and resistant to detergent extraction, which disrupted the GSL-depleted phase. To exclude the possibility that the domain structure was an artifact caused by the lipid layer support, GUVs were formed from the synthetic and natural lipid mixtures, in which the probe, LAURDAN, was incorporated. The emission spectrum of LAURDAN was examined by two-photon fluorescence microscopy, which allowed identification of regions with high or low order of lipid acyl chain alignment. In GUVs formed from the raft lipid mixture or from brush border membrane lipids an array of more ordered and less ordered domains that were in register in both monolayers could reversibly be formed and disrupted upon cooling and heating. Overall, the notion that in biomembranes selected lipids could laterally aggregate to form more ordered, detergent-resistant lipid rafts into which glycosphingolipids partition is strongly supported by this study.     

The effects of temperature, pressure and peptide incorporation on ternary model raft mixtures--a Laurdan fluorescence spectroscopy study

Recently, an increasing evidence accumulated for the existence of lipid microdomains, called lipid rafts, in cell membranes, which may play an important role in many important membrane-associated biological processes. Suitable model systems for studying biophysical properties of lipid rafts are lipid vesicles composed of three-component lipid mixtures, such as POPC/SM/cholesterol, which exhibit a rich phase diagram, including raft-like liquid-ordered/liquid-disordered phase coexistence regions. We explored the temperature, pressure and concentration-dependent phase behavior of such canonical model raft mixtures using the Laurdan fluorescence spectroscopic technique. Hydrostatic pressure has not only been used as a physical parameter for studying the stability and energetics of these systems, but also because high pressure is an important feature of certain natural membrane environments. We show that the liquid-disordered/liquid-ordered phase coexistence regions of POPC/SM/cholesterol model raft mixtures extends over a very wide temperature range of about 50 degrees C. Upon pressurization, an overall ordered membrane state is reached at pressures of approximately 1,000 bar at 20 degrees C, and of approximately 2,000 bar at 40 degrees C. Incorporation of 5 mol% gramicidin as a model ion channel slightly increases the overall order parameter profile in the l(o)+l(d) two-phase coexistence region, probably by selectively partitioning into l(d) domains, does not change the overall phase behavior, however. This behavior is in contrast to the effect of the peptide incorporation into simple, one-component phospholipid bilayer systems.     

Identification of a critical lipid ratio in raft-like phases exposed to nitric oxide: An AFM study

Lipid rafts are discrete, heterogeneous domains of phospholipids, sphingolipids, and sterols that are present in the cell membrane. They are responsible for conducting cell signaling and maintaining lipid-protein functionality. Redox-stress-induced modifications to any of their components can severely alter the mechanics and dynamics of the membrane causing impairment to the lipid-protein functionality. Here, we report on the effect of sphingomyelin (SM) in controlling membrane permeability and its role as a regulatory lipid in the presence of nitric oxide (NO). Force spectroscopy and atomic force microscopy imaging of raft-like phases (referring here to the coexistence of “liquid-ordered” and “liquid-disordered” phases in model bilayer membranes) prepared from lipids: 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC):SM:cholesterol (CH) (at three ratios) showed that the adhesion forces to pull the tip out of the membrane increased with increasing SM concentration, indicating decreased membrane permeability. However, in the presence of NO radical (1 and 5 μM), the adhesion forces decreased depending on SM concentration. The membrane was found to be stable at the ratio POPC:SM:CH (2:1:1) even when exposed to 1 μM NO. We believe that this is a critical ratio needed by the raft-like phases to maintain homeostasis under stress conditions. The stability could be due to an interplay existing between SM and CH. However, at 5 μM NO, membrane deteriorations were detected. For POPC:SM:CH (2:2:1) ratio, NO displayed a pro-oxidant behavior and damaged the membrane at both radical concentrations. These changes were reflected by the differences in the height profiles of the raft-like phases observed by atomic force microscopy imaging. Malondialdehyde (a peroxidation product) detection suggests that lipids may have undergone lipid nitroxidation. The changes were instantaneous and independent of radical concentration and incubation time. Our study underlines the need for identifying appropriate ratios in the lipid rafts of the cell membranes to withstand redox imbalances caused by radicals such as NO.     

Cholesterol displacement by ceramide in sphingomyelin-containing liquid-ordered domains, and generation of gel regions in giant lipidic vesicles

Fluorescence confocal microscopy and differential scanning calorimetry are used in combination to study the phase behaviour of bilayers composed of PC:PE:SM:Chol equimolecular mixtures, in the presence or absence of 10mol% egg ceramide. In the absence of ceramide, separate liquid-ordered and liquid-disordered domains are observed in giant unilamellar vesicles. In the presence of ceramide, gel-like domains appear within the liquid-ordered regions. The melting properties of these gel-like domains resemble those of SM:ceramide binary mixtures, suggesting Chol displacement by ceramide from SM:Chol-rich liquid-ordered regions. Thus three kinds of domains coexist within a single vesicle in the presence of ceramide: gel, liquid-ordered, and liquid-disordered. In contrast, when 10mol% egg diacylglycerol is added instead of ceramide, homogeneous vesicles, consisting only of liquid-disordered bilayers, are observed.     

Pro-inflammatory protein S100A9 alters membrane organization by dispersing ordered domains

Pro-inflammatory, calcium-binding protein S100A9 is localized in the cytoplasm of many cells and regulates several intracellular and extracellular processes. S100A9 is involved in neuroinflammation associated with the pathogenesis of Alzheimer's disease (AD). The number of studies on the impact of S100A9 in co-aggregation processes with amyloid-like proteins is increasing. However, there is still a lack of data on how this protein interacts with lipid membranes. We employed atomic force microscopy (AFM), dynamic light scattering (DLS), and fluorescence measurements (Laurdan and Thioflavin-T) to study the interaction between protein and the membrane surface. We used lipid vesicles in bulk and planar tethered lipid bilayers as biomimetic membrane models. We demonstrated that the protein accumulates on negatively charged lipid bilayers but with no further loss of the bilayer's integrity. The most important result is that the initial adsorption and accumulation of apo-form of S100A9 on the lipid membrane surface is lipid phase-sensitive. The breaking down of raft-like and disappearance of gel-like domains indicate that protein incorporates into the hydrophobic part of the lipid bilayer. We observed the most noticeable loss of integrity in lipid bilayers constructed from a lipid mixture (brain total lipid extract). Understanding the function and interactions of these proteins in cellular environments might expand the development of new diagnostic and therapeutic approaches for AD or other related diseases.     

Phase separation dynamics and lateral organization of two-component lipid membranes.

The non-equilibrium dynamic ordering process of coexisting phases has been studied for two-component lipid bilayers composed of saturated di-acyl phospholipids with different acyl chain lengths, such as DC14PC-DC18PC and DC12PC-DC18PC. By means of a microscopic interaction model and computer-simulation techniques the non-equilibrium properties of these two mixtures have been determined with particular attention paid to the effects of the non-equilibrium ordering process on membrane heterogeneity in terms of local and global lateral membrane organization. The results reveal that a sudden temperature change that takes the lipid mixture from the fluid one-phase region into the gel-fluid phase-coexistence region leads to the formation of a large number of small lipid domains which slowly are growing in time. The growth of the lipid domains, which is limited by long-range diffusion of the lipid molecules within the two-dimensional membrane plane, gives rise to the existence of a highly heterogeneous percolative-like structure with a network of interfacial regions that have properties different from those of the phase-separated gel and fluid bulk phases. The results, which are discussed in relation to recent experimental observations interpreted in terms of a percolative-like membrane structure within the two phase region (Almeida, P.F.F., Vaz, W.L.C., and T.E. Thompson. 1992. Biochemistry 31:7198-7210), suggest that non-equilibrium effects may influence lipid domain formation and membrane organization on various length and time scales. Such effects might be of importance in relation to membrane processes that require molecular mobility of the membrane components in restricted geometrical environments of the compartmentalized lipid membrane.     

Oleic and docosahexaenoic acid differentially phase separate from lipid raft molecules: a comparative NMR, DSC, AFM, and detergent extraction study

We have previously suggested that the omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA) may in part function by enhancing membrane lipid phase separation into lipid rafts. Here we further tested for differences in the molecular interactions of an oleic (OA) versus DHA-containing phospholipid with sphingomyelin (SM) and cholesterol (CHOL) utilizing (2)H NMR spectroscopy, differential scanning calorimetry, atomic force microscopy, and detergent extractions in model bilayer membranes. (2)H NMR and DSC (differential scanning calorimetry) established the phase behavior of the OA-containing 1-[(2)H(31)]palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1PE-d(31))/SM (1:1) and the DHA-containing 1-[(2)H(31)]palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (16:0-22:6PE-d(31))/SM (1:1) in the absence and presence of equimolar CHOL. CHOL was observed to affect the OA-containing phosphatidylethanolamine (PE) more than the DHA-containing PE, as exemplified by >2 x greater increase in order measured for the perdeuterated palmitic chain in 16:0-18:1PE-d(31)/SM (1:1) compared to 16:0-22:6PE-d(31)/SM (1:1) bilayers in the liquid crystalline phase. Atomic force microscopy (AFM) experiments showed less lateral phase separation between 16:0-18:1PE-rich and SM/CHOL-rich raft domains in 16:0-18:1PE/SM/CHOL (1:1:1) bilayers than was observed when 16:0-22:6PE replaced 16:0-18:1PE. Differences in the molecular interaction of 16:0-18:1PE and 16:0-22:6PE with SM/CHOL were also found using biochemical detergent extractions. In the presence of equimolar SM/CHOL, 16:0-18:1PE showed decreased solubilization in comparison to 16:0-22:6PE, indicating greater phase separation with the DHA-PE. Detergent experiments were also conducted with cardiomyocytes fed radiolabeled OA or DHA. Although both OA and DHA were found to be largely detergent solubilized, the amount of OA that was found to be associated with raft-rich detergent-resistant membranes exceeded DHA by almost a factor of 2. We conclude that the OA-PE phase separates from rafts far less than DHA-PE, which may have implications for cellular signaling.     

N- and O-methylation of sphingomyelin markedly affects its membrane properties and interactions with cholesterol

We have prepared palmitoyl sphingomyelin (PSM) analogs in which either the 2-NH was methylated to NMe, the 3-OH was methylated to OMe, or both were methylated simultaneously. The aim of the study was to determine how such modifications in the membrane interfacial region of the molecules affected interlipid interactions in bilayer membranes. Measuring DPH anisotropy in vesicle membranes prepared from the SM analogs, we observed that methylation decreased gel-phase stability and increased fluid phase disorder, when compared to PSM. Methylation of the 2-NH had the largest effect on gel-phase instability (T(m) was lowered by ~7°C). Atomistic molecular dynamics simulations showed that fluid phase bilayers with methylated SM analogs were more expanded but thinner compared to PSM bilayers. It was further revealed that 3-OH methylation dramatically attenuated hydrogen bonding also via the amide nitrogen, whereas 2-NH methylation did not similarly affect hydrogen bonding via the 3-OH. The interactions of sterols with the methylated SM analogs were markedly affected. 3-OH methylation almost completely eliminated the capacity of the SM analog to form sterol-enriched ordered domains, whereas the 2-NH methylated SM analog formed sterol-enriched domains but these were less thermostable (and thus less ordered) than the domains formed by PSM. Cholestatrienol affinity to bilayers containing methylated SM analogs was also markedly reduced as compared to its affinity for bilayers containing PSM. Molecular dynamics simulations revealed further that cholesterol's bilayer location was deeper in PSM bilayers as compared to the location in bilayers made from methylated SM analogs. This study shows that the interfacial properties of SMs are very important for interlipid interactions and the formation of laterally ordered domains in complex bilayers.     

Effect of sphingomyelin headgroup size on molecular properties and interactions with cholesterol

Sphingomyelins (SMs) and sterols are important constituents of the plasma membrane and have also been identified as major lipid components in membrane rafts. Using SM analogs with decreasing headgroup methylation, we systemically analyzed the effect of headgroup size on membrane properties and interactions with cholesterol. An increase in headgroup size resulted in a decrease in the main phase transition. Atom-scale molecular-dynamics simulations were in agreement with the fluorescence anisotropy experiments, showing that molecular areas increased and acyl chain order decreased with increasing headgroup size. Furthermore, the transition temperatures were constantly higher for SM headgroup analogs compared to corresponding phosphatidylcholine headgroup analogs. The sterol affinity for phospholipid bilayers was assessed using a sterol-partitioning assay and an increased headgroup size increased sterol affinity for the bilayer, with a higher sterol affinity for SM analogs as compared to phosphatidylcholine analogs. Moreover, the size of the headgroup affected the formation and composition of cholesterol-containing ordered domains. Palmitoyl-SM (the largest headgroup) seemed to attract more cholesterol into ordered domains than the other SM analogs with smaller headgroups. The ordering and condensing effect of cholesterol on membrane lipids was also largest for palmitoyl-SM as compared to the smaller SM analogs. The results show that the size of the SM headgroup is crucially important for SM-SM and SM-sterol interactions. Our results further emphasize that interfacial electrostatic interactions are important for stabilizing cholesterol interactions with SMs.     

Compared effects of cholesterol and 7-dehydrocholesterol on sphingomyelin-glycerophospholipid bilayers studied by ESR

The ESR of 7- and 16-doxylstearic spin-labeled fatty acids (7NS and 16NS, respectively) reveal the distinct influence of cholesterol or cholesterol precursor analogue, delta7-dehydrocholesterol, on the molecular ordering and the fluidity of lipid mixtures containing sphingomyelin (SM). The phase-separation of sphingomyelin domains mixed within fluid glycerophospholipids (phosphatidylethanolamine and phosphatidylserine) can be followed by ESR as a function of the temperature and in the presence of sterols [cholesterol (CHOL) or 7-dehydrocholesterol (DHCHOL)]. The time scale of spin-label exchange among phases is appropriate to follow the occurrence of the specific sphingomyelin/sterol association forming liquid ordered (Lo) microdomains which separate from the fluid surrounding phase Lalpha. Sphingomyelin embedded within the fluid bilayer associates with both sterols below 36 degrees C to give a phase Lo traceable by ESR in the form of a highly anisotropic component. Above 36 degrees C, the contribution in the ESR spectrum, of the Lo phase formed by 7-dehydrocholesterol with sphingomyelin is reduced by contrast with cholesterol forming a temperature-stable liquid ordered phase up to 42 degrees C. The consequences of this destabilization of the SM/sterol microdomains are envisioned in the biosynthesis defect where the precursor 7-dehydrocholesterol substitutes, for a significant part, the embryonic cell cholesterol.     

Elastic deformation and failure of lipid bilayer membranes containing cholesterol.

Giant bilayer vesicles were reconstituted from several lipids and lipid/cholesterol (CHOL) mixtures: stearolyloleoylphosphatidylcholine (SOPC), bovine sphingomyelin (BSM), diarachidonylphosphatidylcholine (DAPC), SOPC/CHOL, BSM/CHOL, DAPC/CHOL, and extracted red blood cell (RBC) lipids with native cholesterol. Single-walled vesicles were manipulated by micropipette suction and several membrane material properties were determined. The properties measured were the elastic area compressibility modulus K, the critical areal strain alpha c, and the tensile strength tau lys, from which the failure energy or membrane toughness Tf was calculated. The elastic area expansion moduli for these lipid and lipid/cholesterol bilayers ranged from 57 dyn/cm for DAPC to 1,734 dyn/cm for BSM/CHOL. The SOPC/CHOL series and RBC lipids had intermediate values. The results indicated that the presence of cholesterol is the single most influential factor in increasing bilayer cohesion, but only for lipids where both chains are saturated, or mono- or diunsaturated. Multiple unsaturation in both lipid chains inhibits the condensing effect of cholesterol in bilayers. The SOPC/CHOL system was studied in more detail. The area expansion modulus showed a nonlinear increase with increasing cholesterol concentration up to a constant plateau, indicating a saturation limit for cholesterol in the bilayer phase of approximately 55 mol% CHOL. The membrane compressibility was modeled by a property-averaging composite theory involving two bilayer components, namely, uncomplexed lipid and a lipid/cholesterol complex of stoichiometry 1/1.22. The area expansion modulus of this molecular composite membrane was evaluated by a combination of the expansion moduli of each component scaled by their area fractions in the bilayer. Bilayer toughness, which is the energy stored in the bilayer at failure, showed a maximum value at approximately 40 mol% CHOL. This breakdown energy was found to be only a fraction of the available thermal energy, implying that many molecules (approximately 50-100) may be involved in forming the defect structure that leads to failure. The area expansion modulus of extracted RBC lipids with native cholesterol was compared with recent measurements of intact RBC membrane compressibility. The natural membrane was also modeled as a simple composite made up to a compressible lipid/cholesterol matrix containing relatively incompressible transmembrane proteins. It appears that the interaction of incompressible proteins with surrounding lipid confers enhanced compressibility on the composite structure.     

The role of different P-glycoproteins in hepatobiliary secretion of fluorescently labeled short-chain phospholipids.

Class III P-glycoproteins (Pgps) mediate biliary phosphatidylcholine (PC) secretion. Recent findings that class I P-glycoproteins are able to transport several short-chain phospholipid analogues raises questions about the role of these Pgps in physiological lipid transport. We investigated the biliary secretion of C6-7-nitro-2,1, 3-benzoxadiazol-4-yl (NBD)-labeled ceramide and its metabolites in Mdr1a/b and Mdr2 knockout mice compared to control mice. Biliary secretion of these NBD-lipids was unaffected in Mdr1a/b -/- mice. Thus neither Mdr1a nor Mdr1b Pgp mediates biliary secretion of these lipids. In contrast, secretion of all three NBD-labeled short-chain phospholipids was significantly reduced in Mdr2 -/- mice. As in vitro studies revealed that Mdr2 Pgp is not able to translocate these lipid analogues, we hypothesized that Mdr2 -/- mice had a reduced PC content of the exoplasmic canalicular membrane leaflet so that extraction of the short-chain lipid probes from this membrane by canalicular bile salts was impaired. To investigate this possibility we studied the bile salt-mediated extraction of natural sphingomyelin (SM) and NBD-labeled short-chain SM from small unilamellar vesicles of different lipid composition. Natural SM could be extracted by the bile salt tauroursodeoxycholate from vesicles containing PC, cholesterol (CHOL), and SM (1:2:2) but not from vesicles containing only SM and CHOL (3:2). NBD-labeled short-chain SM could be extracted from vesicles containing PC while its extraction from pure SM:CHOL vesicles was reduced by 65%.These data confirm that the efficiency of NBD-SM extraction depends on the lipid composition and suggest that the canalicular membrane outer leaflet of Mdr2 -/- mice has a reduced PC content.