Aspirin inhibits formation of cholesterol rafts in fluid lipid membranes

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

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

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

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

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

Phospholipid/cholesterol model membranes: formation of cholesterol crystallites

Experimental data that define conditions under which cholesterol crystallites form in cholesterol/phospholipid model membranes are reviewed. Structural features of the phospholipids that determine cholesterol crystallization include the length and degree of unsaturation of the acyl chains, the presence of charge on the headgroups and interheadgroup hydrogen bonds.     

Detergent-resistant membranes and the protein composition of lipid rafts

The plasma membrane of eukaryotic cells contains lipid rafts with protein and lipid compositions differing from the bulk plasma membrane. Several recent proteomic studies have addressed the composition of lipid rafts, but the different definitions used for lipid rafts need scrutinizing before results can be evaluated.     

Phospholipid/cholesterol model membranes: formation of cholesterol crystallites

Experimental data that define conditions under which cholesterol crystallites form in cholesterol/phospholipid model membranes are reviewed. Structural features of the phospholipids that determine cholesterol crystallization include the length and degree of unsaturation of the acyl chains, the presence of charge on the headgroups and interheadgroup hydrogen bonds.     

Investigating the competitive effects of cholesterol and melatonin in model lipid membranes

We have studied the impact of cholesterol and/or melatonin on the static and dynamical properties of bilayers made of DPPC or DOPC utilizing neutron scattering techniques, Raman spectroscopy and molecular dynamics simulations. While differing in the amplitude of the effect due to cholesterol or melatonin when comparing their interactions with the two lipids, their addition ensued recognizable changes to both types of bilayers. As expected, based on the two-component systems of lipid/cholesterol or lipid/melatonin studied previously, we show the impact of cholesterol and melatonin being opposite and competitive in the case of three-component systems of lipid/cholesterol/melatonin. The effect of cholesterol appears to prevail over that of melatonin in the case of structural properties of DPPC-based bilayers, which can be explained by its interactions targeting primarily the saturated lipid chains. The dynamics of hydrocarbon chains represented by the ratio of trans/gauche conformers reveals the competitive effect of cholesterol and melatonin being somewhat more balanced. The additive yet opposing effects of cholesterol and melatonin have been observed also in the case of structural properties of DOPC-based bilayers. We report that cholesterol induced an increase in bilayer thickness, while melatonin induced a decrease in bilayer thickness in the three-component systems of DOPC/cholesterol/melatonin. Commensurately, by evaluating the projected area of DOPC, we demonstrate a lipid area decrease with an increasing concentration of cholesterol, and a lipid area increase with an increasing concentration of melatonin. The demonstrated condensing effect of cholesterol and the fluidizing effect of melatonin appear in an additive manner upon their mutual presence.     

Evidence for the involvement of lipid rafts localized at the ER-mitochondria associated membranes in autophagosome formation

Mitochondria-associated membranes (MAMs) are subdomains of the endoplasmic reticulum (ER) that interact with mitochondria. This membrane scrambling between ER and mitochondria appears to play a critical role in the earliest steps of autophagy. Recently, lipid microdomains, i.e. lipid rafts, have been identified as further actors of the autophagic process. In the present work, a series of biochemical and molecular analyses has been carried out in human fibroblasts with the specific aim of characterizing lipid rafts in MAMs and to decipher their possible implication in the autophagosome formation. In fact, the presence of lipid microdomains in MAMs has been detected and, in these structures, a molecular interaction of the ganglioside GD3, a paradigmatic “brick” of lipid rafts, with core-initiator proteins of autophagy, such as AMBRA1 and WIPI1, was revealed. This association seems thus to take place in the early phases of autophagic process in which MAMs have been hypothesized to play a key role. The functional activity of GD3 was suggested by the experiments carried out by knocking down ST8SIA1 gene expression, i.e., the synthase that leads to the ganglioside formation. This experimental condition results in fact in the impairment of the ER-mitochondria crosstalk and the subsequent hindering of autophagosome nucleation. We thus hypothesize that MAM raft-like microdomains could be pivotal in the initial organelle scrambling activity that finally leads to the formation of autophagosome.     

Magainin 2 channel formation in planar lipid membranes: the role of lipid polar groups and ergosterol

Magainin 2, a polycationic peptide, displays bactericidal and tumoricidal activity, presumably interacting with negatively charged phospholipids in the membrane hosts. In this work, we investigate the role played by the lipid head-group in the interactions and self-association of magainin 2 during pore formation in lipid bilayers. Two methods are used: single-channel and macroscopic incorporation into planar lipid membranes. Single-channel incorporation showed that magainin 2 did not interact with zwitterionic membranes, while the addition of negatively charged dioleoylphosphatidylglycerol to the membrane leads to channel formation. On the other hand, magainin 2 did not form channels in membranes made up of dioleoylphosphatidylserine (DOPS), although the addition of ergosterol to DOPS membranes leads to channel formation. This finding could indicate that ergosterol may be a possible target of magainin 2 in fungal membranes. Further support for this hypothesis comes from experiments in which the addition of ergosterol to palmitoyloleoylphosphatidylcholine membranes induced channel formation. Besides the role of negatively charged membranes, this study has shown that magainin 2 also forms channels in membranes lacking heads, such as monoolein and oxidized cholesterol, indicating an interaction of magainin 2 with acyl chains and cholesterol, respectively. This finding provides further evidence that peptide binding and assembly in lipid membranes is a complex process driven by electrostatic and/or hydrophobic interactions, depending on the structure of the peptide and the membrane composition.     

Lipid peroxidation induces cholesterol domain formation in model membranes

Numerous reports have established that lipid peroxidation contributes to cell injury by altering the basic physical properties and structural organization of membrane components. Oxidative modification of polyunsaturated phospholipids has been shown, in particular, to alter the intermolecular packing, thermodynamic, and phase parameters of the membrane bilayer. In this study, the effects of oxidative stress on membrane phospholipid and sterol organization were measured using small angle x-ray diffraction approaches. Model membranes enriched in dilinoleoylphosphatidylcholine were prepared at various concentrations of cholesterol and subjected to lipid peroxidation at physiologic conditions. At cholesterol-to-phospholipid mole ratios (C/P) as low as 0.4, lipid peroxidation induced the formation of discrete, membrane-restricted cholesterol domains having a unit cell periodicity or d-space value of 34 A. The formation of cholesterol domains correlated directly with lipid hydroperoxide levels and was inhibited by treatment with vitamin E. In the absence of oxidative stress, similar cholesterol domains were observed only at C/P ratios of 1.0 or higher. In addition to changes in sterol organization, lipid peroxidation also caused reproducible changes in overall membrane structure, including a 10 A reduction in the width of the surrounding, sterol-poor membrane bilayer. These data provided direct evidence that lipid peroxidation alters the essential organization and structure of membrane lipids in a manner that may contribute to changes in membrane function during aging and oxidative stress-related disorders.     

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.     

Selective cholesterol dynamics between lipoproteins and caveolae/lipid rafts

Although low-density lipoprotein (LDL) receptor-mediated cholesterol uptake through clathrin-coated pits is now well understood, the molecular details and organizing principles for selective cholesterol uptake/efflux (reverse cholesterol transport, RCT) from peripheral cells remain to be resolved. It is not yet completely clear whether RCT between serum lipoproteins and the plasma membrane occurs primarily through lipid rafts/caveolae or from non-raft domains. To begin to address these issues, lipid raft/caveolae-, caveolae-, and non-raft-enriched fractions were resolved from purified plasma membranes isolated from L-cell fibroblasts and MDCK cells by detergent-free affinity chromatography and compared with detergent-resistant membranes isolated from the same cells. Fluorescent sterol exchange assays between lipoproteins (VLDL, LDL, HDL, apoA1) and these enriched domains provided new insights into supporting the role of lipid rafts/caveolae and caveolae in plasma membrane/lipoprotein cholesterol dynamics: (i) lipids known to be translocated through caveolae were detected (cholesteryl ester, triacylglycerol) and/or enriched (cholesterol, phospholipid) in lipid raft/caveolae fractions; (ii) lipoprotein-mediated sterol uptake/efflux from lipid rafts/caveolae and caveolae was rapid and lipoprotein specific, whereas that from non-rafts was very slow and independent of lipoprotein class; and (iii) the rate and lipoprotein specificity of sterol efflux from lipid rafts/caveolae or caveolae to lipoprotein acceptors in vitro was slower and differed in specificity from that in intact cells-consistent with intracellular factors contributing significantly to cholesterol dynamics between the plasma membrane and lipoproteins.     

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.     

Role of gangliosides in adhesion and conductance changes in large spherical model membranes

The formation of two spherical model membranes at the tips of two syringes has allowed us to study the role of gangliosides in membrane adhesion and look for changes in conductance between two such membranes during the process of adhesion. Membranes were formed in aqueous 100 mM NaCl, 10 mM KCl, 1 mM CaCl2 from 1% (w/v) egg phosphatidylcholine in n-decane, with or without mixed bovine brain gangliosides. After thinning to the 'black' bilayer state, two membranes were moved into contact. With gangliosides, the contact area and conductance increased colinearly with time over a 5 to 20 min period of adhesion. The role of electrostatic bridging by calcium was investigated. In the absence of calcium or in the presence of 2 mM EDTA, adhesion proceeded after a longer lag time at about one-half the normal rate. As the ganglioside concentration was increased from 0 to 15 mol%, the electrical conductance of individual membranes decreased 3-fold from 48 +/- 30 nS/cm2 to 17 +/- 13 nS/cm2. The conductance was pH dependent with a minimum at neutral values. At neutral pH, when two membranes containing 4.1 mol% gangliosides adhered, the region of adhesion had a specific conductance three times that of the nonadhering regions of membranes. Without gangliosides, the specific conductance of the contact region was the same as that of non-adhering regions of the membrane. These data suggest that mixed gangliosides can mediate an adhesion-dependent increase in conductance.     

Association of gamma-secretase with lipid rafts in post-Golgi and endosome membranes

Alzheimer's disease-associated beta-amyloid peptides (Abeta) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases. There is growing evidence that cholesterol- and sphingolipid-rich membrane microdomains are involved in regulating trafficking and processing of APP. BACE1, the major beta-secretase in neurons is a palmitoylated transmembrane protein that resides in lipid rafts. A subset of APP is subject to amyloidogenic processing by BACE1 in lipid rafts, and this process depends on the integrity of lipid rafts. Here we describe the association of all four components of the gamma-secretase complex, namely presenilin 1 (PS1)-derived fragments, mature nicastrin, APH-1, and PEN-2, with cholesterol-rich detergent insoluble membrane (DIM) domains of non-neuronal cells and neurons that fulfill the criteria of lipid rafts. In PS1(-/-)/PS2(-/-) and NCT(-/-) fibroblasts, gamma-secretase components that still remain fail to become detergent-resistant, suggesting that raft association requires gamma-secretase complex assembly. Biochemical evidence shows that subunits of the gamma-secretase complex and three TGN/endosome-resident SNAREs cofractionate in sucrose density gradients, and show similar solubility or insolubility characteristics in distinct non-ionic and zwitterionic detergents, indicative of their co-residence in membrane microdomains with similar protein-lipid composition. This notion is confirmed using magnetic immunoisolation of PS1- or syntaxin 6-positive membrane patches from a mixture of membranes with similar buoyant densities following Lubrol WX extraction or sonication, and gradient centrifugation. These findings are consistent with the localization of gamma-secretase in lipid raft microdomains of post-Golgi and endosomes, organelles previously implicated in amyloidogenic processing of APP.     

Probing the stability of S-layer-supported planar lipid membranes

Isolated protein subunits of the crystalline bacterial cell surface layer (S-layer) of Bacillus coagulans E38-66 have been recrystallized on one side of planar black lipid membranes (BLMs) and their influence on the electrical properties, rupture kinetics and mechanical stability of the BLM was investigated. The effect on the boundary potential, the capacitance or the conductance of the membrane was negligible whereas the mechanical properties were considerably changed. The mechanical stability was characterized by applying voltage pulses or ramps to induce irreversible rupture. The amplitude of the voltage pulse leading to rupture allows conclusions on the ability of membranes to resist external forces. Surprisingly, these amplitudes were significantly lower for composite S-layer/lipid membranes compared to undecorated BLMs. In contrast, the delay time between the voltage pulse and the appearance of the initial defect was found to be drastically longer for the S-layer-supported lipid bilayer. Furthermore, the kinetics of the rupture process was recorded. Undecorated membranes show a fast linear increase of the pore conductance in time, indicating an inertia-limited defect growth. The attachment of an S-layer causes a slow exponential increase in the conductance during rupture, indicating a viscosity-determined widening of the pore. In addition, the mechanical properties on a longer time scale were investigated by applying a hydrostatic pressure across the BLMs. This causes the BLM to bulge, as monitored by an increase in capacitance. Compared to undecorated BLMs, a significantly higher pressure gradient has to be applied on the S-layer face of the composite BLMs to observe any change in capacitance. Received: 4 May 1999 / Revised version: 1 July 1999 / Accepted: 1 July 1999     

Distribution of ganglioside GM1 in L-alpha-dipalmitoylphosphatidylcholine/cholesterol monolayers: a model for lipid rafts

The distribution of low concentrations of ganglioside GM1 in L-alpha-dipalmitoylphosphatidylcholine (DPPC) and DPPC/cholesterol monolayers supported on mica has been studied using atomic force microscopy (AFM). The monolayers studied correspond to a pure gel phase and a mixture of liquid-expanded (LE) and liquid-condensed (LC) phases for DPPC and to a single homogeneous liquid-ordered phase for 2:1 DPPC/cholesterol. The addition of 2.5-5% GM1 to phase-separated DPPC monolayers resulted in small round ganglioside-rich microdomains in the center and at the edges of the LC domains. Higher amounts of GM1 (10%) give numerous filaments in the center of the LC domains and larger patches at the edges. A gel phase DPPC monolayer containing GM1 showed large domains containing a network of GM1-rich filaments. The addition of GM1 to a liquid-ordered 2:1 DPPC/cholesterol monolayer gives small, round domains that vary in size from 50 to 150 nm for a range of surface pressures. Larger amounts of GM1 lead to coalescence of the small, round domains to give longer filaments that cover 30-40% of the monolayer surface for 10 mol % GM1. The results indicate that biologically relevant GM1 concentrations lead to submicron-sized domains in a cholesterol-rich liquid-ordered phase that is analogous to that found in detergent-insoluble membrane fractions, and are thought to be important in membrane microdomains or rafts. This demonstrates that AFM studies of model monolayers and bilayers provide a powerful method for the direct detection of microdomains that are too small for study with most other techniques.     

Role of lipid rafts in agrin-elicited acetylcholine receptor clustering

Emerging concepts of membrane organization point to the compartmentalization of the plasma membrane into distinct lipid microdomains. This lateral segregation within cellular membranes is based on cholesterol-sphingolipid-enriched microdomains or lipid rafts which can move laterally and assemble into large-scale domains to create plasma membrane specialized cellular structures at specific cell locations. Such domains are likely involved in the genesis of the postsynaptic specialization at the neuromuscular junction, which requires the accumulation of acetylcholine receptors (AChRs), through activation of the muscle specific kinase MuSK by the neurotropic factor agrin and the reorganization of the actin cytoskeleton. We used C2C12 myotubes as a model system to investigate whether agrin-elicited AChR clustering correlated with lipid rafts. In a previous study, using two-photon Laurdan confocal imaging, we showed that agrin-induced AChR clusters corresponded to condensed membrane domains: the biophysical hallmark of lipid rafts [F. Stetzkowski-Marden, K. Gaus, M. Recouvreur, A. Cartaud, J. Cartaud, Agrin elicits membrane condensation at sites of acetylcholine receptor clusters in C2C12 myotubes, J. Lipid Res. 47 (2006) 2121-2133]. We further demonstrated that formation and stability of AChR clusters depend on cholesterol. We also reported that three different extraction procedures (Triton X-100, pH 11 or isotonic Ca++, Mg++ buffer) generated detergent resistant membranes (DRMs) with similar cholesterol/GM1 ganglioside content, which are enriched in several signalling postsynaptic components, notably AChR, the agrin receptor MuSK, rapsyn and syntrophin. Upon agrin engagement, actin and actin-nucleation factors such as Arp2/3 and N-WASP were transiently recovered within raft fractions suggesting that the activation by agrin can trigger actin polymerization. Taken together, the present data suggest that AChR clustering at the neuromuscular junction relies upon a mechanism of raft coalescence driven by agrin-elicited actin polymerization.     

Adriamycin inhibits the formation of non-bilayer lipid structures in cardiolipin-containing model membranes

The effect of adriamycin on cardiolipin-containing model membrane systems have been studied by ³¹P-NMR, freeze-fracture electron microscopy and binding experiments. Adriamycin effectively inhibits the formation of non-bilayer lipid structures induced by Ca²⁺ and cytochrome c in cardiolipin-containing liposomes. This drug also strongly inhibits the uptake of Ca²⁺ by cardiolipin into an organic phase. These results are discussed in relation to the cardiotoxic effect of adriamycin and the possible importance of non-bilayer lipid structures for the functioning of the mitochondrion.