Cholesterol transfer between lipid vesicles. Effect of phospholipids and gangliosides.

The effect of lipid composition on the rate of cholesterol movement between cellular membranes is investigated using lipid vesicles. The separation of donor and acceptor vesicles required for rate measurement is achieved by differential centrifugation so that the lipid effect can be quantified in the absence of a charged lipid generally used for ion-exchange-based separation. The rate of cholesterol transfer from small unilamellar vesicles (SUVs) containing 50 mol% cholesterol to a common large unilamellar vesicle (LUV) acceptor containing 20 mol% cholesterol decreases with increasing mol% of sphingomyelin in the SUVs, while phosphatidylethanolamine and phosphatidylserine have no appreciable effect at physiologically relevant levels. There is a large decrease in rate when phosphatidylethanolamine constitutes 50 mol% of donor phospholipids. Interestingly, gangliosides which have the same hydrocarbon moiety as sphingomyelin exert an opposite effect. The effect of spingomyelin seems to be mediated by its ability to decrease the fluidity of the lipid matrix, while that of gangliosides may arise from a weakening of phosphatidylcholine-cholesterol interactions or from a more favourable (less polar) microenvironment for the desorption of cholesterol provided by the head-group interactions involving sugar residues. If the effect of asymmetric transbilayer distribution of lipids is taken into consideration, the observed composition-dependent rate changes could partly account for the large difference in the rates of cholesterol desorption from the inner and outer layers of plasma membrane. Such rate differences may be responsible for an unequal steady-state distribution of cholesterol among various cellular membranes and lipoproteins.     

Interaction of calcium and cholesterol sulphate induces membrane destabilization and fusion: implications for the acrosome reaction

Cholesterol sulphate is a potent stabilizer of membrane bilayer structure in both dielaidoylphosphatidylethanolamine and egg phosphatidylethanolamine model membranes, however, the addition of calcium abolishes this bilayer stabilization. Calcium also induces fusion and leakage of egg phosphatidylethanolamine large unilamellar vesicles containing cholesterol sulphate, but has no effect on fusion or leakage of egg phosphatidylcholine large unilamellar vesicles containing cholesterol sulphate. With egg phosphatidylethanoiamine liposomes, the initial rate, and extent of fusion, at constant calcium concentration, vary inversely with the mol percentage of cholesterol sulphate present in the vesicle membrane. The interaction of calcium and cholesterol sulphate, which causes membrane destabilization and fusion in phosphatidylethanolamine containing model systems, may play a role in the acrosome reaction in human sperm.     

Factors contributing to the distribution of free fatty acids among phospholipid vesicles

The distribution of free fatty acids at equilibrium after incubation of small sonicated unilamellar vesicles (SUV) with large unilamellar vesicles (LUV) of different lipid composition has been determined. Stearic acid (SA) and oleic acid (OA) showed similar preferences for SUV and LUV of egg yolk phosphatidylcholine (EYPC). Both ionized and protonated forms of the free fatty acids (FFAs) behaved similarly with respect to the equilibrium distribution between EYPC of different size. The charge of the vesicles was found, however, to be important, since both FFAs in their ionized form preferentially associated to vesicles of phosphatidylcholine (PC) as compared with vesicles of phosphatidylglycerol (PC). While SA preferred membranes in the gel state, OA showed preference for the membrane in fluid state. The insertion of both OA and SA in phosphatidylethanolamine (PE)/phosphatidylcholine vesicles is less favourable than in vesicles of pure PC. All these data suggest that membrane lipid content may play a role in determining the distribution of free fatty acids among the membranes of a cell.     

Preparation of Artificial Plasma Membrane Mimicking Vesicles with Lipid Asymmetry

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

Sorting of streptavidin protein coats on phase-separating model membranes

Heterogeneities in cell membranes due to the ordering of lipids and proteins are thought to play an important role in enabling protein and lipid trafficking throughout the secretory pathway and in maintaining cell polarization. Protein-coated vesicles provide a major mechanism for intracellular transport of select cargo, which may be sorted into lipid microdomains; however, the mechanisms and physical constraints for lipid sorting by protein coats are relatively unexplored. We studied the influence of membrane-tethered protein coats on the sorting, morphology, and phase behavior of liquid-ordered lipid domains in a model system of giant unilamellar vesicles composed of dioleoylphosphatidylcholine, sphingomyelin, and cholesterol. We created protein-coated membranes by forming giant unilamellar vesicles containing a small amount of biotinylated lipid, thereby creating binding sites for streptavidin and avidin proteins in solution. We found that individual tethered proteins colocalize with the liquid-disordered phase, whereas ordered protein domains on the membrane surface colocalize with the liquid-ordered phase. These observations may be explained by considering the thermodynamics of this coupled system, which maximizes its entropy by cosegregating ordered protein and lipid domains. In addition, protein ordering inhibits lipid domain rearrangement and modifies the morphology and miscibility transition temperature of the membrane, most dramatically near the critical point in the membrane phase diagram. This observation suggests that liquid-ordered domains are stabilized by contact with ordered protein domains; it also hints at an approach to the stabilization of lipid microdomains by cross-linked protein clusters or ordered protein coats.     

Interaction of cholesterol with sphingomyelin in bilayer membranes: evidence that the hydroxy group of sphingomyelin does not modulate the rate of cholesterol exchange between vesicles

Cholesterol undergoes exchange between membranes containing sphingomyelin at a much slower rate than between membranes lacking sphingomyelin. To investigate the role of the hydroxy group at the 3-position of sphingomyelin in the interaction between sphingomyelin and cholesterol, we have measured the rates of [4-14C]cholesterol exchange between unilamellar vesicles prepared with N-stearoylsphingomyelin or with synthetic analogues in which the hydroxy group is replaced with an O-alkyl group or with hydrogen. Vesicles prepared from 3-deoxy- and 3-O-methyl-N-stearoylsphingomyelin had the same rate of [14C]-cholesterol desorption. The half-times for exchange from vesicles prepared with 3-O-methyl- and 3-deoxy-N-stearoylsphingomyelins and 10 mol % of cholesterol were only slightly faster (a factor of only 1.5) than that found from vesicles prepared from N-stearoylsphingomyelin and 10 mol % cholesterol. The rate of cholesterol desorption from vesicles could be accelerated by preparing vesicles from bulky 3-O-alkyl analogues of sphingomyelin. Vesicles containing 3-O-ethyl-N-stearoylsphingomyelin and 3-O-tetrahydropyranyl egg sphingomyelin gave rate enhancements of approximately 14 and 35, compared with the rates observed in vesicles made from N-stearoyl- and egg sphingomyelin, respectively. These data indicate that insertion of sterically bulky groups at the 3-position of sphingomyelin (such as ethoxy and tetrahydropyranyloxy) in place of hydroxy interferes markedly with the molecular packing of cholesterol and sphingomyelin in bilayer membranes; however, the hydroxy group of sphingomyelin is not critical for the strong interaction of cholesterol with sphingomyelin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sphingomyelinase D activity in model membranes: structural effects of in situ generation of ceramide-1-phosphate

The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1) ceramide-1-phosphate (particularly lauroyl ceramide-1-phosphate) can be incorporated into sphingomyelin bilayers in a concentration-dependent manner and generates coexistence of liquid disordered/solid ordered domains, 2) the activity of sphingomyelinase D is clearly influenced by the supramolecular organization of its substrate in membranes and, 3) in situ ceramide-1-phosphate generation by enzymatic activity profoundly alters the lateral structure and morphology of the target membranes.     

Equinatoxin II permeabilizing activity depends on the presence of sphingomyelin and lipid phase coexistence

Equinatoxin II is a pore-forming protein of the actinoporin family. After membrane binding, it inserts its N-terminal α-helix and forms a protein/lipid pore. Equinatoxin II activity depends on the presence of sphingomyelin in the target membrane; however, the role of this specificity is unknown. On the other hand, sphingomyelin is considered an essential ingredient of lipid rafts and promotes liquid-ordered/liquid-disordered phase separation in model membranes that mimic raft composition. Here, we used giant unilamellar vesicles to simultaneously investigate the effect of sphingomyelin and phase separation on the membrane binding and permeabilizing activity of Equinatoxin II. Our results show that Equinatoxin II binds preferentially to the liquid-ordered phase over the liquid-disordered one and that it tends to concentrate at domain interfaces. In addition, sphingomyelin strongly enhances membrane binding of the toxin but is not sufficient for membrane permeabilization. Under the same experimental conditions, Equinatoxin II formed pores in giant unilamellar vesicles containing sphingomyelin only when liquid-ordered and -disordered phases coexisted. Our observations demonstrate the importance of phase boundaries for Equinatoxin II activity and suggest a double role of sphingomyelin as a specific receptor for the toxin and as a promoter of the membrane organization necessary for Equinatoxin II action.     

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

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

Kinetics of cholesterol extraction from lipid membranes by methyl-β-cyclodextrin—A surface plasmon resonance approach

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

Lipid phase coexistence favors membrane insertion of equinatoxin-II, a pore-forming toxin from Actinia equina

Equinatoxin-II is a eukaryotic pore-forming toxin belonging to the family of actinoporins. Its interaction with model membranes is largely modulated by the presence of sphingomyelin. We have used large unilamellar vesicles and lipid monolayers to gain further information about this interaction. The coexistence of gel and liquid-crystal lipid phases in sphingomyelin/phosphatidylcholine mixtures and the coexistence of liquid-ordered and liquid-disordered lipid phases in phosphatidylcholine/cholesterol or sphingomyelin/phosphatidylcholine/cholesterol mixtures favor membrane insertion of equinatoxin-II. Phosphatidylcholine vesicles are not permeabilized by equinatoxin-II. However, the localized accumulation of phospholipase C-generated diacylglycerol creates conditions for toxin activity. By using epifluorescence microscopy of transferred monolayers, it seems that lipid packing defects arising at the interfaces between coexisting lipid phases may function as preferential binding sites for the toxin. The possible implications of such a mechanism in the assembly of a toroidal pore are discussed.     

Vesicles with charged domains

This work summarizes results obtained on membranes composed of the ternary mixture dioleoylphosphatidylglycerol (DOPG), egg sphingomyelin (eSM) and cholesterol (Chol). The membrane phase state as a function of composition is characterized from data collected with fluorescence microscopy on giant unilamellar vesicles. The results suggest that the presence of the charged DOPG significantly decreases the composition region of coexistence of liquid ordered and liquid disordered phases as compared to that in the ternary mixture of dioleoylphosphatidycholine, sphingomyelin and cholesterol. The addition of calcium chloride to DOPG:eSM:Chol vesicles, and to a lesser extent the addition of sodium chloride, leads to the stabilization of the two-phase coexistence region, which is expressed in an increase in the miscibility temperature. On the other hand, addition of the chelating agent EDTA has the opposite effect, suggesting that impurities of divalent cations in preparations of giant vesicles contribute to the stabilization of charged domains. We also explore the behavior of these membranes in the presence of extruded unilamellar vesicles made of the positively charged lipid dioleoyltrimethylammoniumpropane (DOTAP). The latter can induce domain formation in DOPG:eSM:Chol vesicles with initial composition in the one-phase region.     

Stability of giant unilamellar vesicles and large unilamellar vesicles of liquid-ordered phase membranes in the presence of Triton X-100

We have investigated the stability of giant unilamellar vesicles (GUVs) and large unilamellar vesicles (LUVs) of lipid membranes in the liquid-ordered phase (lo phase) against a detergent, Triton X-100. We found that in the presence of high concentrations of Triton X-100, the structure of GUVs and LUVs of dipalmitoyl-PC (DPPC)/cholesterol (chol) and sphingomyelin (SM)/chol membranes in the lo phase was stable and no leakage of fluorescent probes from the vesicles occurred. We also found that ether-linked dihexadecylphosphatidylcholine (DHPC) membranes containing more than 20 mol% cholesterol were in the lo phase, and that DHPC/chol-GUV and DHPC/chol-LUV in the lo phase were stable and no leakage of internal contents occurred in the presence of Triton X-100. In contrast, octylglucoside solution could easily break these GUVs and LUVs of the lo phase membranes and induced internal contents leakage. These data indicate that GUVs and LUVs of the lo phase membranes are very valuable for practical use.     

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

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

Cholesterol modulates curcumin partitioning and membrane effects

Curcumin, a polyphenol molecule, presents a wide range of biological activities as antioxidant, anticancer, anti-inflammatory, antimicrobial and wound healing. Although some strengths attributed to curcumin derive from promiscuous biological activity, possibly because curcumin can interfere on many membrane located processes, knowledge of underlying interactions are lacking. Mammalian cell membranes characteristically contain 25 to 50% cholesterol/phospholipid ratio; however, most studies involving lipid bilayers and curcumin consider pure phosphatidylcholine and compare effects of curcumin on membranes with those of cholesterol. We investigated the interaction of curcumin with lipid bilayers containing cholesterol mimicking mammalian cells, and used spectroscopy techniques to determine partition coefficients, rigidity parameters and lytic activity. We found that curcumin partitions into different lipid bilayers (10⁴ order coefficients that vary by less than a factor of two), containing cholesterol or not, and in the presence of sphingomyelin or phosphatidylserine. Curcumin decreases rigidity in all tested compositions, except that containing 40% cholesterol in which it increases the lipid packing order. In addition, curcumin induces leakage from giant unilamellar vesicles on a cholesterol concentration dependent way. Our results are compatible with the hypothesis of curcumin interaction with membranes being modulated by the liquid disordered phase and by the coexistence of liquid-ordered/liquid disordered phases. In bilayers containing cholesterol, curcumin assumes a more superficial location, drastically stiffens the 40% cholesterol bilayer and decreases the lytic effect. Our study may help researchers in the analysis of the biological effects of curcumin and curcumin-derived formulations by calling the attention to the discriminating role of the cholesterol content.     

Differential interaction of equinatoxin II with model membranes in response to lipid composition

Equinatoxin II is a 179-amino-acid pore-forming protein isolated from the venom of the sea anemone Actinia equina. Large unilamellar vesicles and lipid monolayers of different lipid compositions have been used to study its interaction with membranes. The critical pressure for insertion is the same in monolayers made of phosphatidylcholine or sphingomyelin (approximately 26 mN m(-1)) and explains why the permeabilization of large unilamellar vesicles by equinatoxin II with these lipid compositions is null or moderate. In phosphatidylcholine-sphingomyelin (1:1) monolayers, the critical pressure is higher (approximately 33 mN m(-1)), thus permitting the insertion of equinatoxin II in large unilamellar vesicles, a process that is accompanied by major conformational changes. In the presence of vesicles made of phosphatidylcholine, a fraction of the protein molecules remains associated with the membranes. This interaction is fully reversible, does not involve major conformational changes, and is governed by the high affinity for membrane interfaces of the protein region comprising amino acids 101-120. We conclude that although the presence of sphingomyelin within the membrane creates conditions for irreversible insertion and pore formation, this lipid is not essential for the initial partitioning event, and its role as a specific receptor for the toxin is not so clear-cut.     

Exposure of phosphatidylinositol transfer proteins to sphingomyelin-cholesterol membranes suggests transient but productive interactions with raft-like, liquid-ordered domains

Both isoforms of rat phosphatidylinositol transfer protein (PITP) mediate the intermembrane transfer of sphingomyelin (CerPCho). In the plasma membrane, CerPCho often segregates with cholesterol into microdomains such as lipid rafts and caveolae. To test the hypothesis that PITP exhibits a preference for CerPCho- and cholesterol-rich membranes, we prepared unilamellar vesicles containing variable amounts of these two lipids. We also used CerPCho species with different acyl composition and treated vesicles with agents known to sequester and remove cholesterol. We observed that the beta isoform of rat PITP was more sensitive to membrane cholesterol than was the alpha isoform, as shown by increases in specific activities of lipid transfer of 2-6-fold. A relatively high membrane content of cholesterol (mole fraction > 0.4) was required to elicit such enhancements. Treatment of cholesterol-rich membranes with a series of beta cyclodextrins demonstrated that, upon depletion of cholesterol from participating membranes, the PITPbeta activity changes were fully reversible. We finally noted that the mechanism by which cholesterol enhances the activity of PITPbeta appeared to involve a decreased affinity of the protein for the membrane surface, in a manner that was independent of vesicle size and membrane microviscosity. We conclude that PITPbeta interacts transiently but productively with the liquid-ordered phase formed by CerPCho and cholesterol and discuss the possibility of PITP interactions in vivo with sphingolipid- and cholesterol-rich membrane microdomains.     

Asymmetry of lipid organization in cholinergic synaptic vesicle membranes.

The lipid composition of purified Torpedo cholinergic synaptic vesicles was determined and their distribution between the inner and outer leaflets of the vesicular membrane was investigated. The vesicles contain cholesterol and phospholipids at a molar ratio of 0.63. The vesicular phospholipids are (mol% of total phospholipids): phosphatidylcholine (40.9); phosphatidylethanolamine (24.6); plasmenylethanolamine (11.5); sphingomyelin (12); phosphatidylserine (7.3); phosphatidylinositol (3.7). The asymmetry of the synaptic vesicle membranes was investigated by two independent approaches: (a) determining accessibility of the amino lipids to the chemical label trinitrobenzenesulphonic acid (TNBS); (b) determining accessibility of the vesicular glycerophospholipids to phospholipase C (Bacillus cereus). TNBS was found to render the vesicles leaky and thus cannot be used reliably to determine the asymmetry of Torpedo synaptic vesicle membranes. Incubation of the vesicles with phospholipase C (Bacillus cereus) results in biphasic hydrolysis of the vesicular glycerophospholipids. About 45% of the phospholipids are hydrolysed in less than 1 min, during which no vesicular acetylcholine is released. In the second phase, the hydrolysis of the phospholipids slows down markedly and is accompanied by loss of all the vesicular acetylcholine. These findings suggest that the lipids hydrolysed during the first phase are those comprising the outer leaflet. Analysis of the results thus obtained indicate that the vesicular membrane is asymmetric: all the phosphatidylinositol, 77% of the phosphatidylethanolamine, 47% of the plasmenylethanolamine and 58% of the phosphatidylcholine were found to reside in the outer leaflet. Since phosphatidylserine is a poor substrate for phospholipase C (B. cereus), its distribution between the two leaflets of the synaptic vesicle membrane is only suggestive.     

Lipid dynamics and domain formation in model membranes composed of ternary mixtures of unsaturated and saturated phosphatidylcholines and cholesterol

In recent years, the implication of sphingomyelin in lipid raft formation has intensified the long sustained interest in this membrane lipid. Accumulating evidences show that cholesterol preferentially interacts with sphingomyelin, conferring specific physicochemical properties to the bilayer membrane. The molecular packing created by cholesterol and sphingomyelin, which presumably is one of the driving forces for lipid raft formation, is known in general to differ from that of cholesterol and phosphatidylcholine membranes. However, in many studies, saturated phosphatidylcholines are still considered as a model for sphingolipids. Here, we investigate the effect of cholesterol on mixtures of dioleoyl-phosphatidylcholine (DOPC) and dipalmitoyl-phosphatidylcholine (DPPC) or distearoyl-phosphatidylcholine (DSPC) and compare it to that on mixtures of DOPC and sphingomyelin analyzed in previous studies. Giant unilamellar vesicles prepared from ternary mixtures of various lipid compositions were imaged by confocal fluorescence microscopy and, within a certain range of sterol content, domain formation was observed. The assignment of distinct lipid phases and the molecular mobility in the membrane bilayer was investigated by fluorescence correlation spectroscopy. Cholesterol was shown to affect lipid dynamics in a similar way for DPPC and DSPC when the two phospholipids were combined with cholesterol in binary mixtures. However, the corresponding ternary mixtures exhibited different spatial lipid organization and dynamics. Finally, evidences of a weaker interaction of cholesterol with saturated phosphatidylcholines than with sphingomyelin (with matched chain length) are discussed.     

LUV's lipid composition modulates diffusion of bile acids

Large unilamellar vesicles were prepared from phosphatidylcholine (PC), sphingomyelin (SM), cholesterol (Chol) and cardiolipin (CL) by an extrusion technique (LUVETs). Diffusion of the more hydrophobic lithocholic acid (LCA) and the less hydrophobic chenodeoxycholic acid (CDCA) was investigated by using the pyranine fluorescence method. Membrane permeability was studied by measuring the inclusion of carboxyfluoresceine (CF) into the lipid vesicles, and membrane fluidity was determined with diphenylhexatriene (DPH) and trimethylammonium-diphenylhexatriene (TMA-DPH). All results indicate that, CDCA compared to LCA, exhibits a significantly better penetration into vesicles containing SM. LCA penetrates better into vesicles containing cholesterol. Small amounts of CL influenced the diffusional properties of CDCA more than those of LCA. Since Lamcharfi et al. (1997a) Euro. Biophys. 25, 285-291 have observed differences in the conformational forms of CDCA and LCA in solution, it is suggested that the diffusion rate of bile acids through (model-)membranes is not only dependent on hydrophobicity, but also on bile acid di-(poly-)meric associations and on membrane-lipid composition.