Fluorescence probe partitioning between Lo/Ld phases in lipid membranes

Fluorescence microscopy imaging is an important technique for studying lipid membranes and is increasingly being used for examining lipid bilayer membranes, especially those showing macroscopic coexisting domains. Lipid phase coexistence is a phenomenon of potential biological significance. The identification of lipid membrane heterogeneity by fluorescence microscopy relies on membrane markers with well-defined partitioning behavior. While the partitioning of fluorophores between gel and liquid-disordered phases has been extensively characterized, the same is not true for coexisting liquid phases. We have used fluorescence microscopy imaging to examine a large variety of lipid membrane markers for their liquid phase partitioning in membranes with various lipid compositions. Most fluorescent lipid analogs are found to partition strongly into the liquid-disordered (L_d) phase. In contrast, some fluorescent polycyclic aromatic hydrocarbons with a flat ring system were found to partition equally, but others partition preferentially into liquid-ordered (L_o) phases. We have found these fluorescent markers effective for identification of coexisting macroscopic membrane phases in ternary lipid systems composed of phospholipids and cholesterol.     

Lipid domains in giant unilamellar vesicles and their correspondence with equilibrium thermodynamic phases: A quantitative fluorescence microscopy imaging approach

We report a novel analytical procedure to measure the surface areas of coexisting lipid domains in giant unilamellar vesicles (GUVs) based on image processing of 3D fluorescence microscopy data. The procedure involves the segmentation of lipid domains from fluorescent image stacks and reconstruction of 3D domain morphology using active surface models. This method permits the reconstruction of the spherical surface of GUVs and determination of the area fractions of coexisting lipid domains at the level of single vesicles. Obtaining area fractions enables the scrutiny of the lever rule along lipid phase diagram's tie lines and to test whether or not the coexistence of lipid domains in GUVs correspond to equilibrium thermodynamic phases. The analysis was applied to DLPC/DPPC GUVs displaying coexistence of lipid domains. Our results confirm the lever rule, demonstrating that the observed membrane domains correspond to equilibrium thermodynamic phases (i.e., solid ordered and liquid disordered phases). In addition, the fact that the lever rule is validated from 11 to 14 randomly selected GUVs per molar fraction indicates homogeneity in the lipid composition among the explored GUV populations. In conclusion, our study shows that GUVs are reliable model systems to perform equilibrium thermodynamic studies of membranes.     

Fluorescence probe partitioning between Lo/Ld phases in lipid membranes

Fluorescence microscopy imaging is an important technique for studying lipid membranes and is increasingly being used for examining lipid bilayer membranes, especially those showing macroscopic coexisting domains. Lipid phase coexistence is a phenomenon of potential biological significance. The identification of lipid membrane heterogeneity by fluorescence microscopy relies on membrane markers with well-defined partitioning behavior. While the partitioning of fluorophores between gel and liquid-disordered phases has been extensively characterized, the same is not true for coexisting liquid phases. We have used fluorescence microscopy imaging to examine a large variety of lipid membrane markers for their liquid phase partitioning in membranes with various lipid compositions. Most fluorescent lipid analogs are found to partition strongly into the liquid-disordered (L(d)) phase. In contrast, some fluorescent polycyclic aromatic hydrocarbons with a flat ring system were found to partition equally, but others partition preferentially into liquid-ordered (L(o)) phases. We have found these fluorescent markers effective for identification of coexisting macroscopic membrane phases in ternary lipid systems composed of phospholipids and cholesterol.     

Segregation of saturated chain lipids in pulmonary surfactant films and bilayers

The physical properties of organized system (bilayers and monolayers at the air water interface) composed of bovine lipid extract surfactant (BLES) were studied using correlated experimental techniques. 6-Dodecanoyl-2-dimethylamino-naphthalene (LAURDAN)-labeled giant unilamelar vesicles (mean diameter approximately 30 microm) composed of BLES were observed at different temperatures using two-photon fluorescence microscopy. As the temperature was decreased, dark domains (gel-like) appeared at physiological temperature (37 degrees C) on the surface of BLES giant unilamelar vesicles. The LAURDAN two-photon fluorescent images show that the gel-like domains span the lipid bilayer. Quantitative analysis of the LAURDAN generalized polarization function suggests the presence of a gel/fluid phase coexistence between 37 degrees C to 20 degrees C with low compositional and energetic differences between the coexisting phases. Interestingly, the microscopic scenario of the phase coexistence observed below 20 degrees C shows different domain's shape compared with that observed between 37 degrees C to 20 degrees C, suggesting the coexistence of two ordered but differently organized lipid phases on the bilayer. Epifluorescence microscopy studies of BLES monomolecular films doped with small amounts of fluorescent lipids showed the appearance and growth of dark domains (liquid condensed) dispersed in a fluorescent phase (liquid expanded) with shapes and sizes similar to those observed in BLES giant unilamelar vesicles. Our study suggests that bovine surfactant lipids can organize into discrete phases in monolayers or bilayers with equivalent temperature dependencies and may occur at physiological temperatures and surface pressures equivalent to those at the lung interface.     

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.     

Modulation of concentration fluctuations in phase-separated lipid membranes by polypeptide insertion

The lateral membrane organization and phase behavior of the binary lipid mixture DMPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine) - DSPC (1,2-distearoyl-sn-glycero-3-phosphatidylcholine) without and with incorporated gramicidin D (GD) as a model biomembrane polypeptide was studied by small-angle neutron scattering, Fourier-transform infrared spectroscopy, and by two-photon excitation fluorescence microscopy on giant unilamellar vesicles. The small-angle neutron scattering method allows the detection of concentration fluctuations in the range from 1 to 200 nm. Fluorescence microscopy was used for direct visualization of the lateral lipid organization and domain shapes on a micrometer length scale including information of the lipid phase state. In the fluid-gel coexistence region of the pure binary lipid system, large-scale concentration fluctuations appear. Infrared spectral parameters were used to determine the peptide conformation adopted in the different lipid phases. The data show that the structure of the temperature-dependent lipid phases is significantly altered by the insertion of 2 to 5 mol% GD. At temperatures corresponding to the gel-fluid phase coexistence region the concentration fluctuations drastically decrease, and we observe domains in the giant unilamellar vesicles, which mainly disappear by the incorporation of 2 to 5 mol% GD. Further, the lipid matrix has the ability to modulate the conformation of the inserted polypeptide. The balance between double-helical and helical dimer structures of GD depends on the phospholipid chain length and phase state. A large hydrophobic mismatch, such as in gel phase one-component DSPC bilayers, leads to an increase in population of double-helical structures. Using an effective molecular sorting mechanism, a large hydrophobic mismatch can be avoided in the DMPC-DSPC lipid mixture, which leads to significant changes in the heterogeneous lipid structure and in polypeptide conformation.     

Polypeptide clearing in model membranes: an analysis of the partition of gramicidin a' between cadmium ion induced gel and liquid-crystalline phases in vesicles of phosphatidic acid and phosphatidylcholine

Using a simple model for a biological membrane we examine cation-induced gel phase formation and the depletion of polypeptide from the gel phase. The model system consists of vesicles of phosphatidic acid and phosphatidylcholine which contain gramicidin A'. By use of electron spin resonance to monitor lipid phase behavior, Cd2+ is found to induce gel and liquid-crystal phase coexistence over a wide range of lipid composition. Quenching of gramicidin A' tryptophanyl fluorescence by spin-labeled phosphatidic acid or spin-labeled phosphatidylcholine is analyzed to obtain the partition coefficient, Kp, for gramicidin A' between gel and liquid-crystal phases. The value of Kp = 3 favoring the liquid-crystal phase indicates a partial clearing of the membrane-bound polypeptide from Cd2+-induced gel phase regions of the membrane.     

Compositional domain immiscibility in whole myelin monolayers at the air-water interface and Langmuir-Blodgett films

Monomolecular layers of whole myelin membrane can be formed at the air-water interface from vesicles or from solvent solution of myelin. The films appear microheterogeneous as seen by epifluorescence and Brewster angle microscopy. The pattern consists mainly of two coexisting liquid phases over the whole compression isotherm. The liquid nature of the phases is apparent from the fluorescent probe behavior, domain mobility, deformability and boundary relaxation due to the line tension of the surface domains. The monolayers were transferred to alkylated glass and fluorescently labeled against myelin components. The immunolabeling of two major proteins of myelin (myelin basic protein, proteolipid-DM20) and of 2',3'-cyclic nucleotide 3'-phosphodiesterase shows colocalization with probes partitioning preferentially in liquid-expanded lipid domains also containing ganglioside G(M1). A different phase showing an enrichment in cholesterol, galactocerebroside and phosphatidylserine markers is also found. The distribution of components is qualitatively independent of the lateral surface pressure and is generally constituted by one phase enriched in charged components in an expanded state coexisting with another phase enriched in non-charged constituents of lower compressibility. The domain immiscibility provides a physical basis for the microheterogeneity found in this membrane model system.     

Detection of motional heterogeneities in lipid bilayer membranes by dual probe fluorescence correlation spectroscopy

We report the detection of heterogeneities in the diffusion of lipid molecules for the three-component mixture dipalmitoyl-PC/dilauroyl-PC/cholesterol, a chemically simple lipid model for the mammalian plasma membrane outer leaflet. Two-color fluorescence correlation spectroscopy (FCS) was performed on giant unilamellar vesicles (GUVs) using fluorescent probes that have differential lipid phase partition behavior—DiO-C18:2 favors disordered fluid lipid phases, whereas DiI-C20:0 prefers spatially ordered lipid phases. Simultaneously-obtained fluorescence autocorrelation functions from the same excitation volume for each dye showed that, depending on the lipid composition of this ternary mixture, the two dyes exhibited different lateral mobilities in regions of the phase diagram with previously proposed submicroscopic two-phase coexistence. In one-phase regions, both dyes reported identical diffusion coefficients. Two-color FCS thus may be detecting local membrane heterogeneities at size scales below the optical resolution limit, either due to short-range order in a single phase or due to submicroscopic phase separation.     

Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence.

The organization of lipids surrounding membrane proteins can influence their properties. We have used 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) to study phase coexistence and phase interconversion in membrane model systems. The fluorescence properties of Laurdan provide a unique possibility to study lipid domains because of the different excitation and emission spectra of this probe in the gel and in the liquid-crystalline phase. The difference in excitation spectra allows photoselection of Laurdan molecules in one of the two phases. Using the difference in emission spectra it is then possible to observe interconversion between the two phases. We have performed experiments in dipalmitoyl-phosphatidylcholine (DPPC) vesicles at different temperatures, in particular in the region of the phase transition, where phase coexistence and interconversion between phases is likely to be maximal. We have also studied vesicles of different lipids and mixtures dilauroyl-phosphatidylcholine (DLPC), DPPC, and 50% DLPC in DPPC. Both steady-state fluorescence intensity and polarization data have been collected. To quantitate phase coexistence and interconversion we have introduced the concept of "generalized polarization." We have also performed time-resolved experiments to directly prove the interconversion process. We have found that in DLPC-DPPC mixtures, at 20 degrees C, phase interconversion occurs in approximately 30-40 ns.     

The role of sulfatide lipid domains in the membrane pore-forming activity of cobra cardiotoxin

Cobra CTX A3, the major cardiotoxin (CTX) from Naja atra, is a cytotoxic, basic β-sheet polypeptide that is known to induce a transient membrane leakage of cardiomyocytes through a sulfatide-dependent CTX membrane pore formation and internalization mechanism. The molecular specificity of CTX A3-sulfatide interaction at atomic levels has also been shown by both nuclear magnetic resonance (NMR) and X-ray diffraction techniques to reveal a role of CTX-induced sulfatide conformational changes for CTX A3 binding and dimer formation. In this study, we investigate the role of sulfatide lipid domains in CTX pore formation by various biophysical methods, including fluorescence imaging and atomic force microscopy, and suggest an important role of liquid-disordered (ld) and solid-ordered (so) phase boundary in lipid domains to facilitate the process. Fluorescence spectroscopic studies on the kinetics of membrane leakage and CTX oligomerization further reveal that, although most CTXs can oligomerize on membranes, only a small fraction of CTXs oligomerizations form leakage pores. We therefore suggest that CTX binding at the boundary between the so and so/ld phase coexistence sulfatide lipid domains could form effective pores to significantly enhance the CTX-induced membrane leakage of sulfatide-containing phosphatidylcholine vesicles. The model is consistent with our earlier observations that CTX may penetrate and lyse the bilayers into small aggregates at a lipid/protein molar ratio of about 20 in the ripple P(β)' phase of phosphatidylcholine bilayers and suggest a novel mechanism for the synergistic action of cobra secretary phospholipase A2 and CTXs.     

Detection of motional heterogeneities in lipid bilayer membranes by dual probe fluorescence correlation spectroscopy

We report the detection of heterogeneities in the diffusion of lipid molecules for the three-component mixture dipalmitoyl-PC/dilauroyl-PC/cholesterol, a chemically simple lipid model for the mammalian plasma membrane outer leaflet. Two-color fluorescence correlation spectroscopy (FCS) was performed on giant unilamellar vesicles (GUVs) using fluorescent probes that have differential lipid phase partition behavior--DiO-C18:2 favors disordered fluid lipid phases, whereas DiI-C20:0 prefers spatially ordered lipid phases. Simultaneously-obtained fluorescence autocorrelation functions from the same excitation volume for each dye showed that, depending on the lipid composition of this ternary mixture, the two dyes exhibited different lateral mobilities in regions of the phase diagram with previously proposed submicroscopic two-phase coexistence. In one-phase regions, both dyes reported identical diffusion coefficients. Two-color FCS thus may be detecting local membrane heterogeneities at size scales below the optical resolution limit, either due to short-range order in a single phase or due to submicroscopic phase separation.     

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

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

Comparison of three ternary lipid bilayer mixtures: FRET and ESR reveal nanodomains

Phase diagrams of ternary lipid mixtures containing cholesterol have provided valuable insight into cell membrane behaviors, especially by describing regions of coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases. Fluorescence microscopy imaging of giant unilamellar vesicles has greatly assisted the determination of phase behavior in these systems. However, the requirement for optically resolved Ld + Lo domains can lead to the incorrect inference that in lipid-only mixtures, Ld + Lo domain coexistence generally shows macroscopic domains. Here we show this inference is incorrect for the low melting temperature phosphatidylcholines abundant in mammalian plasma membranes. By use of high compositional resolution Förster resonance energy transfer measurements, together with electron spin resonance data and spectral simulation, we find that ternary mixtures of DSPC and cholesterol together with either POPC or SOPC, do indeed have regions of Ld + Lo coexistence. However, phase domains are much smaller than the optical resolution limit, likely on the order of the Förster distance for energy transfer (R₀, ∼2-8 nm).     

HDLs induce raft domain vanishing in heterogeneous giant vesicles

Cholesterol efflux from the plasma membrane to HDLs is essential for cell cholesterol homeostasis. Recently, cholesterol-enriched ordered membrane domains, i.e. lipid rafts have been proposed to play an important role in this process. Here we introduce a new method to investigate the role of HDL interactions with the raft lipid phase and to directly visualize the effects of HDL-induced cholesterol efflux on rafts in model membranes. Addition of HDLs to giant lipid vesicles containing raft-type domains promoted decrease in size and disappearance of such domains as visualized by fluorescence microscopy. This was interpreted as resulting from cholesterol efflux from the vesicles to the HDLs. The raft vanishing rate was directly related to the HDL concentration. Evidence for a direct interaction of HDLs with the membrane was obtained by observing mutual adhesion of vesicles. It is suggested that the present method can be used to study the selective role of the bilayer lipid phase (raft and non-raft) in cholesterol efflux and membrane-HDL interaction and their underlying mechanisms. Such mechanisms may contribute to cholesterol efflux in vivo.     

Compositional domain immiscibility in whole myelin monolayers at the air-water interface and Langmuir-Blodgett films

Monomolecular layers of whole myelin membrane can be formed at the air-water interface from vesicles or from solvent solution of myelin. The films appear microheterogeneous as seen by epifluorescence and Brewster angle microscopy. The pattern consists mainly of two coexisting liquid phases over the whole compression isotherm. The liquid nature of the phases is apparent from the fluorescent probe behavior, domain mobility, deformability and boundary relaxation due to the line tension of the surface domains. The monolayers were transferred to alkylated glass and fluorescently labeled against myelin components. The immunolabeling of two major proteins of myelin (myelin basic protein, proteolipid-DM20) and of 2′,3′-cyclic nucleotide 3′-phosphodiesterase shows colocalization with probes partitioning preferentially in liquid-expanded lipid domains also containing ganglioside G_M1. A different phase showing an enrichment in cholesterol, galactocerebroside and phosphatidylserine markers is also found. The distribution of components is qualitatively independent of the lateral surface pressure and is generally constituted by one phase enriched in charged components in an expanded state coexisting with another phase enriched in non-charged constituents of lower compressibility. The domain immiscibility provides a physical basis for the microheterogeneity found in this membrane model system.     

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.     

Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy

Confocal fluorescence microscopy and fluorescence correlation spectroscopy (FCS) have been employed to investigate the lipid spatial and dynamic organization in giant unilamellar vesicles (GUVs) prepared from ternary mixtures of dioleoyl-phosphatidylcholine/sphingomyelin/cholesterol. For a certain range of cholesterol concentration, formation of domains with raft-like properties was observed. Strikingly, the lipophilic probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C18) was excluded from sphingomyelin-enriched regions, where the raft marker ganglioside GM1 was localized. Cholesterol was shown to promote lipid segregation in dioleoyl-phosphatidylcholine-enriched, liquid-disordered, and sphingomyelin-enriched, liquid-ordered phases. Most importantly, the lipid mobility in sphingomyelin-enriched regions significantly increased by increasing the cholesterol concentration. These results pinpoint the key role, played by cholesterol in tuning lipid dynamics in membranes. At cholesterol concentrations >50 mol%, domains vanished and the lipid diffusion slowed down upon further addition of cholesterol. By taking the molecular diffusion coefficients as a fingerprint of membrane phase compositions, FCS is proven to evaluate domain lipid compositions. Moreover, FCS data from ternary and binary mixtures have been used to build a ternary phase diagram, which shows areas of phase coexistence, transition points, and, importantly, how lipid dynamics varies between and within phase regions.     

Aqueous two-phase polymer partitioning of lipid vesicles of defined size and composition

The kinetics of the partitioning of lipid vesicles containing acidic phospholipids in aqueous two-phase polymer systems are dependent upon the vesicle size; the larger the vesicles, the more readily they absorb to the interfaces between the two polymer phases and hence are cleared from the top phase as phase separation proceeds. The partitioning of neutral lipid vesicles is principally to the bulk interface and is the same in phase systems of both low and high electrostatic potential difference between the two phases (delta psi). The incorporation of negatively charged lipids has two effects upon partition. First, vesicles with negatively charged lipids exhibit increased bottom phase partitioning in phases of low delta psi due to an enhanced wetting of the charged lipids by the lower phase. Second, the presence of a negatively charged group on the vesicle surface results in increased partition to the interface and top phase in phase systems of high delta psi. Differences observed in the partition of vesicles containing various species of negatively charged lipid thus reflect a competition between these two opposing factors.     

Destabilization exerted by peptides derived from the membrane-proximal external region of HIV-1 gp41 in lipid vesicles supporting fluid phase coexistence

The human immunodeficiency virus (HIV) envelope is enriched in cholesterol and sphingomyelin, two lipids that sustain the formation of laterally segregated liquid-ordered fluid domains in model systems. Several evidences indicate that the high lipid order existing at the envelope may play a role in HIV pathogenesis. A putative mechanism might involve the modulation of the membrane-perturbing function of the gp41 membrane-proximal external region (MPER). To test such hypothesis, we investigate here the effect of lipid phase coexistence on the membrane-restructuring properties of NpreTM and CpreTM, two peptides based on the amino- and carboxy-terminal MPER sequences, respectively. Fluid phase coexistence elicited the fusogenic activity of NpreTM at high membrane doses and stimulated "graded" leakage at low doses. By comparison, the effect on CpreTM was restricted to an enhancement of "all-or-none" leakage that was consistent with the promotion of its surface aggregation. Confocal microscopy of single vesicles revealed the preference of both peptides for liquid-disordered domains. Accordingly, we speculate that confinement into envelope fluid nanodomains might boost the distinct capacities of HIV MPER hydrophobic modules for inducing membrane defects during fusion.