Association of a fluorescent amphiphile with lipid bilayer vesicles in regions of solid-liquid-disordered phase coexistence

The effects of solid-fluid phase separations on the kinetics of association of a single-chain fluorescent amphiphile were investigated in two different systems: pure DMPC (dimyristoylphosphatidylcholine) and a 1:1 mixture of DMPC and DSPC (distearoylphosphatidylcholine). In pure DMPC vesicles, solid (s) and fluid (l(d)) phases coexist at the phase transition temperature, T(m), whereas a 1:1 mixture of DMPC and DSPC shows a stable s-l(d) phase separation over a large temperature interval. We found that in single-component bilayers, within the main phase transition, the experimental kinetics of association are clearly not single-exponential, the deviation from that function becoming maximal at the T(m). This observation can be accounted for by a rate of desorption that is slower than desorption from either fluid or solid phases, leaving the rates of insertion unchanged, but a treatment in terms of stable fluid and solid domains may not be adequate for the analysis of the association of an amphiphile with pure DMPC vesicles at the T(m). In DMPC/DSPC mixtures with solid-fluid phase coexistence, association occurs overall faster than expected based on phase composition. The observed kinetics can be described by an increase in the rate of insertion, leaving the desorption rates unchanged. The fast kinetics of insertion of the amphiphile into two-phase bilayers in two-component vesicles is attributed to a more rapid insertion into defect-rich regions, which are most likely phase boundaries between solid and fluid domains. A two-component mixture of lipids that shows a stable phase separation between l(d)-s phases over a large temperature interval thus behaves very differently from a single-component bilayer at the T(m), with respect to insertion of amphiphiles.     

Direct measurement of phase coexistence in DPPC/cholesterol vesicles using Raman spectroscopy

The phase behavior of bilayers of binary mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol has been studied using Raman spectroscopy. It is observed that the shape of the cholesterol vibrational spectrum in lipid-cholesterol binary mixtures does not vary significantly with either the cholesterol concentration or the temperature. This permits determination of the lipid vibrational signatures of the liquid-disordered (l(d)), solid-ordered (s(o)) and liquid-ordered (l(o)) phases. Within the phase coexistence region, the measured spectra are described very well by a linear combination of the different spectral components, which permits a quantitative analysis of the phase diagram. In contrast to earlier findings, our experiments provide no indication of a phase boundary at low cholesterol concentration. The upper boundary of the phase coexistence region is found at approximately 27 and approximately 22 mol% for l(d)-l(o) and s(o)-l(o) coexistence region, respectively. Within these phase coexistence regions, the partitioning of cholesterol between the cholesterol-poor and the cholesterol-rich phases is in close agreement with the lever rule.     

The effect of cholesterol on the lateral diffusion of phospholipids in oriented bilayers

Pulsed field gradient NMR was utilized to directly determine the lipid lateral diffusion coefficient for the following macroscopically aligned bilayers: dimyristoylphosphatidylcholine (DMPC), sphingomyelin (SM), palmitoyloleoylphosphatidylcholine (POPC), and dioleoylphosphatidylcholine (DOPC) with addition of cholesterol (CHOL) up to approximately 40 mol %. The observed effect of cholesterol on the lipid lateral diffusion is interpreted in terms of the different diffusion coefficients obtained in the liquid ordered (l(o)) and the liquid disordered (l(d)) phases occurring in the phase diagrams. Generally, the lipid lateral diffusion coefficient decreases linearly with increasing CHOL concentration in the l(d) phase for the PC-systems, while it is almost independent of CHOL for the SM-system. In this region the temperature dependence of the diffusion was always of the Arrhenius type with apparent activation energies (E(A)) in the range of 28-40 kJ/mol. The l(o) phase was characterized by smaller diffusion coefficients and weak or no dependence on the CHOL content. The E(A) for this phase was significantly larger (55-65 kJ/mol) than for the l(d) phase. The diffusion coefficients in the two-phase regions were compatible with a fast exchange between the l(d) and l(o) regions in the bilayer on the timescale of the NMR experiment (100 ms). Thus, strong evidence has been obtained that fluid domains (with size of micro m or less) with high molecular ordering are formed within a single lipid bilayer. These domains may play an important role for proteins involved in membrane functioning frequently discussed in the recent literature. The phase diagrams obtained from the analysis of the diffusion data are in qualitative agreement with earlier published ones for the SM/CHOL and DMPC/CHOL systems. For the DOPC/CHOL and the POPC/CHOL systems no two-phase behavior were observed, and the obtained E(A):s indicate that these systems are in the l(d) phase at all CHOL contents for temperatures above 25 degrees C.     

Bilayer polarity and its thermal dependency in the l(o) and l(d) phases of binary phosphatidylcholine/cholesterol mixtures

Diverse variations in membrane properties are observed in binary phosphatidylcholine/cholesterol mixtures. These mixtures are nonideal, displaying single or phase coexistence, depending on chemical composition and other thermodynamic parameters. When compared with pure phospholipid bilayers, there are changes in water permeability, bilayer thickness and thermomechanical properties, molecular packing and conformational freedom of phospholipid acyl chains, in internal dipolar potential and in lipid lateral diffusion. Based on the phase diagrams for DMPC/cholesterol and DPPC/cholesterol, we compare the equivalent polarity of pure bilayers with specific compositions of these mixtures, by using the Py empirical scale of polarity. Besides the contrast between pure and mixed lipid bilayers, we find that liquid-ordered (l(o)) and liquid-disordered (l(d)) phases display significantly different polarities. Moreover, in the l(o) phase, the polarities of bilayers and their thermal dependences vary with the chemical composition, showing noteworthy differences for cholesterol proportions at 35, 40, and 45 mol%. At 20 degrees C, for DMPC/cholesterol at 35 and 45 mol%, the equivalent dielectric constants are 21.8 and 23.8, respectively. Additionally, we illustrate potential implications of polarity in various membrane-based processes and reactions, proposing that for cholesterol containing bilayers, it may also go along with the occurrence of lateral heterogeneity in biological membranes.     

Use of cyclodextrin for AFM monitoring of model raft formation

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

Fluid-fluid membrane microheterogeneity: a fluorescence resonance energy transfer study

Large unilamellar vesicles of dimyristoylphosphatidylcholine/cholesterol mixtures were studied using fluorescence techniques (steady-state fluorescence intensity and anisotropy, fluorescence lifetime, and fluorescence resonance energy transfer (FRET)). Three compositions (cholesterol mole fraction 0.15, 0.20, and 0.25) and two temperatures (30 and 40 degrees C) inside the coexistence range of liquid-ordered (l(o)) and liquid-disordered (l(d)) phases were investigated. Two common membrane probes, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dimyristoylphosphatidylethanolamine (NBD-DMPE) and N-(lissamine(TM)-rhodamine B)-dimyristoylphosphatidylethanolamine (Rh-DMPE), which form a FRET pair, were used. The l(o)/l(d) partition coefficients of the probes were determined by individual photophysical measurements and global analysis of time-resolved FRET decays. Although the acceptor, Rh-DMPE, prefers the l(d) phase, the opposite is observed for the donor, NBD-DMPE. Accordingly, FRET efficiency decreases as a consequence of phase separation. Comparing the independent measurements of partition coefficient, it was possible to detect very small domains (<20 nm) of l(o) in the cholesterol-poor end of the phase coexistence range. In contrast, domains of l(d) in the cholesterol-rich end of the coexistence range have comparatively large size. These observations are probably related to different processes of phase separation, nucleation being preferred in formation of l(o) phase from initially pure l(d), and domain growth being faster in formation of l(d) phase from initially pure l(o).     

Bending elasticities of model membranes: influences of temperature and sterol content

Giant liposomes obtained by electroformation and observed by phase-contrast video microscopy show spontaneous deformations originating from Brownian motion that are characterized, in the case of quasispherical vesicles, by two parameters only, the membrane tension sigma and the bending elasticity k(c). For liposomes containing dimyristoyl phosphatidylcholine (DMPC) or a 10 mol% cholesterol/DMPC mixture, the mechanical property of the membrane, k(c), is shown to be temperature dependent on approaching the main (thermotropic) phase transition temperature T(m). In the case of DMPC/cholesterol bilayers, we also obtained evidence for a relation between the bending elasticity and the corresponding temperature/cholesterol molecular ratio phase diagram. Comparison of DMPC/cholesterol with DMPC/cholesterol sulfate bilayers at 30 degrees C containing 30% sterol ratio shows that k(c) is independent of the surface charge density of the bilayer. Finally, bending elasticities of red blood cell (RBC) total lipid extracts lead to a very low k(c) at 37 degrees C if we refer to DMPC/cholesterol bilayers. At 25 degrees C, the very low bending elasticity of a cholesterol-free RBC lipid extract seems to be related to a phase coexistence, as it can be observed by solid-state (31)P-NMR. At the same temperature, the cholesterol-containing RBC lipid extract membrane shows an increase in the bending constant comparable to the one observed for a high cholesterol ratio in DMPC membranes.     

Observation of the main phase transition of dinervonoylphosphocholine giant liposomes by fluorescence microscopy

The phase heterogeneity of giant unilamellar dinervonoylphosphocholine (DNPC) vesicles in the course of the main phase transition was investigated by confocal fluorescence microscopy observing the fluorescence from the membrane incorporated lipid analog, 1-palmitoyl-2-(N-4-nitrobenz-2-oxa-1,3-diazol)aminocaproyl-sn-glycero-3-phosphocholine (NBDPC). These data were supplemented by differential scanning calorimetry (DSC) of DNPC large unilamellar vesicles (LUV, diameter approximately 0.1 and 0.2 microm) and multilamellar vesicles (MLV). The present data collected upon cooling reveal a lack of micron-scale gel and fluid phase coexistence in DNPC GUVs above the temperature of 20.5 degrees C, this temperature corresponding closely to the heat capacity maxima (T(em)) of DNPC MLVs and LUVs (T(em) approximately 21 degrees C), measured upon DSC cooling scans. This is in keeping with the model for phospholipid main transition inferred from our previous fluorescence spectroscopy data for DMPC, DPPC, and DNPC LUVs. More specifically, the current experiments provide further support for the phospholipid main transition involving a first-order process, with the characteristic two-phase coexistence converting into an intermediate phase in the proximity of T(em). This at least macroscopically homogenous intermediate phase would then transform into the liquid crystalline state by a second-order process, with further increase in acyl chain trans-->gauche isomerization.     

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.     

Permeabilization of raft-containing lipid vesicles by delta-lysin: a mechanism for cell sensitivity to cytotoxic peptides

Delta-lysin is a linear, 26-residue peptide that adopts an alpha-helical, amphipathic structure upon binding to membranes. Delta-lysin preferentially binds to mammalian cell membranes, the outer leaflets of which are enriched in sphingomyelin, cholesterol, and unsaturated phosphatidylcholine. Mixtures including these lipids have been shown to exhibit separation between liquid-disordered (l(d)) and liquid-ordered (l(o)) domains. When rich in sphingomyelin and cholesterol, these ordered domains have been called lipid "rafts". We found that delta-lysin binds poorly to the l(o) (raft) domains; therefore, in mixed-phase lipid vesicles, delta-lysin preferentially binds to the l(d) domains. This leads to the concentration of delta-lysin in l(d) domains, enhancing peptide aggregation and, consequently, the rate of peptide-induced dye efflux from lipid vesicles. The efficient lysis of eukaryotic cells by delta-lysin can thus be attributed not to specific delta-lysin-cholesterol or delta-lysin-sphingomyelin interactions but, rather, to the exclusion of delta-lysin from ordered rafts. The degree to which the kinetics of dye efflux are enhanced in mixed-phase vesicles over those observed in pure, unsaturated phosphatidylcholine vesicles directly reflects the amount of l(d) phase present in mixed-phase systems. This effect of lipid domains has broader consequences, beyond the hemolytic efficiency of delta-lysin. We discuss the hypothesis that bacterial sensitivity to antimicrobial peptides may be determined by a similar mechanism.     

Lipid lateral diffusion and membrane heterogeneity

The pulsed field gradient (pfg)-NMR method for measurements of translational diffusion of molecules in macroscopically aligned lipid bilayers is described. This technique is proposed to have an appreciable potential for investigations in the field of lipid and membrane biology. Transport of molecules in the plane of the bilayer can be successfully studied, as well as lateral phase separation of lipids and their dynamics within the bilayer organizations. Lateral diffusion coefficients depend on lipid packing and acyl chain ordering and investigations of order parameters of perdeuterated acyl chains, using (2)H NMR quadrupole splittings, are useful complements. In this review we summarize some of our recent achievements obtained on lipid membranes. In particular, bilayers exhibiting two-phase coexistence of liquid disordered (l(d)) and liquid ordered (l(o)) phases are considered in detail. Methods for obtaining good oriented lipid bilayers, necessary for the pfg-NMR method to be efficiently used, are also briefly described. Among our major results, besides determinations of l(d) and l(o) phases, belongs the finding that the lateral diffusion is the same for all components, independent of the molecular structure (including cholesterol (CHOL)), if they reside in the same domain or phase in the membrane. Furthermore, quite unexpectedly CHOL seems to partition into the l(d)and l(o) phases to roughly the same extent, indicating that CHOL has no strong preference for any of these phases, i.e. CHOL seems to have similar interactions with all of the lipids. We propose that the lateral phase separation in bilayers containing one high-T(m) and one low-T(m) lipid together with CHOL is driven by the increasing difficulty of incorporating an unsaturated or prenyl lipid into the highly ordered bilayer formed by a saturated lipid and CHOL, i.e. the phase transition is entropy driven to keep the disorder of the hydrocarbon chains of the unsaturated lipid.     

The effect of cholesterol and epicholesterol on the activity and temperature dependence of the purified, phospholipid-reconstituted (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B membranes.

The (Na+ + Mg2+)-ATPase purified from Acholeplasma laidlawii B membranes was reconstituted into large, unilamellar vesicles formed from dimyristoylphosphatidylcholine (DMPC) and varying amounts of cholesterol or epicholesterol. The ATP hydrolytic activity of the reconstituted enzyme was then determined over a range of temperatures and the phase state of the DMPC in the ATPase-containing vesicles was characterized by high-sensitivity differential scanning calorimetry. In the vesicles containing only DMPC, the ATPase activity is higher in association with lipids in the liquid-crystalline state than with gel-state phospholipids, resulting in a curvilinear, biphasic Arrhenius plot with a pronounced change in slope at the elevated gel to liquid-crystalline phase transition temperature of the DMPC. The incorporation of increasing amounts of cholesterol into the DMPC vesicles results in a progressively greater degree of inhibition of ATPase activity at higher temperatures but a stimulation of activity at lower temperatures, thus producing Arrhenius plots with progressively less curvature and without an abrupt change in slope at physiological temperatures. As cholesterol concentration in the ATPase-DMPC vesicles increases, the calorimetric phase transition of the phospholipid is further broadened and eventually abolished. The incorporation of epicholesterol into the DMPC proteoliposomes results in similar but less pronounced effects on ATPase activity, and its effect on the phase behavior of the DMPC-ATPase vesicles is also similarly attenuated in comparison with cholesterol. Moreover, cholesterol added to the purified enzyme in the absence of phospholipid does not show any significant effect on either the activity or the temperature dependence of the detergent-solubilized ATPase. These findings are consistent with the suggestion that cholesterol exerts its effect on the ATPase activity by altering the physical state of the phospholipid, since the ordering effect of cholesterol (or epicholesterol) on liquid-crystalline lipid results in a reduction of ATPase activity while the disordering of gel-state lipid results in an increase in activity.     

Effects of sphingomyelin on melittin pore formation

The effect of sphingomyelin (SM), one of the main lipids in the external monolayer of erythrocyte plasma membrane, on the ability of the hemolytic peptide melittin to permeabilize liposomes was investigated. The peptide induced contents efflux in large unilamellar vesicles (LUV) composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/SM (1:1 mole ratio), at lower (>1:10,000) peptide-to-lipid mole ratios than in pure POPC (>1:1000) or POPC/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) (1:1 mole ratio) (>1:300) vesicles. Analysis of the leakage data according to a kinetic model of pore formation showed a good fit for hexameric-octameric pores in SM-containing vesicles, whereas mediocre fits and lower surface aggregation constants were obtained in POPC and POPC/POPG vesicles. Disturbance of lateral separation into solid (s(o)) and liquid-disordered (l(d)) phases in POPC/SM mixtures increased the peptide-dose requirements for leakage. Inclusion of cholesterol (Chol) in POPC/SM mixtures under conditions inducing lateral separation of lipids into liquid-ordered (l(o)) and l(d) phases did not alter the number of melittin peptides required to permeabilize a single vesicle, but increased surface aggregation reversibility. Partitioning into liposomes or insertion into lipid monolayers was not affected by the presence of SM, suggesting that: (i) melittin accumulated at comparable doses in membranes with different SM content, and (ii) differences in leakage were due to promotion of melittin transmembrane pores under coexistence of s(o)-l(d) and l(o)-l(d) phases. Our results support the notion that SM may regulate the stability of size-defined melittin pores in natural membranes.     

Evidence for a regular distribution of cholesterol in phospholipid bilayers from diphenylhexatriene fluorescence.

Cholesterol/dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles were studied by steady-state fluorescence using diphenylhexatriene (DPH) as a probe. A series of dips were found in the plot of DPH fluorescence intensity versus cholesterol concentration at certain specific cholesterol concentrations. This observation indicates that there are dominant domains in which cholesterol molecules are regularly distributed on a hexagonal superlattice in the acyl chain matrix of DMPC at critical cholesterol concentrations. These concentrations can be predicted by an equation or a mathematical series, except the one at 33 mol %. These dips of DPH fluorescence intensity are temperature dependent. The excellent agreement between experimental data and calculated values as well as similar previous findings of dips and/or kinks in the excimer-over-monomer fluorescence in pyrenephosphatidylcholine/phospholipid mixtures confirm our conclusion about lateral organizations of cholesterol and acyl lipid chains in cholesterol/phospholipid multilamellar vesicles. The regular distribution model at critical concentration is consistent with the phase diagram of cholesterol/DMPC. Using the model of regular distribution, the physical origin of the liquid-disordered (Ld) phase, liquid-ordered phase (Lo), and coexistence of liquid-disordered phase and Lo phase (Lo + Ld) is discussed on the molecular level.     

Fluorescence-based evaluation of the partitioning of lipids and lipidated peptides into liquid-ordered lipid microdomains: a model for molecular partitioning into "lipid rafts"

A fluorescence-quenching assay is described that can directly monitor the relative extents of partitioning of different but structurally homologous fluorescent molecules into liquid-ordered (l(o)) domains in lipid vesicles exhibiting liquid-ordered/liquid-disordered (l(o)/l(d)) phase coexistence. Applying this assay to a series of bimane-labeled diacyl phospholipid probes in cholesterol-containing ternary lipid mixtures exhibiting l(o)/l(d) phase separation, we demonstrate that partitioning into l(o)-phase domains is negligible for diunsaturated species and greatest for long-chain disaturated species. These conclusions agree well with those derived from previous studies of the association of lipids and lipid-anchored molecules with l(o)-phase domains, using methods based on the isolation of a detergent-insoluble fraction from model or biological membranes at low temperatures. However, we also find that monounsaturated and shorter-chain saturated species partition into l(o) phases with significant, albeit modest affinities, and that the level of partitioning of these latter species into l(o)-phase domains is significantly underestimated (relative to that of their long-chain saturated counterparts) by the criterion of low-temperature detergent insolubility. Finally, applying the fluorescence-quenching method to a family of lipid-modified peptides, we demonstrate that the S-palmitoyl/S-isoprenyl dual-lipidation motif found in proteins such as H- and N-ras and yeast Ste18p does not promote significant association with l(o) domains in l(o)/l(d)-phase-separated bilayers.     

Role of cholesterol in the formation and nature of lipid rafts in planar and spherical model membranes

Sterols play a crucial regulatory and structural role in the lateral organization of eukaryotic cell membranes. Cholesterol has been connected to the possible formation of ordered lipid domains (rafts) in mammalian cell membranes. Lipid rafts are composed of lipids in the liquid-ordered (l(o)) phase and are surrounded with lipids in the liquid-disordered (l(d)) phase. Cholesterol and sphingomyelin are thought to be the principal components of lipid rafts in cell and model membranes. We have used fluorescence microscopy and fluorescence recovery after photobleaching in planar supported lipid bilayers composed of porcine brain phosphatidylcholine (bPC), porcine brain sphingomyelin (bSM), and cholesterol to map the composition-dependence of l(d)/l(o) phase coexistence. Cholesterol decreases the fluidity of bPC bilayers, but disrupts the highly ordered gel phase of bSM, leading to a more fluid membrane. When mixed with bPC/bSM (1:1) or bPC/bSM (2:1), cholesterol induces the formation of l(o) phase domains. The fraction of the membrane in the l(o) phase was found to be directly proportional to the cholesterol concentration in both phospholipid mixtures, which implies that a significant fraction of bPC cosegregates into l(o) phase domains. Images reveal a percolation threshold, i.e., the point where rafts become connected and fluid domains disconnected, when 45-50% of the total membrane is converted to the l(o) phase. This happens between 20 and 25 mol % cholesterol in 1:1 bPC/bSM bilayers and between 25 and 30 mol % cholesterol in 2:1 bPC/bSM bilayers at room temperature, and at approximately 35 mol % cholesterol in 1:1 bPC/bSM bilayers at 37 degrees C. Area fractions of l(o) phase lipids obtained in multilamellar liposomes by a fluorescence resonance energy transfer method confirm and support the results obtained in planar lipid bilayers.     

Imaging and shape analysis of GUVs as model plasma membranes: effect of trans DOPC on membrane properties

Unsaturated trans fatty acids have been linked to a higher incidence of coronary artery disease, but not enough is known about the effect of trans lipids on membrane properties. Liquid-ordered (l(o)) and liquid-disordered (l(d)) membrane domains are implicated in various biological processes, such as endocytosis, adhesion, signaling, protein transport, apoptosis, and disease pathogenesis. The physical forces that induce domain formation and thus orchestrate cell function need to be further addressed and quantified. Here, we test the effect of trans DOPC (dielaidoyl phosphatidylcholine or DEPC) on the morphology of giant unilamellar vesicles (GUVs, used as a biomembrane model) made by electroformation with varying compositions of egg sphingomyelin, trans DOPC, cis DOPC, and cholesterol. GUVs were imaged by confocal fluorescence microscopy and then analyzed for changes in membrane morphology and properties such as l(o)/l(d) phase coexistence and area fractions, distribution of meridional curvature, and fluorescent-probe intensity distribution. BODIPY-FL-C(12)-sphingomyelin, Lissamine rhodamine B dioleoylphosphatidylethanolamine and BODIPY-TR-C(12)-sphingomyelin were used as fluorescent probes to differentially label the l(o) and l(d) phases. Trans DOPC induces some vesicles to form multidomain, invaginated morphologies that differ from the typical two-domain circular and truncated spherical shapes observed in its absence. Trans DOPC also alters the membrane curvature distribution; this is more pronounced in the l(o) phase near the phase boundary, where significantly negative curvatures (<-0.5 microm(-1)) are observed. A narrower distribution of meridional curvatures in GUVs with trans DOPC is suggestive of higher membrane bending rigidity. The ratio of average fluorescent intensities in the l(d)/l(o) phases indicates a greater concentration or brightness of the probes BODIPY-FL-C(12)-sphingomyelin and BODIPY-TR-C(12)-sphingomyelin in the l(o) phase in the presence of trans DOPC. Addition of trans DOPC does not alter the l(o)/l(d) area fractions, indicating that it does not act like egg sphingomyelin, a saturated lipid. These changes in membrane properties seen in the presence of trans lipids could significantly impact cell function.     

Lipid lateral diffusion in ordered and disordered phases in raft mixtures

Lipid lateral diffusion coefficients in the quarternary system of dioleoylphosphatidylcholine (DOPC), sphingomyelin, cholesterol, and water were determined by the pulsed field gradient NMR technique on macroscopically aligned bilayers. The molar ratio between dioleoylphosphatidylcholine and sphingomyelin was set to 1:1, the cholesterol content was varied between 0 and 45 mol %, the water content was 40 wt %, and the temperature was varied between 293 and 333 K. The diffusion coefficients were separated into fast and slow spectral components by using the CORE method for global analysis of correlated spectral data. A large two-phase region, tentatively assigned to the liquid disordered (l(d)) and the liquid ordered (l(o)) phases, was present in the phase diagram. The l(d) phase was enriched in dioleoylphosphatidylcholine and exhibited diffusion coefficients that were about three to five times larger than for the l(o) phase. Both the diffusion coefficients and the apparent activation energies for the quarternary systems were compatible with earlier reports on ternary phospholipid/cholesterol/water systems. However, in contrast to the latter ternary systems, the exchange of lipids between the l(o) and the l(d) phases was slow on the timescale for the diffusion experiment for the quarternary ones. This means that on the millisecond timescale fluid, ordered domains are floating around in a sea of faster diffusing lipids, assigned to consist of mainly dioleoylphosphatidylcholine.     

Temperature change of the ripple structure in fully hydrated dimyristoylphosphatidylcholine/cholesterol multibilayers.

The ripple structure was studied as a function of temperature in fully hydrated dimyristoylphosphatidylcholine (DMPC)/cholesterol multibilayers using synchrotron x-ray small-angle diffraction and freeze-fracture electron microscopy. In the presence of cholesterol, the ripple structure appears below the pretransition temperature of pure DMPC multibilayers. In this temperature range the ripple periodicity is relatively large (25-30 nm) and rapidly decreases with increasing temperature. In this region, defined as region I, we observed coexistence of the P beta' phase and the L beta' phase. The large ripple periodicity is caused by the formation of the P beta' phase region in which cholesterol is concentrated and the L beta' phase region from which cholesterol is excluded. An increase in ripple periodicity also takes place in the narrow temperature range just below the main transition temperature. We define this temperature region as region III, where the ripple periodicity increases dramatically toward the main transition temperature. In region II, between regions I and III, the ripple periodicity decreases gradually with temperature. This behavior is quite similar to that of pure DMPC. Temperature-versus-ripple periodicity curves are parallel among pure DMPC and DMPCs with various cholesterol contents. We explain this behavior in terms of a model proposed by other workers.     

Complexity of lipid domains and rafts in giant unilamellar vesicles revealed by combining imaging and microscopic and macroscopic time-resolved fluorescence

The application of fluorescence lifetime imaging microscopy to study gel/fluid and raftlike lipid domains in giant unilamellar vesicles (GUVs) is demonstrated here. Different regions of the ternary dipalmitoylphosphatidylcholine/dioleoylphosphatidylcholine/cholesterol phase diagram were studied. The head-labeled phospholipid Rhodamine-dioleoylphosphatidylethanolamine (Rhod-DOPE) was used as a fluorescent probe. Gel/fluid and liquid-ordered (l(o))/liquid-disordered (l(d)) phase separation were clearly visualized upon two-photon excitation. Fluorescence intensity decays in different regions of a GUV were also obtained with the microscope in fixed laser-beam configuration. The ensemble behavior of the system was studied by obtaining fluorescence intensity decays of Rhod-DOPE in nongiant vesicle suspensions. The fingerprints for gel/fluid coexistence and for the presence of l(o) raftlike phase, based on fluorescence lifetime imaging microscopy histograms and images, and on the fluorescence intensity decay parameters of Rhod-DOPE, are presented. The presence of three lipid phases in one single GUV is detected unequivocally. From the comparison of lifetime parameters, it can be concluded that the l(o) phase is formed in the binary dipalmitoylphosphatidylcholine/cholesterol but not in the dioleoylphosphatidylcholine/cholesterol mixture. The domains apparent in fluorescence intensity images have a more complex substructure revealed by analysis of the lifetime data. The potential applications of this combined imaging/microscopic/macroscopic methodology are discussed.