Gel-fluid Transition Research Articles

Detergent-Resistant, Ceramide-Enriched Domains in Sphingomyelin/Ceramide Bilayers

When cell membranes are treated with Triton X-100 or other detergents at 4°C, a nonsolubilized fraction can often be recovered, the “detergent-resistant membranes”, that is not found when detergent treatment takes place at 37°C. Detergent-resistant membranes may be related in some cases to membrane “rafts”. However, several basic aspects of the formation of detergent-resistant membranes are poorly understood. To answer some of the relevant questions, a simple bilayer composition that would mimic detergent-resistant membranes was required. The screening of multiple lipid compositions has shown that the binary mixture egg sphingomyelin/egg ceramide (SM/Cer) exhibits the required detergent resistance. In detergent-free membranes composed of different mixtures of SM and Cer (5–30mol % of Cer) differential scanning calorimetry, fluorescence spectroscopy, and fluorescence microscopy experiments reveal the presence of discrete, Cer-enriched gel domains in a broad temperature range. In particular, at temperatures below SM phase transition (≈ 40°C) two gel (respectively Cer-rich and SM-rich) phases are directly observed using fluorescence microscopy. Although pure SM membranes are fully solubilized by Triton X-100 at room temperature, 5mol % Cer is also enough to induce detergent resistance, even with a large detergent excess and lengthy equilibration times. Short-chain Cers do not give rise to detergent resistance. SM/Cer mixtures containing up to 30mol % Cer become fully soluble at ∼50°C, i.e., well above the gel-fluid transition temperature of SM. The combined results of temperature-dependent solubilization and differential scanning calorimetry reveal that SM-rich domains are preferentially solubilized over the Cer-rich ones as soon as the former melt (i.e., at ∼40°C). As a consequence, at temperatures allowing only partial solubilization, the nonsolubilized residue is enriched in Cer with respect to the original bilayer composition. Fluorescence microscopy of giant unilamellar vesicles at room temperature clearly shows that SM-rich domains are preferentially solubilized over the Cer-rich ones and that the latter become more rigid and extensive as a consequence of the detergent effects. These observations may be relevant to the phenomena of sphingomyelinase-dependent signaling, generation of “raft platforms”, and detergent-resistant cell membranes.

Characterization of cationic liposomes based on dimethyldioctadecylammonium and synthetic cord factor from M. tuberculosis (trehalose 6,6′-dibehenate)—A novel adjuvant inducing both strong CMI and antibody responses

Incorporation of the glycolipid trehalose 6,6′-dibehenate (TDB) into cationic liposomes composed of the quaternary ammonium compound dimethyldioctadecylammonium (DDA) produce an adjuvant system which induces a powerful cell-mediated immune response and a strong antibody response, desirable for a high number of disease targets. We have used differential scanning calorimetry (DSC) to investigate the effect of TDB on the gel-fluid phase transition of DDA liposomes and to demonstrate that TDB is incorporated into DDA liposome bilayers. Transmission Electron Microscopy (TEM) and cryo-TEM confirmed that liposomes were formed when a lipid film of DDA containing small amounts of TDB was hydrated in an aqueous buffer solution at physiological pH. Furthermore, time development of particle size and zeta potential of DDA liposomes incorporating TDB during storage at 4 °C and 25 °C, indicates that TDB effectively stabilizes the DDA liposomes. Immunization of mice with the mycobacterial fusion protein Ag85B–ESAT-6 in DDA–TDB liposomes induced a strong, specific Th1 type immune response characterized by substantial production of the interferon-γ cytokine and high levels of IgG2b isotype antibodies. The lymphocyte subset releasing the interferon-γ was identified as CD4 T cells.

Direct Visualization of Asymmetric Behavior in Supported Lipid Bilayers at the Gel-Fluid Phase Transition

We utilize in situ, temperature-dependent atomic force microscopy to examine the gel-fluid phase transition behavior in supported phospholipid bilayers constructed from 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. The primary gel-fluid phase transition at Tm occurs through development of anisotropic cracks in the gel phase, which develop into the fluid phase. At ∼5°C above Tm, atomic force microscopy studies reveal the presence of a secondary phase transition in all three bilayers studied. The secondary phase transition occurs as a consequence of decoupling between the two leaflets of the bilayer due to enhanced stabilization of the lower leaflet with either the support or the water entrained between the support and the bilayer. Addition of the transmembrane protein gramicidin A or construction of a highly defected gel phase results in elimination of this decoupling and removal of the secondary phase transition.

Different Effects of Long- and Short-Chain Ceramides on the Gel-Fluid and Lamellar-Hexagonal Transitions of Phospholipids: A Calorimetric, NMR, and X-Ray Diffraction Study

The effects on dielaidoylphosphatidylethanolamine (DEPE) bilayers of ceramides containing different N-acyl chains have been studied by differential scanning calorimetry small angle x-ray diffraction and (31)P-NMR spectroscopy. N-palmitoyl (Cer16), N-hexanoyl (Cer6), and N-acetyl (Cer2) sphingosines have been used. Both the gel-fluid and the lamellar-inverted hexagonal transitions of DEPE have been examined in the presence of the various ceramides in the 0-25 mol % concentration range. Pure hydrated ceramides exhibit cooperative endothermic order-disorder transitions at 93 degrees C (Cer16), 60 degrees C (Cer6), and 54 degrees C (Cer2). In DEPE bilayers, Cer16 does not mix with the phospholipid in the gel phase, giving rise to high-melting ceramide-rich domains. Cer16 favors the lamellar-hexagonal transition of DEPE, decreasing the transition temperature. Cer2, on the other hand, is soluble in the gel phase of DEPE, decreasing the gel-fluid and increasing the lamellar-hexagonal transition temperatures, thus effectively stabilizing the lamellar fluid phase. In addition, Cer2 was peculiar in that no equilibrium could be reached for the Cer2-DEPE mixture above 60 degrees C, the lamellar-hexagonal transition shifting with time to temperatures beyond the instrumental range. The properties of Cer6 are intermediate between those of the other two, this ceramide decreasing both the gel-fluid and lamellar-hexagonal transition temperatures. Temperature-composition diagrams have been constructed for the mixtures of DEPE with each of the three ceramides. The different behavior of the long- and short-chain ceramides can be rationalized in terms of their different molecular geometries, Cer16 favoring negative curvature in the monolayers, thus inverted phases, and the opposite being true of the micelle-forming Cer2. These differences may be at the origin of the different physiological effects that are sometimes observed for the long- and short-chain ceramides.

Liposome Fluidization and Melting Point Depression by Pressurized CO2 Determined by Fluorescence Anisotropy

The influence of CO2 on the bilayer fluidity of liposomes, which are representative of model cellular membranes, was examined for the first time at the elevated pressures (up to 13.9 MPa) associated with CO2-based processing of liposomes and microbial sterilization. Fluidization and melting point depression of aqueous dipalmitoylphosphatidylcholine (DPPC) liposomes by pressurized CO2 (present as an excess phase) were studied by steady-state fluorescence anisotropy using the membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Isothermal experiments revealed reversible, pressure-dependent fluidization of DPPC bilayers at temperatures corresponding to near-gel (295 K) and fluid (333 K) phases at atmospheric pressure, where the gel-to-fluid phase transition (Tm) occurs at approximately 315 K. Isobaric measurements (PCO2 =1.8, 7.0, and 13.9 MPa) of DPH anisotropy demonstrate substantial melting point depression (DeltaTm = -4.8 to -18.5 K) and a large broadening of the gel-fluid phase transition region, which were interpreted using conventional theories of melting point depression. Liposome fluidity is influenced by CO2 accumulation in the hydrocarbon core and polar headgroup region, as well as the formation of carbonic acid and/or the presence of buffering species under elevated CO2 pressure.

Structural characterization of diC 14-amidine, a pH-sensitive cationic lipid used for transfection

The structure of N-t-butyl- N′-tetradecyl-3-tetradecylaminopropionamidine (diC 14-amidine) cationic vesicles, used for transfection, was investigated at different pH values and ionic strengths, through the analysis of the electron spin resonance (ESR) spectra of spin labels. Phospholipid derivatives, spin labeled at the 5th and 16th C-atoms along the hydrocarbon chain, incorporated in diC 14-amidine bilayers, show that the bilayer structure is highly sensitive to the pH value of the medium, due to the two titratable groups present in the amphiphile. Compared with samples at higher pH values, the double charged diC 14-amidine at pH 3 presents a rather non-organized bilayer gel phase, and a much lower gel-fluid temperature transition, in accord with a strong headgroup electrostatic repulsion. In addition, the structure was found to be highly dependent on the ionic strength of the medium. However, pH 3 diC 14-amidine bilayer, in the fluid phase, was found to be slightly more closely packed than those at pH 7.4 or 9.0, which are less charged. Parallel to that, the larger isotropic hyperfine splitting measured for nitroxides in the center of the pH 3 diC 14-amidine bilayer suggests a higher membrane polarity for the highly charged low pH sample.

Cholesterol modulation of sphingomyelinase activity at physiological temperatures

Bacillus cereus sphingomyelinase activity was assayed on large unilamellar vesicles composed of sphingomyelin (SM)/cholesterol (Ch) mixtures at varying proportions. Natural (egg) SM was used with a gel–fluid transition temperature at ca. 40 °C. When the enzyme was assayed at 37 °C, the activity on pure SM was exceedingly low, but a small increase was observed as soon as some Ch was added, and a large enhancement of activity occurred with Ch proportions above 25 mol%. The data were interpreted in terms of sphingomyelinase activity being higher in the cholesterol-induced liquid-ordered phase than in the gel phase. The abrupt increase in activity above 25 mol% Ch would occur as a result of a change in domain connectivity, when the Ch-rich liquid-ordered domains coalesced. In equimolar SM/Ch mixtures, that were in the liquid-ordered state in a wide range of temperatures, sphingomyelinase activity was virtually constant in the 30–70 °C range. The results demonstrate that at the mammalian and bird physiological temperatures Ch modulates sphingomyelinase activity, and that this can occur precisely because most SM have a gel–fluid transition temperature above the physiological temperature range. In addition, Ch activation of sphingomyelinase and the strong affinity of Ch for SM allow the rapid, localised and self-contained production of the metabolic signal ceramide in specific microdomains (rafts).

Farnesylated peptides in model membranes: a biophysical investigation.

Protein prenylation plays an important role in signal transduction, protein-protein interactions, and the localization and association of proteins with membranes. Using three different techniques, this study physically characterizes the interactions between model dimyristoylphosphatidylcholine membranes and a series of farnesylated peptides. Magic angle spinning nuclear Overhauser enhancement spectroscopy and differential scanning calorimetry reveal that both charged [Ac-Asn-Lys-Asn-Cys-(farnesyl)-OMe and Ac-Asn-Lys-Asn-Cys-(farnesyl)-NH(2)] and uncharged [Ac-Cys-(farnesyl)-OMe and farnesol] species partition into dimyristoylphosphatidylcholine bilayers. Calorimetry and vesicle fluctuation analysis of giant unilamellar vesicles show that the charged peptides modestly decrease the main gel-fluid phase transition and markedly increase the bending rigidity of large unilamellar vesicles. Uncharged species, on the other hand, dramatically decrease the main phase transition and modestly decrease the bending rigidity. No difference with carboxyl methylation is detected.

Comment on “Gel-Fluid Transition in Dilute versus Concentrated DMPG Aqueous Dispersions”

Comment on “Gel-Fluid Transition in Dilute versus Concentrated DMPG Aqueous Dispersions”

Hydrophilic/Hydrophobic Balance Determines Morphology of Glycolipids with Oligolactose Headgroups

The morphology of synthetic glycolipids with lactose oligomers (Lac N, the number of lactose units, N = 1, 2, 3) was studied in lamellar phase. By a systematic combination of differential scanning calorimetry and small- and wide-angle x-ray scattering experiments, the effects of hydrophilic/hydrophobic balance on their thermotropic phase behaviors were discussed. The dispersion of Lac 1 exhibited a crystalline-fluid phase transition, dominated by the strong van der Waals interaction between dihexadecyl chains. In the case of Lac 2, the hydrophilic/hydrophobic balance between the headgroup and the alkyl chains is shifted to the hydrophilic side, resulting in a gel-fluid phase transition with a decreased transition temperature and phase transition enthalpy. Different from the first two systems, the differential scanning calorimetry trace of Lac 3 showed much less remarkable peaks. The small- and wide-angle x-ray diffraction patterns did not reveal any transition in the chain ordering, suggesting that the correlation between the hexasaccharide headgroups is so strong that the melting of the alkyl chains was not allowed. Such dominant effects of the hydrophilic/hydrophobic balance on the morphology of Lac N lipids can be attributed to the small sterical mismatch between the alkyl chains and the linear, cylindrical oligolactose groups.

The peculiar thermo-structural behavior of the anionic lipid DMPG

Aqueous dispersions of the anionic phospholipid dimyristoyl phosphatidylglycerol (DMPG), around 100 mM ionic strength, are known to exhibit a thermal behavior similar to that of the largely studied lipid dimyristoyl phosphatidylcholine (DMPC), which undergoes a gel to liquid crystalline phase transition at 23 °C, well characterized by differential scanning calorimetry (DSC), and other methods. However, at low ionic strength, DMPG has been shown to present a large gel–fluid transition region, ranging from 18 to 35 °C. This intermediate phase is optically transparent and characterized by a continuous change in membrane packing. Structural properties of the DMPG gel–fluid transition region will be discussed, based on results obtained by several techniques: electron spin resonance (ESR) of spin labels at the membrane surface and intercalated at different depths in the bilayer; light scattering; DSC; small angle X-ray scattering (SAXS); and fluorescence spectroscopy of probes in the bilayer.

Exploring the ripple phase of biomembranes

Monte Carlo simulations are used to study the rippled Pβ′ phase in biomembranes conceived as a lamellar stack of lipid bilayers, each represented by a two-dimensional hexagonal lattice, in aqueous environment. The appearance of this intermediate phase between the ordered Lβ′ phase and the fluid Lα phase in the temperature driven gel–fluid transition is attributed to the coexistence of gel–fluid rich domains in the pre-transition phase. The interfacial energy, that plays a crucial role in domain formation, is governed by a delicate balance between the hydrophobic interaction within the hydrocarbon chains and the headgroup repulsion at the interface. The latter gets disturbed by the increase in the cross-sectional area per molecule at increased temperature and hydration. Periodic surface ripples appear to compensate the packing frustration between the headgroups and the hydrocarbon chains. The Cv plots, produced by the simulation work, confirm the presence of the pre-transition phase. A typical configuration of the Pβ′ phase also shows the corrugated surface profile. The essential presence of certain minimum hydration for the appearance of the Pβ′ phase has also been observed.

Lipid Bilayer Thickness and Surface Area in Lamellar Phases of Hydrated Mixtures of Dipalmitoylphosphatidylcholine and Homologs of Local Anesthetic Heptacaine

The lipid bilayer thickness and the surface area in fluid lyotropic lamellar phases of dipalmitoylphosphatidylcholine (DPPC) containing amphiphilic monohydrochlorides of [2-(alkyloxy)phenyl]-2-(1-piperidinyl)ethyl esters of carbamic acid (CnA, n=number of alkyloxy chain carbons) at DPPC:CnA=1:1 and HO:DPPC≈20:1 molar ratios have been estimated from small angle synchrotron X-ray diffractograms. CnAs intercalate between DPPC molecules resulting in a lateral bilayer expansion and in a decrease of . At the reduced temperature =(-)/=0.035 where is the gel-fluid phase transition temperature, the values of () equal to 3.289±0.012 nm (1.060±0.012 nm), 3.391±0.011 nm (1.055±0.013 nm), 3.403±0.013 nm (1.080±0.012 nm), 3.418±0.012 nm (1.090±0.013 nm) and 3.730±0.012 nm (1.025±0.012 nm) were obtained for C5A, C7A, C9A, C10A and C12A containing phases, respectively. The increase of with increasing n is caused by the decrease in the difference in DPPC acyl and CnA alkyloxy chains lengths. The values of =3.733±0.011 nm and =0.902±0.012 nm obtained for C3A indicate that its location in the phase is different from that of the other CnAs studied. The non-linear dependencies of and on n can contribute to CnAs biological (local anesthetic, antimicrobial, antiphotosynthetic) effects that display similar dependencies on n.

Gel−Fluid Transition in Dilute versus Concentrated DMPG Aqueous Dispersions

Aqueous dispersions of the anionic phospholipid dimyristoyl phosphatidylglycerol (DMPG) in 100 mM ionic strength, at pH values higher than 4, are known to exhibit a thermal behavior rather similar to that of the zwitterionic lipid dimyristoyl phosphatidylcholine (DMPC), which undergoes a gel-to-liquid crystalline phase transition at 23 °C. However, in aqueous medium of low ionic strength, DMPG was shown to present a large gel-fluid transition region, ranging from 18 to 35 °C. This intermediate phase is optically transparent and characterized by a continuous change in membrane packing. The present work shows that, below a certain lipid concentration, which we denote c′ (c′ ) 0.4 ( 0.2 mM), this anomalous behavior vanishes, being replaced by a sharper phase transition centered at a unique transition temperature Tm. Interestingly, Tm increases as the lipid concentration is further decreased, reaching a limiting value of about 41 °C. Below c′, opposite to the effect observed for DMPG at higher concentrations, the value of Tm decreases as the ionic strength is increased. Above 500 mM NaCl, the thermal behavior of DMPG aqueous dispersions is roughly independent of the lipid concentration. Furthermore, increasing sample pH significantly decreases Tm in the low concentration regime, whereas in concentrated DMPG dispersions, Tm is not affected by changes in pH above 6. The dependence of the thermal behavior of dilute DMPG aqueous dispersions on pH and ionic strength is discussed here in the light of the dependence of the headgroup protonation on DMPG concentration.

Simulation of bilayer membranes in soft confinement

The basic interactions underlying the bilayer membranes have been studied in the biologically relevant soft confinement regime of interlayer spacing of 5– 30 A ̊ . Due to its unique molecular structure, a lipid membrane shows both liquid-like and liquid crystalline (elastic) characteristics. Monte Carlo simulation of interacting membranes, conceived as a lamellar stack of lipid bilayers in aqueous environment, yields results in agreement with experimental findings. Temperature driven structural transitions involving fully anisotropic gel phase to disordered fluid phase have been confirmed by simulation. The change induced in the gel–fluid transition due to the presence of excess cholesterol in the lipid bilayer is also been observed.

Monte Carlo simulation towards ripple phase modelling

We present a novel approach to analysis of the gel-fluid transition of lipid membrane. The method is based on the Pink's model but in contrast to its standard version the dipole character of the lipid molecules polar part is considered. Moreover, less constrained movement of entire molecules is allowed. Such an approach includes into the model conditions imposed by the adjacent medium such as ionic strength, pH, and other factors affecting biological membranes via the polar part. The results obtained contribute to the explanation of the ripple phase phenomenon.

Monte Carlo Simulation of Two-Component Bilayers: DMPC/DSPC Mixtures

In this paper, we describe a relatively simple lattice model of a two-component, two-state phospholipid bilayer. Application of Monte Carlo methods to this model permits simulation of the observed excess heat capacity versus temperature curves of dimyristoylphosphatidylcholine (DMPC)/distearoylphosphatidylcholine (DSPC) mixtures as well as the lateral distributions of the components and properties related to these distributions. The analysis of the bilayer energy distribution functions reveals that the gel–fluid transition is a continuous transition for DMPC, DSPC, and all DMPC/DSPC mixtures. A comparison of the thermodynamic properties of DMPC/DSPC mixtures with the configurational properties shows that the temperatures characteristics of the configurational properties correlate well with the maxima in the excess heat capacity curves rather than with the onset and completion temperatures of the gel–fluid transition. In the gel–fluid coexistence region, we also found excellent agreement between the threshold temperatures at different system compositions detected in fluorescence recovery after photobleaching experiments and the temperatures at which the percolation probability of the gel clusters is 0.36. At every composition, the calculated mole fraction of gel state molecules at the fluorescence recovery after photobleaching threshold is 0.34 and, at the percolation threshold of gel clusters, it is 0.24. The percolation threshold mole fraction of gel or fluid lipid depends on the packing geometry of the molecules and the interchain interactions. However, it is independent of temperature, system composition, and state of the percolating cluster.

Ceramides in Phospholipid Membranes: Effects on Bilayer Stability and Transition to Nonlamellar Phases

The effects of ceramides of natural origin on the gel-fluid and lamellar-inverted hexagonal phase transitions of phospholipids (mainly dielaidoylphosphatidylethanolamine) have been studied by differential scanning calorimetry, with additional support from infrared and 31P nuclear magnetic resonance (NMR) spectroscopy. In the lamellar phase, ceramides do not mix ideally with phospholipids, giving rise to the coexistence of domains that undergo the gel-fluid transition at different temperatures. The combination of differential scanning calorimetry and infrared spectroscopy, together with the use of deuterated lipids, allows the demonstration of independent melting temperatures for phospholipid and ceramide in the mixtures. In the lamellar-hexagonal phase transitions, ceramides (up to 15 mol %) decrease the transition temperature, without significantly modifying the transition enthalpy, thus facilitating the inverted hexagonal phase formation. 31P-NMR indicates the coexistence, within a certain range of temperatures, of lamellar and hexagonal phases, or hexagonal phase precursors. Ceramides from egg or from bovine brain are very similar in their effects on the lamellar-hexagonal transition. They are also comparable to diacylglycerides in this respect, although ceramides are less potent. These results are relevant in the interpretation of certain forms of interfacial enzyme activation and in the regulation and dynamics of the bilayer structure of cell membranes.

Thermal acclimation of phase behavior in plasma membrane lipids of rainbow trout hepatocytes.

The fluorescent probes laurdan (6-dodecanoyl-2-dimethylaminonapthalene) and N-[7-nitrobenz-2-oxa-1, 3-diazol-4-yl] dipalmitoyl-L-alpha-phosphatidylethanolamine (NBD-PE) in addition to Fourier transform infrared spectroscopy (FTIR) were employed to measure the phase behavior and physical properties of hepatocyte plasma membranes isolated from the livers of thermally acclimated (5 and 20 degreesC) rainbow trout (Oncorhynchus mykiss). The primary objective was to determine the extent to which the phase behavior of membrane lipids is conserved at different growth temperatures. Arrhenius plots of laurdan-generalized polarization revealed a single discontinuity believed to reflect either the onset of the gel-fluid phase transition or the formation of gel phase microdomains, and this discontinuity occurred at significantly higher temperatures in membranes of 20 degrees C (13.2 +/- 0.7 degrees C)- than 5 degrees C (7.2 +/- 0.1 degrees C)-acclimated trout. Similarly, acclimation from 5 to 20 degrees C increased both the onset temperature (from 2.0 +/- 0.3 to 7.2 +/- 0.6 degrees C) and the thermal range (from 10.9 +/- 0.5 to 16.0 +/- 1.0) of the gel-fluid transition as assessed by FTIR. The gel-fluid transition midpoint (approximately -2 degrees C) and completion temperatures (-9 degrees C) were unchanged by thermal acclimation. The anisotropy of NBD-PE fluorescence displayed a distinct minimum in membranes of both warm- and cold-acclimated trout (reflecting alterations in lipid packing that in pure lipid membranes ultimately lead to the formation of nonlamellar phases) in the range of 56-58 degrees C; only membranes of 5 degrees C-acclimated trout displayed an additional minimum at significantly lower temperatures (24.5 +/- 1.7 degrees C). Collectively, these data suggest that the regulation of both the temperature at which gel phase lipids begin to form in response to cooling as well as the propensity of membrane lipids to form nonlamellar phases at higher temperatures may be key features of membrane organization subject to adaptive regulation.

The effect of amphiphilic counterions on the gel-fluid phase transition of the lipid bilayer

Monte Carlo simulation based on the ten-state Pink's model has been applied to study the effect of amphiphilic counterions on the gel-fluid phase transition of lipid bilayers. Based on the theoretical results obtained, one can explain the lack of measurable effect of alkylsulphonate on lipid membrane permeability. An anionic compound with long alkyl chain when applied to a membrane modified with a cationic compound of the same chain length can cancel the effect of the latter due to the formation of an ionic pair that resembles the lipid molecules that form the membrane. The results obtained were compared with experimental results and proved helpful in giving them a more satisfactory interpretation.