Calcium-induced fusion of didodecylphosphate vesicles: The lamellar to hexagonal II (HII) phase transition

Summary Electron microscopic techniques have been employed to investigate the ability of didodecylphosphate vesicles (diameter approx. 900 Å) to fuse in the presence of Ca²⁺. As revealed by negative staining, Ca²⁺ induces extensive fusion and large vesicles with diameters up to 7000 Å are formed. In a processsecondary to fusion, the fused vesicles display a tendency to flatten and are subsequently transformed into extended tubular structures. Freeze-fracture electron microscopy, in conjunction with³¹P NMR and selected area electron diffraction measurements indicate that the tubes are packed in a hexagonal (H_II) array and that the amphiphiles are converted from the lamellar to the hexagonal H_II phase.The relationship between membrane fusion and the lamellar-to-hexagonal phase transition is discussed in terms of formation and abundance of transiently stable inverted micellar intermediates at contact regions between two interacting membranes. A model for the conversion of the (vesicular) lamellar into the (tubular) hexagonal H_II phase is presented, taking into account the molecular shape of the amphiphile. The relevance of using simple synthetic amphiphiles as models for phospholipid bilayers and complex biomembrane behavior is briefly discussed.     

Modulation of a membrane lipid lamellar-nonlamellar phase transition by cationic lipids: A measure for transfection efficiency

Synthetic cationic lipids can be used as DNA carriers and are regarded to be the most promising non-viral gene carriers. For this investigation, six novel phosphatidylcholine (PC) cationic derivatives with various hydrophobic moieties were synthesized and their transfection efficiencies for human umbilical artery endothelial cells (HUAEC) were determined. Three compounds with relatively short, myristoleoyl or myristelaidoyl 14:1 chains exhibited very high activity, exceeding by ∼ 10 times that of the reference cationic derivative dioleoyl ethylPC (EDOPC). Noteworthy, cationic lipids with 14:1 hydrocarbon chains have not been tested as DNA carriers in transfection assays previously. The other three lipids, which contained oleoyl 18:1 and longer chains, exhibited moderate to weak transfection activity. Transfection efficiency was found to correlate strongly with the effect of the cationic lipids on the lamellar-to-inverted hexagonal, L_α → H_II, phase conversion in dipalmitoleoyl phosphatidylethanolamine dispersions (DPoPE). X-ray diffraction on binary DPoPE/cationic lipid mixtures showed that the superior transfection agents eliminated the direct L_α → H_II phase transition and promoted formation of an inverted cubic phase between the L_α and H_II phases. In contrast, moderate and weak transfection agents retained the direct L_α → H_II transition but shifted to higher temperatures than that of pure DPoPE, and induced cubic phase formation at a later stage. On the basis of current models of lipid membrane fusion, promotion of a cubic phase by the high-efficiency agents may be considered as an indication that their high transfection activity results from enhanced lipoplex fusion with cellular membranes. The distinct, well-expressed correlation established between transfection efficiency of a cationic lipid and the way it modulates nonlamellar phase formation of a membrane lipid could be useful as a criterion to assess the quality of lipid carriers and for rational design of new and superior nucleotide delivery agents.     

Sugars favour formation of hexagonal (HII) phase at the expense of lamellar liquid-crystalline phase in hydrated phosphatidylethanolamines

The disaccharides, sucrose and trehalose, markedly decreased (up to 17-13C°) the temperature of the lamellar to hexagonal (L_α →H_II) phase transition and simultaneously increase by 2–4 C° the temperature of the lamellar gel to lamellar liquid-crystal (L_β →L_α) phase transition in hydrated dihexadecylphosphatidylethanolamine and distearoylphosphatidylethanolamine. These two transitions merge and convert into a single L_β-H_II phase transition when dispersed in 2.4 M sucrose. These results are inconsistent with recent reports by (8) and (9)) which suggest that trehalose stabilizes the L_α phase relative to the H_II phase and shifts upwards beyond detectability the L_α-H_II transition. The present results are considered as a manifestation of the Hofmeister effect in which the sugars act as kosmotropic reagents stabilizing the structure of bulk water. This tends to decrease the area of contact between the lipid and the aqueous phases and favours the H_II and L_β phases relative to L_α phase. This hypothesis is consistent with the effects of chaotropic reagents on the L_α-H_II phase transition (Yeagle and Sen (1986) Biochemistry 25, 7518–7522) and on the stability of the lamellar phase of dipalmitoylphosphatidylcholine (Oku and MacDonald (1983) J. Biol. Chem. 258, 8733–8738).     

The relationship between the bilayer to hexagonal phase transition temperature in membranes and protein kinase C activity

A number of substances affect the activity of protein kinase C. Among uncharged and zwitterionic compounds, those which activate protein kinase C also lower the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine while substances which inhibit protein kinase C raise this transition temperature. Using this criteria, we have identified 3-chloro-5-cholestene, 5-cholan-24-ol and eicosane as new protein kinase C activators and have shown that Z-Ser-Leu-NH₂, Z-Gly-Leu-NH₂, Z-Tyr-Leu-NH₂, cyclosporin A and cholestan-3, 5, 6-triol are protein kinase C inhibitors.     

Analysis of the hexagonal II phase and its relations to lipidic particles and the lamellar phase A freeze-fracture study

Model systems of phosphatidylethanolamine (PE) and cardiolipin (DPG), as pure components and in binary mixtures with phosphatidylcholine (PC) have been morphologically analysed. The relation between the hexagonal_II (H_II) phase and lipidic particles as well as between the H_II phase and the lamellar phase has been studied. Moreover, the periodicity of the various H_II tubes was determined. (1) The periodicity of the H_II phase of cardiolipin is dependent on the cation involved. DPG-Ca exhibits the smallest tube to tube distance when compared to Mg²⁺ and Mn²⁺. Moreover, the DPG-Ca tubes are quite straight, in contrast to the Mg²⁺ and Mn²⁺ tubes, which appear to be frequently curved. (2) H_II tubes with two distinct diameters have been observed in H_II phase containing lipid mixtures. The thickness of the H_II tube is related to the composition of the tube. In the cardiolipin-lecithin system, structural separation of the pure cardiolipin H_II phase has been suggested with Mg²⁺ and Mn²⁺, but not with Ca²⁺. (3) Models for the H_II to lamellar phase transition and for the H_II phase to the lipidic particles are presented. (4) Lipidic particles are exclusively found in lipid model systems, which contain H_II phase favouring lipids. Morphological evidence is presented which suggests these lipidic particles represent inverted micelles. These observations include: (${}_{\mathrm{<mtext>i</mtext>}}$) there is a strong topological and quantitative relation between H_II tubes and lipidic particles, (${}_{\mathrm{<mtext>ii</mtext>}}$) lipidic particles occur densely packed in conglomerates without the presence of a smooth layer.     

Hydration and the lamellar to hexagonal II phase transition of phosphatidylethanolamine

The effects of chaotropic agents on the lamellar to hexagonal II phase transition of soy phosphatidylethanolamine were examined. Guanidine hydrochloride, urea, and NaSCN were used as chaotropic agents. In each case, the lamellar phase was stabilized by the presence of the chaotropic agent. In the case of NaSCN, the temperature of the lamellar to hexagonal phase transition of soy phosphatidylethanolamine was increased by more than 60 degrees C. Guanidine hydrochloride was capable of substantially reducing the aggregation of phosphatidylethanolamine vesicles. These data lead to a thermodynamic understanding of the lamellar to hexagonal phase transition.     

Light scattering measurement and Avrami analysis of the lamellar to inverse hexagonal phase transition kinetics of the lipid DEPE

The lamellar to inverse hexagonal phase transition of lipids is much studied as a model for understanding cellular processes such as membrane fusion and pore formation. Much remains unknown, including a theoretical understanding and a definitive value of the phase transition temperature for DEPE, as literature values vary over 10°C. Avrami theory has been commonly used to analyze phase transition kinetics. However, to the best of our knowledge, Avrami theory has not been used to analyze the lamellar to inverse hexagonal transition in lipids until now. We used laser light scattering to measure phase transition temperature of the lipid DEPE (1,2-dielaidoyl-sn-phosphatidylethanolamine) and found it to be 61.0 ± 0.5°C. We found the hysteresis, |T(measured)-T(equilibrium)|, scaled as r(β), where r is the ramp rate and β=0.29 ± 0.02. This is the same power law behavior found by others for an isomer of DEPE known as DOPE (1,2-dioleoyl-sn-glycero-3 ethanolamine); however, DEPE exhibits roughly half the hysteresis of DOPE. An analysis of DEPE kinetics yields Avrami exponents ranging from 1 to 7, suggesting the transition propagates one dimensionally and is initiated by a widely varying nucleation rate.     

Effect of egg‐phosphatidylcholine on the chilling sensitivity and lipid phase transition of Asian elephant (Elephas maximus) spermatozoa

This study was conducted in an effort to improve our understanding of the response of Asian elephant (Elephas maximus, Em) spermatozoa to chilling. Semen was collected from two elephant bulls by means of the manual rectal stimulation method. Five out of seven semen collections were deemed to be suitable for use based on motility (ranging from 20% to 60%) and membrane integrity. We evaluated the chilling sensitivity by incubating the sperm with a fluorescent dye (5‐carboxyfluorescein diacetate (cFDA)) at 16°C, 12°C, 4°C, and 22°C (control). Cells with an intact membrane retained the dye and were identified as viable. The membrane lipid phase transition (LPT) temperature curve was determined with a Fourier transform infrared (FTIR) spectrometer connected to an FTIR microscope. The LPT center, T_m, was determined by statistical analysis. The LPT and T_m were also assessed in fresh spermatozoa and spermatozoa incubated with egg yolk or egg‐phosphatidylcholine (EPC) liposomes at 16°C, 12°C, 4°C, and 26°C (control). The results show that the membrane integrity of spermatozoa incubated at 16°C, 12°C, and 4°C decreased by 39%, 62%, and 67%, respectively, compared to the control. The LPT temperatures were between room temperature (26°C) and 10°C, with T_m at 14–16°C. The T_m for sperm incubated with liposomes or egg‐yolk extender was below the measured range (2°C). Chilling sensitivity was found at a wide range of temperatures and transition temperatures, suggesting the presence of a wide variety of fatty acids (FAs) in the membrane with a high ratio of saturated‐to‐polyunsaturated FAs. Here we show that the protection afforded by the presence of egg yolk or liposomes in the extender is accomplished by shifting the T_m to below the 4°C point at which chilled semen is maintained for transport, or the point at which fast freezing begins to minimize cellular damage. Zoo Biol 0:1–13, 2005. © 2005 Wiley‐Liss, Inc.     

Farnesol and geranylgeraniol modulate the structural properties of phosphatidylethanolamine model membranes

The biological activity of farnesol (FN) and geranylgeraniol (GG) and their isoprenyl groups is related to membrane-associated processes. We have studied the interactions of FN and GG with 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes using DSC and X-ray diffraction. Storage of samples at low temperature for a long time favors a multidomain system formed by a lamellar crystalline (Lc) phase and isoprenoids (ISPs) aggregates. We demonstrate that ISPs alter the thermotropic behavior of DEPE, thereby promoting a H_II growth in a lamellar Lc phase with a reduced degree of hydration. The H_II phase occurs with the same repeat distance (d_HII=5.4 nm) as the Lc phase and upon heating it expands considerably (δd/δT≈0.22 nm/°C). The dimensional stabilization of this H_II phase coincides with the transition temperature of the Lc to L_α phase. Thereafter, the system DEPE/ISP will progress by increasing the nonlamellar-forming propensity and reaching a single H_II phase at high temperature. The cooling scan followed a similar structural path, except that the system went into a stable gel phase L_β with a repeat distance, d_Lβ=6.5 nm, in co-existence with a H_II phase. The formation of ISP microdomains in model PE membranes substantiates the importance of the isoprenyl group in the binding of isoprenylated proteins to membranes and in lipid–lipid interactions through modulation of the membrane structure.     

PEG blocking of single pores arising on phase transitions in unmodified lipid bilayers

Changes in ionic permeability of bilayer lipid membranes (BLM) from dipalmitoyl phosphatidylcholine at temperature of phase transition in 1 M LiCl solution in the presence of polyethyleneglycols (PEG) of various molecular masses are studied. The transition of ionic membrane channels from conducting to blocked nonconducting state using polymers makes it possible to calibrate lipid pores. It is shown that low-molecular weight glycerol and PEG with molecular weights of 300 and 600 decrease the amplitude of current fluctuations through the membrane, the decrease being proportional to the size of the polymer molecule incorporated. The addition of PEG with molecular masses of 1450, 2000, and 3350 decrease the current fluctuations to the basal noise level. The result is considered as a complete blockade of ion channel conductivity. In the presence of rather large polymers, such as PEG with molecular masses of 6000 and 20000, which are hardly incorporated in the pore, single current fluctuations occur again; however, their amplitudes are somewhat smaller than in the absence of PEG. It is assumed that a complete blockade of the conductivity of lipid ionic channels by PEG with molecular masses of 1450, 2000, and 3350 is due to dehydration of the pore gap and the conversion of the hydrophilic pore to a hydrophobic one.     

The lamellar to hexagonal phase transition in phosphatidylethanolamine liposomes: a fluorescence anisotropy study

The steady-state anisotropy of trimethylammonium diphenylhexatriene fluorescence has been used to monitor the thermotropic lamellar to HII hexagonal phase transition in an unsaturated phosphatidylethanolamine. The transition is observed in lipid aggregates when they are heated above the transition temperature Th, as well as in diluted liposomes after aggregation above Th. Changes in fluorescence anisotropy are not observed with Ca(++)-induced fusion of phosphatidylserine vesicles, a process not involving hexagonal phase formation.     

Lipid phase transition in the plasma membrane of the goat epididymal maturing spermatozoa

Microviscosity of the highly purified plasma membranes isolated from the maturing goat caput, corpus and cauda epididymal sperm, was measured using l,6-diphenyl-l,3,5-hexatriene as the lipophilic probe at varying temperatures (12–42°C). As shown by the Arrhenius plot of the data each of the maturing sperm membranes had two distinct lipid phase transitions in the temperature zones 19–25°C and 34–37°C. The low-temperature transitions for the immature caput- and mature cauda-sperm membranes were noted at 19–20°C, and 24–25°C, respectively, whereas both these membranes showed high temperature transition at 36–37°C. The maturing corpus-sperm membrane had phase transitions at 21–22°C and 35–36°C that were significantly different from those of the immature/mature sperm membranes. The data implicate significant alteration of the sperm membrane structure during epididymal maturation. The phase transition of the mature male gametes at 36–37°C may have a great impact on the subsequent events of the sperm life cycle since the mature spermatozoa that are stored in the epididymis a few degrees below the body temperature, experience higher temperature when ejaculated into the female reproductive tract.     

Soft perforation of planar bilayer lipid membranes of dipalmitoylphosphatidylcholine at the temperature of the phase transition from the liquid crystalline to the gel state

In contrast to the widely used method of electroporation, the method of soft perforation of lipid bilayers is proposed. It is based on the structural rearrangement of the lipid bilayer formed from disaturated phospholipids at the temperature of the phase transition from the liquid crystalline state to the gel state. This allows us to obtain a lipid pore population without the use of a strong electric field. It is shown that the planar lipid bilayer membrane (pBLM) formed from dipalmitoylphosphatidylcholine in 1 M LiCl aqueous solution exhibits the appearance of up to 50 lipid pores per 1 mm² of membrane surface, with an average single pore conductivity of 31±13 nS. The estimation of a single pore radius carried out with water-soluble poly(ethylene glycol)s (PEGs) showed that the average pore radius ranged between 1.0–1.7 nm. It was found experimentally that PEG-1450, PEG-2000, and PEG-3350 should be in a position to block the single pore conductivity completely, while PEG-6000 fully restored the ionic conductivity. The similarity of these PEG effects to ionic conductivity in protein pores makes it possible to suggest that the partition of the PEG molecules between the pore and the bulk solution does not depend on the nature of the chemical groups located in the pore wall.     

Virus replication inhibitory peptide inhibits the conversion of phospholipid bilayers to the hexagonal phase

Virus replication inhibitory peptide (carbobenzoxy-D-Phe-L-PheGly) was shown to be a potent specific inhibitor of the replication of paramyxovirus and myxovirus (Richardson, Scheid and Choppin (1980), Virology105, 205–222). This peptide inhibits the membrane fusing activity of a viral glycoprotein.Many agents which promote the formation of the hexagonal phase in membranes also accelerate membrane fusion. At a mole fraction of 0.1, viral replication inhibitory peptide can raise the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine by almost 10°. Two related peptides, carbobenzoxy-L-PheGly and carbobenzoxy-L-GlyPhe, are less potent in raising the bilayer to hexagonal phase transition temperature, with the latter peptide being the least effective of the three. This order of potency is the same as the order of potency in inhibiting viral replication. Substances which inhibit hexagonal phase formation of pure lipids may also inhibit membrane fusion.Abbreviations DEPE dielaidoylphosphatidyethanolamine - Z carbobenzoxy - DSC differential scanning calorimetry - VRIP virus replication inhibitory peptide (Z-D-Phe-L-PheGly)     

Morphology and Phase Behavior of Two-Component Lipid Membranes

The stability and shapes of domains with different bending rigidities in lipid membranes are investigated. These domains can be formed from the inclusion of an impurity in a lipid membrane or from the phase separation within the membrane. We show that, for weak line tensions, surface tensions and finite spontaneous curvatures, an equilibrium phase of protruding circular domains or striped domains may be obtained. We also predict a possible phase transition between the investigated morphologies.     

Lateral diffusion study of excimer-forming lipids in lamellar to inverted hexagonal phase transition of unsaturated phosphatidylethanolamine

Using multi-frequency cross-correlation fluorometry, the monomer fluorescence lifetime of 1-palmitoyl-2-[10-(1-pyrenyl)decanoyl)phosphatidylcholine (Py-PC) was employed to determine the lateral diffusion constant (DT) of dioleoylphosphatidylethanolamine (DOPE) in both the lamellar (L alpha) and the inverted hexagonal (HII) phases. The values of DT increased with temperature in both phases. However, the rate of increase of DT declined abruptly at approximately 10-13 degrees C (L alpha -HII transition temperature), as indicated by the existence of an inflection point in the log (DT/T) vs. 1/T plot. This observation suggests that the translational motion of lipids in the HII phase is lower than that in the L alpha phase upon temperature extrapolation. Lipid perturbants, cholesterol and diacylglycerol, were found to destabilize the L alpha phase of DOPE. This was demonstrated by a down-shift of the inflection point in the log(DT/T) vs. 1/T plot in the presence of the perturbants. Both cholesterol and 1,2-dioleoyl-sn-glycerol (diolein) decreased the lateral diffusion constant in both phases. Diolein promoted the HII phase more effectively than did the cholesterol. This is explained by an intrinsic wedge-shape geometry of diolein which strongly favors the formation of inverted cylindrical packing of the lipids.     

Thermal acoustic radiation from multilamellar vesicles in lipid phase transition

A new acoustical method for the investigation of lipid phase transition is introduced based on the measurement of the thermal acoustic radiation (TAR) inherent in lipids. The TAR of multilamellar vesicles from dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) was measured in the megahertz range and the variations in the radiation intensity during the lipid phase transition were recorded. Two types of variations are possible: if the temperature of the vesicles decreases (in the process of transition from the liquid crystalline state to the gel state) then the TAR intensity increases, and if the temperature increases (in the reverse transition) then the TAR intensity decreases. These effects are connected with an increase in the ultrasonic absorption in the vesicles under lipid phase transition. Basing on the results of the TAR investigation, a new theoretical estimate has been developed of the variation in the absorption coefficient during the lipid phase transition. In this estimate, the variation is equated to the ratio of the phase transition entropy to the gas constant.     

Role of curvature and phase transition in lipid sorting and fission of membrane tubules

We have recently developed a minimal system for generating long tubular nanostructures that resemble tubes observed in vivo with biological membranes. Here, we studied membrane tube pulling in ternary mixtures of sphingomyelin, phosphatidylcholine and cholesterol. Two salient results emerged: the lipid composition is significantly different in the tubes and in the vesicles; tube fission is observed when phase separation is generated in the tubes. This shows that lipid sorting may depend critically on both membrane curvature and phase separation. Phase separation also appears to be important for membrane fission in tubes pulled out of giant liposomes or purified Golgi membranes.