The shape of lipid molecules and monolayer membrane fusion

The contact between two bilayer membranes results in their monolayer fusion comprising the formation of a trilaminar structure (a single bilayer connected to two bilayers over the whole perimeter) in the contact region. The time required for monolayer fusion was measured and irreversible electrical breakdown was studied for membranes of different compositions. A theoretical model of the monolayer fusion is suggested to explain the results. It assumes that the structural reorganization underlying the process involves the formation of a stalk between bilayers as a result of local bending of the interacting monolayers. This structural reorganization is similar to the hydrophilic pore formation in a bilayer under irreversible breakdown. However, the directions of the monolayer bending are different in the two processes and, therefore, the bending energies depend oppositely on the effective shape of lipid molecules. Theoretical predictions agree well with experimental data. The applicability of the suggested mechanism to biomembrane fusion is discussed.     

The hemifusion intermediate and its conversion to complete fusion: regulation by membrane composition.

To fuse, membranes must bend. The energy of each lipid monolayer with respect to bending is minimized at the spontaneous curvature of the monolayer. Two lipids known to promote opposite spontaneous curvatures, lysophosphatidylcholine and arachidonic acid, were added to different sides of planar phospholipid membranes. Lysophosphatidylcholine added to the contacting monolayers of fusing membranes inhibited the hemifusion we observed between lipid vesicles and planar membranes. In contrast, fusion pore formation depended upon the distal monolayer of the planar membrane; lysophosphatidylcholine promoted and arachidonic acid inhibited. Thus, the intermediates of hemifusion and fusion pores in phospholipid membranes involve different membrane monolayers and may have opposite net curvatures, Biological fusion may proceed through similar intermediates.     

The dispersion of cholesterol with phospholipids and glycolipids

Of the polar lipids studied (phospholipids and glycolipids), only phosphatidylcholine and sphingomyelin can disperse in water with up to 2 mol cholesterol/mol polar lipid. However, mixtures of phosphatidylethanolamine with small amounts of phosphatidylcholine and mixed lipids from mitochondria and myelin will also form sterol-rich dispersions. Steroids in which the 3β-OH group is replaced by an oxo function do not form such steroid-rich dispersions. Electron microscopy and optical rotatory dispersion (ORD) show that sterols disperse with cerebrosides and gangliosides to form cylindrical structures with the regions around C atoms 3 and 7 of the sterol in less polar environments than those they occupy in phospholipid liposomes.

It is proposed that choline-containing phospholipids facilitate entry of sterol molecules into the outer leaflet of cell surface membranes but that the phospholipid composition itself will not give rise to an asymmetric distribution of sterol in membranes with a high cholesterol content.     

Molecular characteristics of astaxanthin and beta-carotene in the phospholipid monolayer and their distributions in the phospholipid bilayer.

The molecular characteristics of the monolayers of astaxanthin with polar group on the beta-ionone ring in the molecule and beta-carotene without polar group and their interactions in mixed carotenoid-phospholipid monolayers and the effects of carotenoids on the phase behavior of the phospholipid bilayers were examined by the monolayer technique and differential scanning calorimetry (DSC). We found from the monolayer study that beta-carotene had an amphiphilic nature. The molecular assembly of astaxanthin in the monolayer at the hydrophobic/hydrophilic interface was more stable than that of beta-carotene. Dimyristoylphosphatidylcholine (DMPC) in the monolayer was miscible with astaxanthin in the range of 0-0.4 mol fractions of astaxanthin, but not fully miscible with beta-carotene even at low concentrations below 0.1 mol fraction of beta-carotene. Surface potential and compression/expansion cycles of beta-carotene monolayer indicated the formation of molecular aggregates by itself. DSC study showed that when small amount of astaxanthin was added, the transition temperature of dipalmitoylphosphatidylcholine (DPPC) was markedly shifted to lower temperatures and that the transition peak was asymmetrically broadened, indicative of a significant depression in cooperativity of the gel to liquid-crystalline transition. The asymmetric DSC endothermic bands of DPPC incorporating small amounts of astaxanthin were well fit by deconvolution into two to three domains containing different concentrations of astaxanthin. On the contrary, the incorporation of beta-carotene resulted in a small depression of the main transition temperature with a slight broadening of the transition peak, suggesting a small miscibility of beta-carotene with the phospholipid bilayer or a formation of aggregates of beta-carotene in the membranes. These results suggest that there would be a high localized concentration in the phase separated membrane for astaxanthin or beta-carotene to function effectively as scavenger.     

Membrane fusion: overcoming of the hydration barrier and local restructuring

The early stages of membrane fusion have been investigated theoretically. It has been shown that the hydration repulsion, operating between apposed membranes, is overcome locally under the action of out-of-plane thermal fluctuations of the bilayers. The fluctuations lead to the formation of close (less than 0.5 nm) contact between the membranes within a small area (approximately 10 nm2). Increasing hydration repulsion between apposed polar heads of lipid molecules in this area causes the rupture of interacting monolayers. The rupture results in monolayer fusion of the membranes, i.e. in the formation of a bridge connecting the monolayers, which is usually named the monolayer stalk. The influence of degree of hydration of the monolayers and their spontaneous curvature on conditions of monolayer fusion have been analysed. The proposed mechanism of early stages of fusion process can proceed without preliminary formation of tight dehydrated contact between the membranes and even without any dehydration.     

Influenza virus hemagglutinin-induced cell-planar bilayer fusion: quantitative dissection of fusion pore kinetics into stages

Cells expressing the influenza virus hemagglutinin (HA) fuse to planar bilayer membranes under acidic conditions. After an electrically quiescent perfusion stage (Q), a fusion pore forms that enlarges in three subsequent stages. A repetitively flickering pore stage (R) develops into a securely open stage (S) that exhibits conductances ranging from a few to tens of nS. The pore then expands to a terminal stage (T) with a large conductance on the order of one microSiemens. We have studied how virus strain, HA receptors in the target bilayer membrane, and cytoskeleton affect the time a fusion pore remains in each stage. These intervals are referred to as waiting times. In the quiescent stage, waiting times were very sensitive to the virus strain and presence of gangliosides (HA receptors) in the bilayer. When bilayers contained gangliosides, the waiting times in the Q stage for cells infected with the PR/8/34 strain of virus were exponentially distributed, whereas waiting times for cells infected with the Japan/305/57 strain were not so distributed. Without gangliosides, the waiting time distribution for PR/8/34 infected cells was complex. The waiting times in the R and S stages of pore growth were exponentially distributed under all tested conditions. Within the R stage, we analyzed the kinetics of the flickering pore by fitting the open and closed time distributions with a sum of two exponentials. Neither the open and closed time distributions nor the flickering pore conductance distributions were appreciably affected by virus strain or gangliosides. Colchicine and cytochalasin B increased the flicker rates, without affecting the waiting time in the R stage. We conclude that variations in amino acid sequences of the HAs and the presence of gangliosides as receptors within the target membrane critically affect the kinetics of fusion pore formation, but do not affect subsequent stages.     

Effects of proteins on phospholipid vesicle aggregation and lipid vesicle-monolayer interactions

The effects of proteins on divalent cation-induced phospholipid vesicle aggregation and phospholipid vesicle-monolayer membrane interactions (fusion) were examined. Glycophorin (from human erythrocytes) suppressed the membrane interactions more than N-2 protein (from human brain myelin) when these proteins were incorporated into acidic phospholipid vesicle membranes. The threshold concentrations of divalent cations which induced vesicle aggregation were increased by protein incorporation, and the rate of vesicle aggregation was reduced. A similar inhibitory effect by the proteins, incorporated into lipid vesicle membranes, was observed for Ca²⁺-induced lipid vesicle-monolayer interactions. However, when these proteins were incorporated only in the acidic phospholipid monolayers, the interaction (fusion) of the lipid vesicle-monolayer membranes, induced by divalent cations, was not appreciably altered by the presence of the proteins.In contrast to these two proteins, the presence of synexin in the solution did enhance the Ca²⁺-induced aggregation of phosphatidylserine vesicles, but did not seem to affect the degree of Ca²⁺-induced fusion between phosphatidylserine/phosphatidylcholine (1:1) and phosphatidylserine vesicles and monolayer membranes.     

Regulation of transbilayer distribution of a fluorescent sterol in tumor cell plasma membranes

The lipid composition and transbilayer distribution of plasma membrane isolated from primary tumor (L-929, LM, A-9 and C3H) and nine metastatic cell lines cultured under identical conditions was examined. Cultured primary tumor and metastatic cells differed two-fold in sterol/phospholipid molar ratios. There was a direct correlation between plasma membrane anionic phospholipid (phosphatidylinositol and phosphatidylserine) content and plasma membrane sterol/phospholipid ratio. This finding may bear on the possible link between oncogenes and inositol lipids. The fluorescent sterol, dehydroergosterol, was incorporated into primary tumor and metastatic cell lines. Selective quenching of outer monolayer fluorescence by covalently linked trinitrophenyl groups demonstrated an asymmetric transbilayer distribution of sterol in the plasma membranes. The inner monolayer of the plasma membranes from both cultured primary and metastatic tumor cells was enriched in sterol as compared with the outer monolayer. Consistent with this, the inner monolayer was distinctly more rigid as determined by the limiting anisotropy of 1,6-diphenyl-1,3,5-hexatriene. Dehydroergosterol fluorescence was temperature dependent and sensitive to lateral phase separations in phosphatidylcholine vesicles and in LM cell plasma membranes. Dehydroergosterol detected phase separations near 24 degrees C in the outer monolayer and at 21 degrees C and 37 degrees C in the inner monolayer of LM plasma membranes. Yet, no change in transbilayer sterol distribution was detected in ascending or descending temperature scans between 4 and 45 degrees C. Alterations in plasma membrane phospholipid polar head group composition by choline analogues (N,N-dimethylethanolamine, N-methylethanolamine, and ethanolamine) also did not perturb transbilayer sterol asymmetry. Treatment with phenobarbital or prilocaine, drugs that selectively fluidize the outer and inner monolayer of LM plasma membranes, respectively, did not change dehydroergosterol transbilayer distribution.     

Channel-forming activity of syringomycin E in two mercury-supported biomimetic membranes

The lipodepsipeptide syringomycin E (SR-E) interacts with two mercury-supported biomimetic membranes, which consist of a self-assembled phospholipid monolayer (SAM) and of a tethered bilayer lipid membrane (tBLM) separated from the mercury surface by a hydrophilic tetraethyleneoxy (TEO) spacer that acts as an ionic reservoir. SR-E interacts more rapidly and effectively with a SAM of dioleoylphosphatidylserine (DOPS) than with one of dioleoylphosphatidylcholine (DOPC). The proximal lipid monolayer of the tBLM has no polar head region, being linked to the TEO spacer via an ether bond, while the distal monolayer consists of either a DOPC or a DOPS leaflet. The ion flow into or out of the spacer through the lipid bilayer moiety of the tBLM was monitored by potential step chronocoulometry and cyclic voltammetry. With the distal monolayer bathed by aqueous 0.1 M KCl and 0.8 μM SR-E, an ion flow in two stages was monitored with DOPC at pH 3 and 5.4 and with DOPS at pH 3, while a single stage was observed with DOPS at pH 5.4. This behavior was compared with that already described at conventional bilayer lipid membranes. The sigmoidal shape of the chronocoulometric charge transients points to an aggregation of SR-E monomers forming an ion channel via a mechanism of nucleation and growth. The ion flow is mainly determined by potassium ions, and is inhibited by calcium ions. The contribution to the transmembrane potential from the distal leaflet depends more on the nature of the lipid than that of the ion channel.     

Steric hindrance of SNARE transmembrane domain organization impairs the hemifusion‐to‐fusion transition

SNAREs fuse membranes in several steps. Trans‐SNARE complexes juxtapose membranes, induce hemifused stalk structures, and open the fusion pore. A recent penetration model of fusion proposed that SNAREs force the hydrophilic C‐termini of their transmembrane domains through the hydrophobic core of the membrane(s). In contrast, the indentation model suggests that the C‐termini open the pore by locally compressing and deforming the stalk. Here we test these models in the context of yeast vacuole fusion. Addition of small hydrophilic tags renders bilayer penetration by the C‐termini energetically unlikely. It preserves fusion activity, however, arguing against the penetration model. Addition of large protein tags to the C‐termini permits SNARE activation, trans‐SNARE pairing, and hemifusion but abolishes pore opening. Fusion proceeds if the tags are detached from the membrane by a hydrophilic spacer or if only one side of the trans‐SNARE complex carries a protein tag. Thus, both sides of a trans‐SNARE complex can drive pore opening. Our results are consistent with an indentation model in which multiple SNARE C‐termini cooperate in opening the fusion pore by locally deforming the inner leaflets.     

Influenza hemagglutinin-mediated fusion pores connecting cells to planar membranes: flickering to final expansion

We have studied the fusion between voltage-clamped planar lipid bilayers and influenza virus infected MDCK cells, adhered to one side of the bilayer, using measurements of electrical admittance and fluorescence. The changes in currents in-phase and 90 degrees out-of- phase with respect to the applied sinusoidal voltage were used to monitor the addition of the cell membrane capacitance to that of the lipid bilayer through a fusion pore connecting the two membranes. When ethidium bromide was included in the solution of the cell-free side of the bilayer, increases in cell fluorescence accompanied tee admittance changes, independently confirming that these changes were due to formation of a fusion pore. Fusion required acidic pH on the cell- containing side and depended on temperature. For fusion to occur, the influenza hemagglutinin (HA) had to be cleaved into HA1 and HA2 subunits. The incorporation of gangliosides into the planar bilayers greatly augmented fusion. Fusion pores developed in four distinct stages after acidification: (a) a pre-pore, electrically quiescent stage; (b) a flickering stage, with 1-2 nS pores opening and closing repetitively; (c) an irreversibly opened stage, in which pore conductances varied between 2 and 100 nS and exhibited diverse kinetics; (d) a fully opened stage, initiated by an instantaneous, time- resolution limited, increase in conductance leveling at approximately 500 nS. The expansion of pores by stages has also been shown to occur during exocytosis in mast cells and fusion of HA-expressing cells and erythrocytes. We conclude that essential features of fusion pores are produced with proteins in just one of the two fusing membranes.     

Membrane permeability changes at early stages of influenza hemagglutinin-mediated fusion

While biological membrane fusion is classically defined as the leak-free merger of membranes and contents, leakage is a finding in both experimental and theoretical studies. The fusion stages, if any, that allow membrane permeation are uncharted. In this study we monitored membrane ionic permeability at early stages of fusion mediated by the fusogenic protein influenza hemagglutinin (HA). HAb2 cells, expressing HA on their plasma membrane, fused with human red blood cells, cultured liver cells PLC/PRF/5, or planar phospholipid bilayer membranes. With a probability that depended upon the target membrane, an increase of the electrical conductance of the fusing membranes (leakage) by up to several nS was generally detected. This leakage was recorded at the initial stages of fusion, when fusion pores formed. This leakage usually accompanied the "flickering" stage of the early fusion pore development. As the pore widened, the leakage reduced; concomitantly, the lipid exchange between the fusing membranes accelerated. We conclude that during fusion pore formation, HA locally and temporarily increases the permeability of fusing membranes. Subsequent rearrangement in the fusion complex leads to the resealing of the leaky membranes and enlargement of the pore.     

Electromechanical stability of planar lipid membranes from bipolar lipids of the thermoacidophilic archebacterium Sulfolobus acidocaldarius

Stable planar membranes have been obtained from the bipolar lipid glycerol dialkyl nonitol tetraether (GDNT) extracted from the thermoacidophilic archebacterium Sulfolobus acidocaldarius. The electric capacity Cm, the resistance Rm and tension sigma of these membranes were measured. The dependence of the bipolar lipid membranes mean life time tau 1 on voltage was investigated. It was shown that the irreversible electric breakdown of membranes from GDNT and usual phospholipids is due to the same mechanism, viz., due to formation of a hydrophilic pore with an overcritical radius. Under electric field the GDNT molecules take U-shape, and the polar headgroups of such molecules cover the pore's interior.     

Minimum Membrane Bending Energies of Fusion Pores

Membranes fuse by forming highly curved intermediates, culminating in structures described as fusion pores. These hourglass-like figures that join two fusing membranes have high bending energies, which can be estimated using continuum elasticity models. Fusion pore bending energies depend strongly on shape, and the present study developed a method for determining the shape that minimizes bending energy. This was first applied to a fusion pore modeled as a single surface and then extended to a more realistic model treating a bilayer as two monolayers. For the two-monolayer model, fusion pores were found to have metastable states with energy minima at particular values of the pore diameter and bilayer separation. Fusion pore energies were relatively insensitive to membrane thickness but highly sensitive to spontaneous curvature and membrane asymmetry. With symmetrical bilayers and monolayer spontaneous curvatures of ?0.1 nm^?1 (a typical value) separated by 6 nm (closest distance determined by repulsive hydration forces), fusion pore formation required 43–65 kT. The pore radius of ~2.25 nm fell within the range estimated from conductance measurements. With bilayer separation >6 nm, fusion pore formation required less energy, suggesting that protein scaffolds can promote fusion by bending membranes toward one another. With nonzero spontaneous monolayer curvature, the shape that minimized the energy change during fusion pore formation differed from the shape that minimized its energy after it formed. Thus, a nascent fusion pore will relax spontaneously to a new shape, consistent with the experimentally observed expansion of nascent fusion pores during viral fusion.     

The role of N-acetylneuraminic (sialic) acid in the pH dependence of influenza virion fusion with planar phospholipid membranes

It is known that fusion of influenza virus to host cell membranes is strongly promoted by acidic pH. We have determined conditions required to obtain pH-dependent fusion of influenza virus to planar bilayer membranes. The rate of viral fusion was determined from the flash rate of R18-labeled virions delivered to the surface of the planar membrane by pressure-ejection from a pipette. For a bilayer formed only of phospholipids and cholesterol, the fusion rate was independent of pH and unaffected by the phospholipid composition. When the gangliosides GD1a + GT1b were included in the planar membrane, however, the fusion rate varied steeply with pH. The rate at pH 7.4 in the presence of the gangliosides was about an order of magnitude less than in their absence. At pH less than approximately 5.5, the rate was about an order of magnitude greater in the presence of gangliosides than in their absence. The fusion rate with planar membranes containing globoside, a ceramide-backboned glycolipid, was also independent of pH, indicating that the pH dependence required sialic acid on the carbohydrate moiety of the glycolipid. The gangliosides GM1a and GM3, both of which possess sialic acid, produced the same pH-dependent fusion rate as seen with GD1a + GT1b, indicating that the presence, but not the location, of terminal sialic acids is critical. Incubating virus with soluble sialyllactose blocked fusion to both ganglioside-free and ganglioside-containing planar membranes. These results show that the pH dependence of influenza virion fusion arises from the interaction of the sialic acid receptor with the influenza hemagglutinin. A model for sialic acid-hemagglutinin interactions accounting for pH-dependent fusion is presented.     

Probing the mechanism of fusion in a two-dimensional computer simulation

A two-dimensional (2D) model of lipid bilayers was developed and used to investigate a possible role of membrane lateral tension in membrane fusion. We found that an increase of lateral tension in contacting monolayers of 2D analogs of liposomes and planar membranes could cause not only hemifusion, but also complete fusion when internal pressure is introduced in the model. With a certain set of model parameters it was possible to induce hemifusion-like structural changes by a tension increase in only one of the two contacting bilayers. The effect of lysolipids was modeled as an insertion of a small number of extra molecules into the cis or trans side of the interacting bilayers at different stages of simulation. It was found that cis insertion arrests fusion and trans insertion has no inhibitory effect on fusion. The possibility of protein participation in tension-driven fusion was tested in simulation, with one of two model liposomes containing a number of structures capable of reducing the area occupied by them in the outer monolayer. It was found that condensation of these structures was sufficient to produce membrane reorganization similar to that observed in simulations with "protein-free" bilayers. These data support the hypothesis that changes in membrane lateral tension may be responsible for fusion in both model phospholipid membranes and in biological protein-mediated fusion.     

Orientation of anthracyclines in lipid monolayers and planar asymmetrical bilayers: a surface-enhanced resonance Raman scattering study.

The interaction of anthracyclines (daunorubicin and idarubicin) with monolayers of zwitterionic palmitoyloleoylphosphatidylcholine (POPC) and anionic dipalmitoylphosphatidic acid (POPC-DPPA 80-20 mol%) was studied by surface pressure measurements and compared with previous results obtained with other anthracyclines (pirarubicin and adriamycin). These anthracycline/phospholipid monolayers were next transferred by a Langmuir-Blodgett technique onto planar supports and studied by surface-enhanced resonance Raman scattering (SERRS), which gave information about the orientation of anthracycline in the monolayers. On the whole, the adsorption of anthracyclines in zwitterionic monolayers increases with the anthracycline hydrophobic/hydrophilic balance, which underlines the role of the hydrophobic component of the interaction. On the contrary, the anthracyclines remain adsorbed on the polar headgroups of the phospholipids in the presence of DPPA and form a screen that limits a deeper penetration of other anthracycline molecules. To study by SERRS measurements the crossing of pirarubicin through a phospholipid bilayer used as a membrane model, asymmetrical POPC-DPPA/POPC or POPC/POPC-DPPA bilayers were transferred by the Langmuir-Schäfer method, thanks to a laboratory-built set-up, and put in contact with a pirarubicin aqueous solution. It has been shown that the presence of anionic DPPA in the first monolayer in contact with pirarubicin would limit its crossing. This limiting effet is not observed if the first monolayer is zwitterionic.     

Simulations of skin barrier function: free energies of hydrophobic and hydrophilic transmembrane pores in ceramide bilayers

Transmembrane pore formation is central to many biological processes such as ion transport, cell fusion, and viral infection. Furthermore, pore formation in the ceramide bilayers of the stratum corneum may be an important mechanism by which penetration enhancers such as dimethylsulfoxide (DMSO) weaken the barrier function of the skin. We have used the potential of mean constraint force (PMCF) method to calculate the free energy of pore formation in ceramide bilayers in both the innate gel phase and in the DMSO-induced fluidized state. Our simulations show that the fluid phase bilayers form archetypal water-filled hydrophilic pores similar to those observed in phospholipid bilayers. In contrast, the rigid gel-phase bilayers develop hydrophobic pores. At the relatively small pore diameters studied here, the hydrophobic pores are empty rather than filled with bulk water, suggesting that they do not compromise the barrier function of ceramide membranes. A phenomenological analysis suggests that these vapor pores are stable, below a critical radius, because the penalty of creating water-vapor and tail-vapor interfaces is lower than that of directly exposing the strongly hydrophobic tails to water. The PMCF free energy profile of the vapor pore supports this analysis. The simulations indicate that high DMSO concentrations drastically impair the barrier function of the skin by strongly reducing the free energy required for pore opening.     

Stalk mechanism of vesicle fusion

Å mechanism for rupture of a separating bilayer, resulting from vesicle monolayer fusion is investigated theoretically. The stalk mechanism of monolayer fusion, assuming the formation and expansion of a stalk between two interacting membranes is considered. The stalk evolution leads to formation of a separating bilayer and mechanical tension appearance in the system. This tension results in rupture of the separating bilayer and hydrophilic pore formation. Competition between the mechanical tension and hydrophilic pore energy defines the criteria of contacting bilayer rupture. The tension increases with an increase of the absolute value of the negative spontaneous curvature of the outer membrane monolayer, K _s ^o . The pore edge energy decreases with an increase of the positive spontaneous curvature of the inner membrane monolayer, K _s ⁱ . The relations of spontaneous curvatures of outer and inner monolayers, leading to separating bilayer rupture, is calculated. It is demonstrated that his process is possible, provided spontaneous curvatures of membrane monolayers have opposite signs: K _s ^o <0, K _s ⁱ <0. Experimental data concerning the fusion process are analysed.     

The interaction of 2,4-dinitrophenol with phospholipids at phospholipid-water interfaces

The effect of 2, 4-dinitrophenol, DNP, on monolayers of egg lecithin, hydrogenated egg lecithin, dipalmitoyl lecithin and mitochondrial lipids has been examined. Both the undissociated and dissociated forms of DNP bind to the phospholipid polar groups. Binding of the acid form leads to a decrease in monolayer surface potential and an expansion of the monolayer. The amount of penetration of the acid form into lecithin monolayers appears to depend on the London-Van der Waals attractions between the lecithin hydrocarbon chains. Binding of the 2,4-dinitro-phenolate anion is reflected in a decrease in surface potential for lecithin monolayers, and an increase in surface potential for mitochondrial lipid monolayers. The adsorption of dinitro-phenolate to egg lecithin has been further investigated by micro-electrophoresis of lecithin liposomes. It is suggested that binding of DNP to phospholipid-water interfaces is important in determining its action as an uncoupler of oxidative phosphorylation, and as a compound that increases the electrical conductance of artificial lipid membranes.