Short-chain alcohols promote an early stage of membrane hemifusion.

Hemifusion, the linkage of contacting lipid monolayers of two membranes before the opening of a fusion pore, is hypothesized to proceed through the formation of a stalk intermediate, a local and strongly bent connection between membranes. When the monolayers' propensity to bend does not support the stalk (e.g., as it is when lysophosphatidylcholine is added), hemifusion is inhibited. In contrast, short-chain alcohols, reported to affect monolayer bending in a manner similar to that of lysophosphatidylcholine, were here found to promote hemifusion between fluorescently labeled liposomes and planar lipid bilayers. Single hemifusion events were detected by fluorescence microscopy. Methanol or ethanol (1.2-1.6 w/w %) added to the same compartment of the planar bilayer chamber as liposomes caused a 5-50 times increase in the number of hemifusion events. Alcohol-induced hemifusion was inhibited by lysophosphatidylcholine. Promotion of membrane hemifusion by short-chain alcohol was also observed for cell-cell fusion mediated by influenza virus hemagglutinin (HA). Alcohol promoted a fusion stage subsequent to the low pH-dependent activation of HA. We propose that binding of short-chain alcohol to the surface of membranes promotes hemifusion by facilitating the transient breakage of the continuity of each of the contacting monolayers, which is required for their subsequent merger in the stalk intermediate.     

Liposome composition effects on lipid mixing between cells expressing influenza virus hemagglutinin and bound liposomes

The involvement of contacting and distal lipid monolayers in different stages of protein-mediated fusion was studied for fusion mediated by influenza virus hemagglutinin. Inclusion of non-bilayer lipids in the composition of the liposomes bound to hemagglutinin-expressing cells affects fusion triggered by low pH. Lysophosphatidylcholine added to the outer membrane monolayers inhibits fusion. The same lipid added to the inner monolayer of the liposomes promotes both lipid and content mixing. In contrast to the inverted cone-shaped lysophosphatidylcholine, lipids of the opposite effective shape, oleic acid or cardiolipin with calcium, present in the inner monolayers inhibit fusion. These results along with fusion inhibition by a bipolar lipid that does not support peeling of one monolayer of the liposomal membrane from the other substantiate the hypothesis that fusion proceeds through a local hemifusion intermediate. The transition from hemifusion to the opening of an expanding fusion pore allows content mixing and greatly facilitates lipid mixing between liposomes and cells.     

Structural intermediates in influenza haemagglutinin-mediated fusion

Fusion pore formation in the haemagglutinin (HA)-mediated fusion is a culmination of a multistep process, which involves low-pH triggered refolding of HA and rearrangement of membrane lipid bilayers. This rearrangement was arrested or slowed down by either altering lipid composition of the membranes, or lowering the density of HA, and/ or temperature. The results suggest that fusion starts with the lateral assembly of activated HA into multimeric complexes surrounding future fusion sites. The next fusion stage involves hemifusion, i.e. merger of only contacting membrane monolayers. Lysophosphatidylcholine reversibly arrests fusion prior to this hemifusion stage. In the normal fusion pathway, hemifusion is transient and is not accompanied by any measurable transfer of lipid probes between the membranes. A temperature of 4degreeC stabilizes this `restricted hemifusion' intermediate. The restriction of lipid flow through the restricted hemifusion site is HA-dependent and can be released by partial cleaving of low pH-forms of HA with mild proteinase K treatment. Lipid effects indicate that fusion proceeds through two different lipid-involving intermediates, which are characterized by two opposite curvatures of the lipid monolayer. Hemifusion involves formation of a stalk, a local bent connection between the outer membrane monolayers. Fusion pore formation apparently involves bending of the inner membrane monolayers, which come together in hemifusion. To couple low pH-induced refolding of HA with lipid rearrangements, it is proposed that the extension of the alpha -helical coiled coil of HA pulls fusion peptides inserted into the HA-expressing membrane and locally bends the membrane into a saddle-like shape. Elastic energy drives self-assembly of these HA-containing membrane elements into a ring-like complex and causes the bulging of the host membrane into a dimple growing towards the target membrane. Bending stresses in the lipidic top of the dimple facilitate membrane fusion.     

Structural intermediates in influenza haemagglutinin-mediated fusion.

Fusion pore formation in the haemagglutinin (HA)-mediated fusion is a culmination of a multistep process, which involves low-pH triggered refolding of HA and rearrangement of membrane lipid bilayers. This rearrangement was arrested or slowed down by either altering lipid composition of the membranes, or lowering the density of HA, and/or temperature. The results suggest that fusion starts with the lateral assembly of activated HA into multimeric complexes surrounding future fusion sites. The next fusion stage involves hemifusion, i.e. merger of only contacting membrane monolayers. Lysophosphatidylcholine reversibly arrests fusion prior to this hemifusion stage. In the normal fusion pathway, hemifusion is transient and is not accompanied by any measurable transfer of lipid probes between the membranes. A temperature of 4 degrees C stabilizes this 'restricted hemifusion' intermediate. The restriction of lipid flow through the restricted hemifusion site is HA-dependent and can be released by partial cleaving of low pH-forms of HA with mild proteinase K treatment. Lipid effects indicate that fusion proceeds through two different lipid-involving intermediates, which are characterized by two opposite curvatures of the lipid monolayer. Hemifusion involves formation of a stalk, a local bent connection between the outer membrane monolayers. Fusion pore formation apparently involves bending of the inner membrane monolayers, which come together in hemifusion. To couple low pH-induced refolding of HA with lipid rearrangements, it is proposed that the extension of the alpha-helical coiled coil of HA pulls fusion peptides inserted into the HA-expressing membrane and locally bends the membrane into a saddle-like shape. Elastic energy drives self-assembly of these HA-containing membrane elements into a ring-like complex and causes the bulging of the host membrane into a dimple growing towards the target membrane. Bending stresses in the lipidic top of the dimple facilitate membrane fusion.     

Effects of Spontaneous Bilayer Curvature on Influenza Virus–mediated Fusion Pores

Cells expressing the hemagglutinin protein of influenza virus were fused to planar bilayer membranes containing the fluorescent lipid probes octadecylrhodamine (R18) or indocarbocyanine (DiI) to investigate whether spontaneous curvature of each monolayer of a target membrane affects the growth of fusion pores. R18 and DiI lowered the transition temperatures for formation of an inverted hexagonal phase, indicating that these probes facilitate the formation of negative curvature structures. The probes are known to translocate from one monolayer of a bilayer membrane to the other in a voltage-dependent manner. The spontaneous curvature of the cis monolayer (facing the cells) or the trans monolayer could therefore be made more negative through control of the polarity of voltage across the planar membrane. Electrical admittance measurements showed that the open times of flickering fusion pores were shorter when probes were in trans monolayers and longer when in cis monolayers compared with times when probe was symmetrically distributed. Open times were the same for probe symmetrically distributed as when probes were not present. Thus, open times were a function of the asymmetry of the spontaneous curvature between the trans and cis monolayers. Enriching the cis monolayer with a negative curvature probe reduced the probability that a small pore would fully enlarge, whereas enriching the trans monolayer promoted enlargement. Lysophosphatidylcholine has positive spontaneous curvature and does not translocate. When lysophosphatidylcholine was placed in trans leaflets of planar membranes, closing of fusion pores was rare. The effects of the negative and positive spontaneous curvature probes do not support the hypothesis that a flickering pore closes from an open state within a hemifusion diaphragm (essentially a “flat” structure). Rather, such effects support the hypothesis that the membrane surrounding the open pore forms a three-dimensional hourglass shape from which the pore flickers shut.     

Lipids in biological membrane fusion

The results reviewed suggest that membrane fusion in diverse biological fusion reactions involves formation of some specific intermediates: stalks and pores. Energy of these intermediates and, consequently, the rate and extent of fusion depend on the propensity of the corresponding monolayers of membranes to bend in the required directions.Proteins and peptides can control the bending energy of membrane monolayers in a number of ways. Monolayer lipid composition may be altered by different phospholipases [50, 85, 90], flipases and translocases [4, 50]. Proteins and peptides can change monolayer spontaneous curvature or hydrophobic void energy by direct interaction with membrane lipids [20, 32, 111]. Proteins may also provide some barriers for lipid diffusion in the plane of the monolayer [83, 141]. If diffusion of lipids at some specific membrane sites (e.g., in the vicinity of fusion protein) is somehow hindered, the energy of the bent fusion intermediates would reflect the elastic properties of these particular sites rather than the spontaneous curvature of the whole monolayers. Proteins may deform membranes while bringing them locally into close contact. The alteration of the geometric (external) curvature will certainly change the elastic energy of the initial state and, thus affect the energetic barriers of the formation of the intermediates [143]. In addition, the area and the energy of the stalk can be reduced by preliminary bending of the contacting membranes [111]. The possible effects of proteins and polymers on local elastic properties and local shapes of the membranes have been recently analyzed [22, 39, 45, 63]. These studies may provide a good basis for future development of theoretical models of protein-mediated fusion.     

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.     

Inner but Not Outer Membrane Leaflets Control the Transition from Glycosylphosphatidylinositol-anchored Influenza Hemagglutinin-induced Hemifusion to Full Fusion

Cells that express wild-type influenza hemagglutinin (HA) fully fuse to RBCs, while cells that express the HA-ectodomain anchored to membranes by glycosylphosphatidylinositol, rather than by a transmembrane domain, only hemifuse to RBCs. Amphipaths were inserted into inner and outer membrane leaflets to determine the contribution of each leaflet in the transition from hemifusion to fusion. When inserted into outer leaflets, amphipaths did not promote the transition, independent of whether the agent induces monolayers to bend outward (conferring positive spontaneous monolayer curvature) or inward (negative curvature). In contrast, when incorporated into inner leaflets, positive curvature agents led to full fusion. This suggests that fusion is completed when a lipidic fusion pore with net positive curvature is formed by the inner leaflets that compose a hemifusion diaphragm. Suboptimal fusion conditions were established for RBCs bound to cells expressing wild-type HA so that lipid but not aqueous dye spread was observed. While this is the same pattern of dye spread as in stable hemifusion, for this “stunted” fusion, lower concentrations of amphipaths in inner leaflets were required to promote transfer of aqueous dyes. Also, these amphipaths induced larger pores for stunted fusion than they generated within a stable hemifusion diaphragm. Therefore, spontaneous curvature of inner leaflets can affect formation and enlargement of fusion pores induced by HA. We propose that after the HA-ectodomain induces hemifusion, the transmembrane domain causes pore formation by conferring positive spontaneous curvature to leaflets of the hemifusion diaphragm.     

Oblique membrane insertion of viral fusion peptide probed by neutron diffraction

Fusion peptides mimic the membrane fusion activities of the larger viral proteins from which they derive their sequences. A possible mode of activity involves their oblique insertion into lipid bilayers, causing membrane disruption by promoting highly curved hemifusion intermediates, leading to fusion. We have determined the location and orientation of the simian immunodeficiency virus (SIV) fusion peptide in planar lipid bilayers using neutron lamellar diffraction. The helical axis of the peptide adopts an angle of 55 degrees relative to the membrane normal, while it positions itself nearest the lipid bilayer surface. This is the first direct observation of the structural interaction between a fusion peptide and a phospholipid bilayer.     

Reversible merger of membranes at the early stage of influenza hemagglutinin-mediated fusion

Fusion mediated by influenza hemagglutinin (HA), a prototype fusion protein, is commonly detected as lipid and content mixing between fusing cells. Decreasing the surface density of fusion-competent HA inhibited these advanced fusion phenotypes and allowed us to identify an early stage of fusion at physiological temperature. Although lipid flow between membranes was restricted, the contacting membrane monolayers were apparently transiently connected, as detected by the transformation of this fusion intermediate into complete fusion after treatments known to destabilize hemifusion diaphragms. These reversible connections disappeared within 10-20 min after application of low pH, indicating that after the energy released by HA refolding dissipated, the final low pH conformation of HA did not support membrane merger. Although the dynamic character and the lack of lipid mixing at 37 degrees C distinguish the newly identified fusion intermediate from the intermediate arrested at 4 degrees C described previously, both intermediates apparently belong to the same family of restricted hemifusion (RH) structures. Because the formation of transient RH structures at physiological temperatures was as fast as fusion pore opening and required less HA, we hypothesize that fusion starts with the formation of multiple RH sites, only a few of which then evolve to become expanding fusion pores.     

Variation of lipid membrane composition caused by strong bending

Dynamic coupling between the morphology and molecular composition of cellular membranes is crucial for formation of the intracellular organelles and transport vesicles. Most of the membrane proteins and lipids discriminate membrane curvatures. However, it remains unclear whether the curvature alone is sufficient to support heterogeneous distribution of lipids. Here we demonstrate that the curvature-driven redistribution of phospholipids, such as dioleoylphosphatidylethanolamine (DOPE), requires strong membrane bending. We used cylindrical lipid nanotubes (NTs) pulled from planar lipid membranes with lateral tension of ∼1 dyn/cm. Such high tensions forced extreme curvatures of the NT membrane, with luminal radius approaching the thickness of the lipid bilayer, 5nm. When the NT contained lipid species with high spontaneous curvature (SC), such as DOPE, we observed slow reduction of its radius. This reduction indicated the redistribution of DOPE between the inner and outer monolayers of the NT. Accordingly, the SC of DOPE was recovered from the measured changes in the radii: the SC value, calculated under the assumption that the DOPE content is coupled to the monolayer curvature, was ∼0.4 nm⁻¹, consistent with the published data. Thus, redistribution of lipids should be taken into account in calculations of composition and material properties of strongly deformed membrane structures, such as intermediate structures arising in the processes of membrane fusion and fission.     

Hemifusion between cells expressing hemagglutinin of influenza virus and planar membranes can precede the formation of fusion pores that subsequently fully enlarge

The chronological relation between the establishment of lipid continuity and fusion pore formation has been investigated for fusion of cells expressing hemagglutinin (HA) of influenza virus to planar bilayer membranes. Self-quenching concentrations of lipid dye were placed in the planar membrane to monitor lipid mixing, and time-resolved admittance measurements were used to measure fusion pores. For rhodamine-PE, fusion pores always occurred before a detectable amount of dye moved into an HA-expressing cell. However, with DiI in the planar membrane, the relationship was reversed: the spread of dye preceded formation of small pores. In other words, by using DiI as probe, hemifusion was clearly observed to occur before pore formation. For hemifused cells, a small pore could form and subsequently fully enlarge. In contrast, for cells that express a glycosylphosphatidylinositol-anchored ectodomain of HA, hemifusion occurred, but no fully enlarged pores were observed. Therefore, the transmembrane domain of HA is required for the formation of fully enlarging pores. Thus, with the planar bilayer membranes as target, hemifusion can precede pore formation, and the occurrence of lipid dye spread does not preclude formation of pores that can enlarge fully.     

Calculating Transition Energy Barriers and Characterizing Activation States for Steps of Fusion

We use continuum mechanics to calculate an entire least energy pathway of membrane fusion, from stalk formation, to pore creation, and through fusion pore enlargement. The model assumes that each structure in the pathway is axially symmetric. The static continuum stalk structure agrees quantitatively with experimental stalk architecture. Calculations show that in a stalk, the distal monolayer is stretched and the stored stretching energy is significantly less than the tilt energy of an unstretched distal monolayer. The string method is used to determine the energy of the transition barriers that separate intermediate states and the dynamics of two bilayers as they pass through them. Hemifusion requires a small amount of energy independently of lipid composition, while direct transition from a stalk to a fusion pore without a hemifusion intermediate is highly improbable. Hemifusion diaphragm expansion is spontaneous for distal monolayers containing at least two lipid components, given sufficiently negative diaphragm spontaneous curvature. Conversely, diaphragms formed from single-component distal monolayers do not expand without the continual injection of energy. We identify a diaphragm radius, below which central pore expansion is spontaneous. For larger diaphragms, prior studies have shown that pore expansion is not axisymmetric, and here our calculations supply an upper bound for the energy of the barrier against pore formation. The major energy-requiring deformations in the steps of fusion are: widening of a hydrophobic fissure in bilayers for stalk formation, splay within the expanding hemifusion diaphragm, and fissure widening initiating pore formation in a hemifusion diaphragm.     

Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.     

Some studies on lipid peroxidation in monomolecular and bimolecular lipid films

Summary Hydrogen peroxide generated from dissolved oxygen through the alloxandialuric acid cycle affected both the permeability and the stability of lipid bilayer membranes. The permeability of the artificial membranes varied directly with the hydrogen peroxide concentration. Membrane stability varied inversely with the hydrogen peroxide concentration. Bilayers formed from solutions containing both phospholipid and the antioxidant vitamin E were less permeable and more stable in the presence of hydrogen peroxide than bilayers generated from solutions containing phospholipid alone. Peroxidation of phospholipid monolayers caused first an expansion of the films presumably through the introduction of peroxide groups. Further oxidation of phospholipid monolayers led to contraction of the films presumably through the formation of water-soluble products. The results of the monolayer studies and a consideration of the possible kinetics for the peroxidation reaction sequence have been used to explain the changes in the permeability and the stability of lipid bilayer membranes. Our data suggest that oxidation of lipid in biological membranes may first increase membrane permeability and then decrease membrane stability.     

Adhesion and merging of lipid bilayers: a method for measuring the free energy of adhesion and hemifusion

Lipid bilayers can be induced to adhere to each other by molecular mediators, and, depending on the lipid composition, such adhesion can lead to merging of the contacting monolayers in a process known as hemifusion. Such bilayer-bilayer reactions have never been systematically studied. In the course of our studies of membrane-active molecules, we encountered such reactions. We believe that they need to be understood whenever bilayer-bilayer interactions take place, such as during membrane fusion. For illustration, we discuss three examples: spontaneous adhesion between phospholipid bilayers induced by low pH, polymer-induced osmotic depletion attraction between lipid bilayers, and anionic lipid bilayers cross-bridged by multicationic peptides. Our purpose here is to describe a general method for studying such interactions. We used giant unilamellar vesicles, each of which was aspirated in a micropipette so that we could monitor the tension of the membrane and the membrane area changes during the bilayer-bilayer interaction. We devised a general method for measuring the free energy of adhesion or hemifusion. The results show that the energies of adhesion or hemifusion of lipid bilayers could vary over 2 orders of magnitude from −1 to −50 × 10⁻⁵ J/m² in these examples alone. Our method can be used to measure the energy of transition in each step of lipid transformation during membrane fusion. This is relevant for current research on membrane fusion, which focuses on how fusion proteins induce lipid transformations.     

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 Pathway of Membrane Fusion Catalyzed by Influenza Hemagglutinin: Restriction of Lipids, Hemifusion, and Lipidic Fusion Pore Formation

The mechanism of bilayer unification in biological fusion is unclear. We reversibly arrested hemagglutinin (HA)-mediated cell–cell fusion right before fusion pore opening. A low-pH conformation of HA was required to form this intermediate and to ensure fusion beyond it. We present evidence indicating that outer monolayers of the fusing membranes were merged and continuous in this intermediate, but HA restricted lipid mixing. Depending on the surface density of HA and the membrane lipid composition, this restricted hemifusion intermediate either transformed into a fusion pore or expanded into an unrestricted hemifusion, without pores but with unrestricted lipid mixing. Our results suggest that restriction of lipid flux by a ring of activated HA is necessary for successful fusion, during which a lipidic fusion pore develops in a local and transient hemifusion diaphragm.     

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.     

Model of membrane fusion: Continuous transition to fusion pore with regard of hydrophobic and hydration interactions

We consider the process of fusion of lipid membranes from the stage of stalk with minimal radius to the stage of fusion pore. We assume that stalk directly developed into the fusion pore, omitting the stage of hemifusion diaphragm. Energy of intermediate stages is calculated on the basis of the classical elasticity theory of liquid crystals adapted for lipid membranes. The trajectory of transition from stalk to pore is obtained with regard to hydrophobic and hydration interactions. Continuous change of orientation of lipids in distal monolayers occurs along the trajectory. The orientation changes from the direction along rotational axis of the system specific to stalk to the direction corresponding to the fusion pore. Dependence of energy of intermediate stages on the value of spontaneous curvature of distal monolayers of the fusing membranes is obtained. We demonstrate that the energy barrier of the stalk-to-pore transition decreases when distal monolayers have positive spontaneous curvature, which is in accordance with available experimental data.