The mechanism of lamellar-to-inverted hexagonal phase transitions in phosphatidylethanolamine: implications for membrane fusion mechanisms

We studied the mechanism of the lamellar-to-inverted hexagonal (L alpha/H[II]) phase transition, using time-resolved cryotransmission electron microscopy (TRC-TEM), 31P-NMR, and differential scanning calorimetry. The transition was initiated in dispersions of large unilamellar vesicles of dipalmitoleoyl phosphatidylethanolamine (DiPoPE). We present evidence that the transition proceeds in three steps. First, many small connections form between apposed membranes. Second, the connections aggregate within the planes of the bilayers, forming arrays with hexagonal order in some projections. Third, these quasihexagonal structures elongate into small domains of H(II) phase, acquiring lipid molecules by diffusion from contiguous bilayers. A previously proposed membrane fusion mechanism rationalizes these results. The modified stalk theory predicts that the L alpha/H(II) phase transition involves some of the same intermediate structures as membrane fusion. The small interbilayer connections observed via TRC-TEM are compatible with the structure of a critical intermediate in the modified stalk mechanism: the trans monolayer contact (TMC). The theory predicts that 1) TMCs should form starting at tens of degrees below TH; 2) when TMCs become sufficiently numerous, they should aggregate into transient arrays like the quasihexagonal arrays observed here by TRC-TEM; and 3) these quasihexagonal arrays can then elongate directly into H(II) phase domains. These predictions rationalize the principal features of our data, which are incompatible with the other transition mechanisms proposed to date. Thus these results support the modified stalk mechanism for both membrane fusion and the L alpha/H(II) phase transition. We also discuss some implications of the modified stalk theory for fusion in protein-containing systems. Specifically, we point out that recent data on the effects of hydrophobic peptides and viral fusion peptides on lipid phase behavior are consistent with an effect of the peptides on TMC stability.     

Acyl-chain correlation in membrane fusion intermediates: x-ray diffraction from the rhombohedral lipid phase

We have studied the acyl-chain conformation in stalk phases of model membranes by x-ray diffraction from oriented samples. As an equilibrium lipid phase induced by dehydration, the stalk or rhombohedral phase exhibits lipidic passages (stalks) between adjacent bilayers, representing a presumed intermediate state in membrane fusion. From the detailed analysis of the acyl-chain correlation peak, we deduce the structural parameters of the acyl-chain fluid above, at, and below the transition from the lamellar to rhombohedral state, at the molecular level.     

A rhombohedral phase of lipid containing a membrane fusion intermediate structure

We constructed the electron density distribution from the x-ray diffraction of a phase of phospholipid that exhibited rhombohedral symmetry. To determine the phases of the diffraction amplitudes, we first extended the well-known one-dimensional swelling method for planar bilayers to a three-dimensional method applicable to a layered system containing in-plane structures, such as rhombohedral structures. The complete phase determination was accomplished by a combination of the swelling method and Luzzati's pattern recognition method. The constructed electron density distribution showed that in each unit cell, two apposed monolayers merged across the water layer and developed into an hourglass structure consistent with a postulated membrane fusion intermediate state called a stalk. The observation of the stalk structure lends a strong support to the stalk hypothesis for membrane fusion and opens a way to measure the structural parameters in the fusion pathway.     

Molecular dynamics simulation of cation-phospholipid clustering in phospholipid bilayers: possible role in stalk formation during membrane fusion

In this study, we performed all-atom long-timescale molecular dynamics simulations of phospholipid bilayers incorporating three different proportions of negatively charged lipids in the presence of K(+), Mg(2+), and Ca(2+) ions to systemically determine how membrane properties are affected by cations and lipid compositions. Our simulations revealed that the binding affinity of Ca(2+) ions with lipids is significantly stronger than that of K(+) and Mg(2+) ions, regardless of the composition of the lipid bilayer. The binding of Ca(2+) ions to the lipids resulted in bilayers having smaller lateral areas, greater thicknesses, greater order, and slower rotation of their lipid head groups, relative to those of corresponding K(+)- and Mg(2+)-containing systems. The Ca(2+) ions bind preferentially to the phosphate groups of the lipids. The complexes formed between the cations and the lipids further assembled to form various multiple-cation-centered clusters in the presence of anionic lipids and at higher ionic strength-most notably for Ca(2+). The formation of cation-lipid complexes and clusters dehydrated and neutralized the anionic lipids, creating a more-hydrophobic environment suitable for membrane aggregation. We propose that the formation of Ca(2+)-phospholipid clusters across apposed lipid bilayers can work as a "cation glue" to adhere apposed membranes together, providing an adequate configuration for stalk formation during membrane fusion.     

A new mechanism of model membrane fusion determined from Monte Carlo simulation

We have carried out extensive Monte Carlo simulations of the fusion of tense apposed bilayers formed by amphiphilic molecules within the framework of a coarse-grained lattice model. The fusion pathway differs from the usual stalk mechanism. Stalks do form between the apposed bilayers, but rather than expand radially to form an axial-symmetric hemifusion diaphragm of the trans leaves of both bilayers, they promote in their vicinity the nucleation of small holes in the bilayers. Two subsequent paths are observed. 1) The stalk encircles a hole in one bilayer creating a diaphragm comprised of both leaves of the other intact bilayer, which ruptures to complete the fusion pore. 2) Before the stalk can encircle a hole in one bilayer, a second hole forms in the other bilayer, and the stalk aligns and encircles them both to complete the fusion pore. Both pathways give rise to mixing between the cis and trans leaves of the bilayer and allow for transient leakage.     

Stalk model of membrane fusion: solution of energy crisis.

Membrane fusion proceeds via formation of intermediate nonbilayer structures. The stalk model of fusion intermediate is commonly recognized to account for the major phenomenology of the fusion process. However, in its current form, the stalk model poses a challenge. On one hand, it is able to describe qualitatively the modulation of the fusion reaction by the lipid composition of the membranes. On the other, it predicts very large values of the stalk energy, so that the related energy barrier for fusion cannot be overcome by membranes within a biologically reasonable span of time. We suggest a new structure for the fusion stalk, which resolves the energy crisis of the model. Our approach is based on a combined deformation of the stalk membrane including bending of the membrane surface and tilt of the hydrocarbon chains of lipid molecules. We demonstrate that the energy of the fusion stalk is a few times smaller than those predicted previously and the stalks are feasible in real systems. We account quantitatively for the experimental results on dependence of the fusion reaction on the lipid composition of different membrane monolayers. We analyze the dependence of the stalk energy on the distance between the fusing membranes and provide the experimentally testable predictions for the structural features of the stalk intermediates.     

Direct Visualization of Large and Protein-Free Hemifusion Diaphragms

Fusion of cellular membranes is a ubiquitous biological process requiring remodeling of two phospholipid bilayers. We believe it is very likely that merging of membranes proceeds via similar sequential intermediates. Contacting membranes form a stalk between the proximal leaflets that expands radially into an hemifusion diaphragm (HD) and subsequently open to a fusion pore. Although considered to be a key intermediate in fusion, direct experimental verification of this structure is difficult due to its transient nature. Using confocal fluorescence microscopy we have investigated the fusion of giant unilamellar vesicles (GUVs) containing phosphatidylserine and fluorescent virus derived transmembrane peptides or membrane proteins in the presence of divalent cations. Time-resolved imaging revealed that fusion was preceded by displacement of peptides and fluorescent lipid analogs from the GUV-GUV adhesion region. A detailed analysis of this area being several μm in size revealed that peptides were completely sequestered as expected for an HD. Lateral distribution of lipid analogs was consistent with formation of an HD but not with the presence of two adherent bilayers. Formation and size of the HD were dependent on lipid composition and peptide concentration.     

Stalk phase formation: effects of dehydration and saddle splay modulus

One of the earliest lipid intermediates forming in the course of membrane fusion is the lipid stalk. Although many aspects of the stalk hypothesis were elaborated theoretically and confirmed by experiments it remained unresolved whether stalk formation is always an energy consuming process or if there are conditions where the stalks are energetically favorable and form spontaneously resulting in an equilibrium stalk phase. Motivated by a recent breakthrough experiments we analyze the physical factors determining the spontaneous stalk formation. We show that this process can be driven by interplay between two factors: the elastic energy of lipid monolayers including a contribution of the saddle splay deformation and the energy of hydration repulsion acting between apposing membranes. We analyze the dependence of stalk formation on the saddle splay (Gaussian) modulus of the lipid monolayers and estimate the values of this modulus based on the experimentally established phase boundary between the lamellar and the stalk phases. We suggest that fusion proteins can induce stalk formation just by bringing the membranes into close contact, and accumulating, at least locally, a sufficiently large energy of the hydration repulsion.     

Molecular Dynamics Simulation Analysis of Membrane Defects and Pore Propensity of Hemifusion Diaphragms

Membrane fusion often exhibits slow dynamics in electrophysiological experiments, involving prespike foot and fusion pore-flickering, but the structural basis of such phenomena remains unclear. Hemifusion intermediates have been implicated in the early phase of membrane fusion. To elucidate the dynamics of formation of membrane defects and pores within the hemifusion diaphragm (HD), atomistic and coarse-grained models of hemifusion intermediates were constructed using dipalmitoylphosphatidylcholine or dioleoylphosphatidylcholine membranes. The work necessary to displace a lipid molecule to the hydrophobic core of the bilayer was measured. For a lipid within the HD with radius of 4 nm, the work was ∼80 kJ/mol, similar to that in a planar bilayer. The work was much less (∼40 kJ/mol) when the HD was surrounded by a steep stalk, i.e., stalk wings forming a large angle at the junction of three bilayers. In the latter case, the lipid displacement engendered formation of a pore contacting the HD rim. The work was similarly small (40 kJ/mol) for a small HD of 1.5 nm radius, where a pore formed and grew rapidly, quickly generating a toroidal structure (<40 ns). Combining the steep stalk and the small HD decreased the work further, although quantitative analysis was difficult because the latter system was not in a stable equilibrium state. Results suggest that fine tuning of fusion dynamics requires strict control of the HD size and the angle between the expanded stalk and HD. In additional free simulations, the steep stalk facilitated widening of a preformed pore contacting the HD rim.     

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.     

The Interaction between Influenza HA Fusion Peptide and Transmembrane Domain Affects Membrane Structure

Viral glycoproteins, such as influenza hemagglutinin (HA) and human immunodeficiency virus gp41, are anchored by a single helical segment transmembrane domain (TMD) on the viral envelope membrane. The fusion peptides (FP) of the glycoproteins insert into the host membrane and initiate membrane fusion. Our previous study showed that the FP or TMD alone perturbs membrane structure. Interaction between the influenza HA FP and TMD has previously been shown, but its role is unclear. We used PC spin labels dipalmitoylphospatidyl-tempo-choline (on the headgroup), 5PC and 14PC (5-C and 14-C positions on the acyl chain) to detect the combined effect of FP-TMD interaction by titrating HA FP to TMD-reconstituted 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phospho-(1’-rac-glycerol)/cholesterol lipid bilayers using electron spin resonance. We found that the FP-TMD increases the lipid order at all positions, which has a greater lipid ordering effect than the sum of the FP or TMD alone, and this effect reaches deeper into the membranes. Although HA-mediated membrane fusion is pH dependent, this combined effect is observed at both pH 5 and pH 7. In addition to increasing lipid order, multiple components are found for 5PC at increased concentration of FP-TMD, indicating that distinct domains are induced. However, the mutation of Gly1 in the FP and L187 in the TMD eliminates the perturbations, consistent with their fusogenic phenotypes. Electron spin resonance on spin-labeled peptides confirms these observations. We suggest that this interaction may provide a driving force in different stages of membrane fusion: initialization, transition from hemifusion stalk to transmembrane contact, and fusion pore formation.     

Atomic force microscope spectroscopy reveals a hemifusion intermediate during soluble N-ethylmaleimide-sensitive factor-attachment protein receptors-mediated membrane fusion

This study investigated the effect of soluble N-ethylmaleimide-sensitive factor-attachment protein (SNAP) receptors (SNAREs) on the fusion of egg L-α-phosphatidylcholine bilayers using atomic force microscope (AFM) spectroscopy. AFM measurements of the fusion force under compression were acquired to reveal the energy landscape of the fusion process. A single main energy barrier governing the fusion process was identified in the absence and presence of SNAREs in the bilayers. Under compression, a significant downward shift in the fusion dynamic force spectrum was observed when cognate v- and t-SNAREs were present in the opposite bilayers. The presence of vesicle-associated membrane protein (VAMP) and binary syntaxin and SNAP 25 in the apposed bilayers resulted in a reduction in the height of the activation potential by ∼1.3 k_BT and a >2-fold increase in the width of the energy barrier. The widening of the energy barrier in the presence SNAREs is interpreted as an increase in the compressibility of the membranes, which translates to a greater ease in the bilayer deformation and subsequently the fusion of the membranes under compression. Facilitation of membrane fusion was observed only when SNAREs were present in both bilayers. Moreover, addition of the soluble cytoplasmic domain of VAMP, which interferes with the interaction between opposing v- and t-SNAREs, prevented such facilitation. These observations implicated the interaction between the cytoplasmic domains of opposing SNAREs in the observed fusion facilitation, possibly by destabilizing the bilayers through pulling on their transmembrane segments. Our AFM compression measurements revealed that SNARE-mediated membrane fusion proceeded through a sequence of two ∼5 nm collapses of the membrane, an observation that is consistent with the existence of a hemifused state during the fusion process.     

Structure and energy of fusion stalks: the role of membrane edges

Fusion of lipid bilayers proceeds via a sequence of distinct structural transformations. Its early stage involves a localized, hemifused intermediate in which the proximal but not yet the distal monolayers are connected. Whereas the so-called stalk model most successfully accounts for the properties of the hemifused intermediate, there is still uncertainty about its microscopic structure and energy. We reanalyze fusion stalks using the theory of membrane elasticity. In our calculations, a short (cylindrical micelle-like) tether connects the two proximal monolayers of the hemifused membranes. The shape of the stalk and the length of the tether are calculated such as to minimize the overall free energy and to avoid the formation of voids within the hydrocarbon core. Our free energy expression is based on three internal degrees of freedom of a perturbed lipid layer: thickness, splay, and tilt deformations. Based on exactly the same model, we compare fusion stalks with and without the ability included to form sharp edges at the interfacial region between the hydrocarbon core and the polar environment. Requiring the interface to be smooth everywhere, our detailed calculations recover previous results: the stalk energies are far too high to account for the experimental observation of fusion intermediates. However, if we allow the interface to be nonsmooth, we find a remarkable reduction of the stalk free energy down to more realistic values. The corresponding structure of a nonsmooth stalk exhibits sharp edges at the transition regions between the bilayer and tether parts. In addition to that, a corner is formed at each of the two distal monolayers. We discuss the mechanism how membrane edges reduce the energy of fusion stalks.     

Lipidic antagonists to SNARE-mediated fusion

SNARE proteins mediate the fusion of lipid bilayers by the directed assembly of coiled-coil domains arising from apposing membranes. We have utilized inverted cone-shaped lipids, antagonists of the necessary membrane deformation during fusion to characterize the extent and range of SNARE assembly up to the moment of stalk formation between bilayers. The inverted cone-shaped lipid family of acyl-CoAs specifically inhibits the completion of fusion in an acyl-chain length-dependent manner. Removal of acyl-CoA from the membrane relieves the inhibition and initiates a burst of membrane fusion with rates exceeding any point in the control curves lacking acyl-CoA. This burst indicates the accumulation of semi-assembled fusion complexes. These preformed complexes are resistant to cleavage by botulinum toxin B and thus appear to have progressed beyond the "loosely zippered" state of docked synaptic vesicles. Surprisingly, application of the soluble domain of VAMP2, which blocks SNARE assembly by competing for binding on the available t-SNAREs, blocks recovery from the acyl-CoA inhibition. Thus, complexes formed in the presence of a lipidic antagonist to fusion are incompletely assembled, suggesting that the formation of tightly assembled SNARE pairs requires progression all the way through to membrane fusion. In this regard, physiologically docked exocytic vesicles may be anchored by a highly dynamic and potentially even reversible SNAREpin.     

Energetics of intermediates in membrane fusion: comparison of stalk and inverted micellar intermediate mechanisms.

To understand the mechanism of membrane fusion, we have to infer the sequence of structural transformations that occurs during the process. Here, it is shown how one can estimate the lipid composition-dependent free energies of intermediate structures of different geometries. One can then infer which fusion mechanism is the best explanation of observed behavior in different systems by selecting the mechanism that requires the least energy. The treatment involves no adjustable parameters. It includes contributions to the intermediate energy resulting from the presence of hydrophobic interstices within structures formed between apposed bilayers. Results of these calculations show that a modified form of the stalk mechanism proposed by others is a likely fusion mechanism in a wide range of lipid compositions, but a mechanism based on inverted micellar intermediates (IMIs) is not. This should be true even in the vicinity of the lamellar/inverted hexagonal phase transition, where IMI formation would be most facile. Another prediction of the calculations is that traces of apolar lipids (e.g., long-chain alkanes) in membranes should have a substantial influence on fusion rates in general. The same theoretical methods can be used to generate and refine mechanisms for protein-mediated fusion.     

Crystallization of antimicrobial pores in membranes: magainin and protegrin

Membrane pores spontaneously formed by antimicrobial peptides in membranes were crystallized for the first time by manipulating the sample hydration and temperature. Neutron diffraction shows that magainins and protegrins form stable pores in fully hydrated fluid membranes. At lower hydration levels or low temperature, the membrane multilayers crystallize. In one crystalline phase, the pores in each bilayer arrange in a regular hexagonal array and the bilayers are stacked into a hexagonal ABC lattice, corresponding to the cubic close-packed structure of spheres. In another crystalline phase, the bilayers are modulated into the rippled multilamellae, corresponding to a 2D monoclinic lattice. The phase diagrams are described. Crystallization of the membrane pores provides possibilities for diffraction studies that might provide useful information on the pore structures.     

Cholesterol-Dependent Nanomechanical Stability of Phase-Segregated Multicomponent Lipid Bilayers

Cholesterol is involved in endocytosis, exocytosis, and the assembly of sphingolipid/cholesterol-enriched domains, as has been demonstrated in both model membranes and living cells. In this work, we explored the influence of different cholesterol levels (5-40 mol %) on the morphology and nanomechanical stability of phase-segregated lipid bilayers consisting of dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/SM/Chol) by means of atomic force microscopy (AFM) imaging and force mapping. Breakthrough forces were consistently higher in the SM/Chol-enriched liquid-ordered domains (L_o) than in the DOPC-enriched fluid-disordered phase (L_d) at a series of loading rates. We also report the activation energies (ΔE_a) for the formation of an AFM-tip-induced fracture, calculated by a model for the rupture of molecular thin films. The obtained ΔE_a values agree remarkably well with reported values for fusion-related processes using other techniques. Furthermore, we observed that within the Chol range studied, the lateral organization of bilayers can be categorized into three distinct groups. The results are rationalized by fracture nanomechanics of a ternary phospholipid/sphingolipid/cholesterol mixture using correlated AFM-based imaging and force mapping, which demonstrates the influence of a wide range of cholesterol content on the morphology and nanomechanical stability of model bilayers. This provides fundamental insights into the role of cholesterol in the formation and stability of sphingolipid/cholesterol-enriched domains, as well as in membrane 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.     

Peptide models for the membrane destabilizing actions of viral fusion proteins.

The fusion of enveloped viruses to target membranes is promoted by certain viral fusion proteins. However, many other proteins and peptides stabilize bilayer membranes and inhibit membrane fusion. We have evaluated some characteristics of the interaction of peptides that are models of segments of measles and influenza fusion proteins with membranes. Our results indicate that these models of the fusogenic domains of viral fusion proteins promote conversion of model membrane bilayers to nonbilayer phases. This is opposite to the effects of peptides and proteins that inhibit viral fusion. A peptide model for the fusion segment of the HA protein of influenza increased membrane leakage as well as promoted the formation of nonbilayer phases upon acidification from pH 7-5. We analyze the gross conformational features of the peptides, and speculate on how these conformational features relate to the structures of the intact proteins and to their role in promoting membrane fusion.