A hemifused complex is the hub in a network of pathways to membrane fusion

Membrane fusion is a critical step for many essential processes, from neurotransmission to fertilization. For over 40 years, protein-free fusion driven by calcium or other cationic species has provided a simplified model of biological fusion, but the mechanisms remain poorly understood. Cation-mediated membrane fusion and permeation are essential in their own right to drug delivery strategies based on cell-penetrating peptides or cation-bearing lipid nanoparticles. Experimental studies suggest calcium drives anionic membranes to a hemifused intermediate that constitutes a hub in a network of pathways, but the pathway selection mechanism is unknown. Here we develop a mathematical model that identifies the network hub as a highly dynamic hemifusion complex. Multivalent cations drive expansion of this high-tension hemifusion interface between interacting vesicles during a brief transient. The fate of this interface determines the outcome, either fusion, dead-end hemifusion, or vesicle lysis. The model reproduces the unexplained finding that calcium-driven fusion of vesicles with planar membranes typically stalls at hemifusion, and we show the equilibrated hemifused state is a novel lens-shaped complex. Thus, membrane fusion kinetics follow a stochastic trajectory within a network of pathways, with outcome weightings set by a hemifused complex intermediate.     

Membrane fusion induced by neuronal SNAREs transits through hemifusion

Synaptic transmission requires the controlled release of neurotransmitter from synaptic vesicles by membrane fusion with the presynaptic plasma membrane. SNAREs are the core constituents of the protein machinery responsible for synaptic membrane fusion. The mechanism by which SNAREs drive membrane fusion is thought to involve a hemifusion intermediate, a condition in which the outer leaflets of two bilayers are combined and the inner leaflets remain intact; however, hemifusion has been observed only as an end point rather than as an intermediate. Here, we examined the kinetics of membrane fusion of liposomes mediated by recombinant neuronal SNAREs using fluorescence assays that monitor both total lipid mixing and inner leaflet mixing. Our results demonstrate that hemifusion is dominant at the early stage of the fusion reaction. Over time, hemifusion transitioned to complete fusion, showing that hemifusion is a true intermediate. We also show that hemifusion intermediates can be trapped, likely as unproductive outcomes, by modulating the surface concentration of the SNARE proteins.     

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.     

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.     

Lipid intermediates in membrane fusion: formation,structure, and decay of hemifusion diaphragm

Lipid bilayer fusion is thought to involve formation of a local hemifusion connection, referred to as a fusion stalk. The subsequent fusion stages leading to the opening of a fusion pore remain unknown. The earliest fusion pore could represent a bilayer connection between the membranes and could be formed directly from the stalk. Alternatively, fusion pore can form in a single bilayer, referred to as hemifusion diaphragm (HD), generated by stalk expansion. To analyze the plausibility of stalk expansion, we studied the pathway of hemifusion theoretically, using a recently developed elastic model. We show that the stalk has a tendency to expand into an HD for lipids with sufficiently negative spontaneous splay, (~)J(s)< 0. For different experimentally relevant membrane configurations we find two characteristic values of the spontaneous splay. (~)J*(s) and (~)J**(s), determining HD dimension. The HD is predicted to have a finite equilibrium radius provided that the spontaneous splay is in the range (~)J**(s)< (~)J(s)<(~)J*(s), and to expand infinitely for (~)J(s)<(~)J**(s). In the case of common lipids, which do not fuse spontaneously, an HD forms only under action of an external force pulling the diaphragm rim apart. We calculate the dependence of the HD radius on this force. To address the mechanism of fusion pore formation, we analyze the distribution of the lateral tension emerging in the HD due to the establishment of lateral equilibrium between the deformed and relaxed portions of lipid monolayers. We show that this tension concentrates along the HD rim and reaches high values sufficient to rupture the bilayer and form the fusion pore. Our analysis supports the hypothesis that transition from a hemifusion to a fusion pore involves radial expansion of the stalk.     

Initiation and dynamics of hemifusion in lipid bilayers

One approach to the understanding of fusion in cells and model membranes involves stalk formation and expansion of the hemifusion diaphragm. We predict theoretically the initiation of hemifusion by stalk expansion and the dynamics of mesoscopic hemifusion diaphragm expansion in the light of recent experiments and theory that suggested that hemifusion is driven by intramembrane tension far from the fusion zone. Our predictions include a square-root scaling of the hemifusion zone size on time as well as an estimate of the minimal tension for initiation of hemifusion. Whereas a minimal amount of pressure is evidently needed for stalk formation, it is not necessarily required for stalk expansion. The energy required for tension-induced fusion is much smaller than that required for pressure-driven fusion.     

Highly Efficient Protein-free Membrane Fusion: A Giant Vesicle Study

Membrane fusion is a ubiquitous process in biology and is a prerequisite for many intracellular delivery protocols relying on the use of liposomes as drug carriers. Here, we investigate in detail the process of membrane fusion and the role of opposite charges in a protein-free lipid system based on cationic liposomes (LUVs, large unilamellar vesicles) and anionic giant unilamellar vesicles (GUVs) composed of different palmitoyloleoylphosphatidylcholine (POPC)/palmitoyloleoylphosphatidylglycerol (POPG) molar ratios. By using a set of optical-microscopy- and microfluidics-based methods, we show that liposomes strongly dock to GUVs of pure POPC or low POPG fraction (up to 10 mol%) in a process mainly associated with hemifusion and membrane tension increase, commonly leading to GUV rupture. On the other hand, docked LUVs quickly and very efficiently fuse with negative GUVs of POPG fractions at or above 20 mol%, resulting in dramatic GUV area increase in a charge-dependent manner; the vesicle area increase is deduced from GUV electrodeformation. Importantly, both hemifusion and full fusion are leakage-free. Fusion efficiency is quantified by the lipid transfer from liposomes to GUVs using fluorescence resonance energy transfer (FRET), which leads to consistent results when compared to fluorescence-lifetime-based FRET. We develop an approach to deduce the final composition of single GUVs after fusion based on the FRET efficiency. The results suggest that fusion is driven by membrane charge and appears to proceed up to charge neutralization of the acceptor GUV.     

Synaptotagmin I and Ca(2+) promote half fusion more than full fusion in SNARE-mediated bilayer fusion

Synaptic membrane fusion, which is necessary for neurotransmitter release, may be mediated by SNAREs and regulated by synaptotagmin (Syt) and Ca(2+). Fusion of liposomes mediated by reconstituted SNAREs produces full fusion and hemifusion, a membrane structure in which outer leaflets are mixed but the inner leaflets remain intact. Here, using the liposome fusion assay, it is shown that Syt promoted both hemifusion and full fusion in a Ca(2+)-dependent manner. Syt.Ca(2+) increased hemifusion more than full fusion, modulating the ratio of hemifusion to full fusion. Unlike the case of neuronal SNAREs, stimulation of fusion by Syt.Ca(2+) was not seen for other SNAREs involved in trafficking in yeast, indicating that the Syt.Ca(2+) stimulation was SNARE-specific. We constructed hybrid SNAREs in which transmembrane domains were swapped between neuronal and yeast SNAREs. With these hybrid SNAREs, we demonstrated that the interaction between the SNARE motifs of neuronal proteins and Syt.Ca(2+) was required for the stimulation of fusion.     

Membrane hemifusion: crossing a chasm in two leaps

During membrane fusion, the outer leaflets of the two membranes merge first, whereas the distal membrane leaflets remain separate until the opening of a fusion pore. This intermediate stage, called hemifusion, is a critical event shared by exocytosis, protein trafficking, and viral entry.     

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.     

Transmembrane organization of yeast syntaxin-analogue Sso1p

Membrane fusion in secretory pathways is thought to be mediated by SNAREs. It is proposed that membrane fusion transits through hemifusion, a condition in which the outer leaflets of the bilayers are mixed, but the inner leaflets are not. Hemifusion then proceeds to the fusion pore that connects the two internal contents. It is believed that the transmembrane domains (TMDs) of the fusion proteins play an essential role in the transition from hemifusion to the fusion pore. In this work, the structure, dynamics, and membrane topology of the TMD of Sso1p, a target membrane (t-) SNARE involved in the trafficking from Golgi to plasma membrane in yeast, was investigated using site-directed spin labeling and EPR spectroscopy. The EPR analysis of spin-labeled mutants showed that the TMD of Sso1p is a well-defined membrane spanning alpha-helix. The results also indicate that there is an equilibrium between the monomers and the oligomers. The oligomerization is mainly mediated through the interaction at the N-terminal half of the TMD, whereas the C-terminal half is free of the tertiary interaction. Additionally, the isotropic hyperfine splitting values were examined for nitroxide-scanning mutants, and it was found that the hyperfine splitting values show a V-shaped profile across the bilayer. Thus, hyperfine splitting may be used as an additional parameter to measure bilayer immersion depths of nitroxide.     

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.     

Field theoretic study of bilayer membrane fusion. I. Hemifusion mechanism

Self-consistent field theory is used to determine structural and energetic properties of metastable intermediates and unstable transition states involved in the standard stalk mechanism of bilayer membrane fusion. A microscopic model of flexible amphiphilic chains dissolved in hydrophilic solvent is employed to describe these self-assembled structures. We find that the barrier to formation of the initial stalk is much smaller than previously estimated by phenomenological theories. Therefore its creation it is not the rate-limiting process. The relevant barrier is associated with the rather limited radial expansion of the stalk into a hemifusion diaphragm. It is strongly affected by the architecture of the amphiphile, decreasing as the effective spontaneous curvature of the amphiphile is made more negative. It is also reduced when the tension is increased. At high tension the fusion pore, created when a hole forms in the hemifusion diaphragm, expands without bound. At very low membrane tension, small fusion pores can be trapped in a flickering metastable state. Successful fusion is severely limited by the architecture of the lipids. If the effective spontaneous curvature is not sufficiently negative, fusion does not occur because metastable stalks, whose existence is a seemingly necessary prerequisite, do not form at all. However if the spontaneous curvature is too negative, stalks are so stable that fusion does not occur because the system is unstable either to a phase of stable radial stalks, or to an inverted-hexagonal phase induced by stable linear stalks. Our results on the architecture and tension needed for successful fusion are summarized in a phase diagram.     

Meta-stability of the hemifusion intermediate induced by glycosylphosphatidylinositol-anchored influenza hemagglutinin.

Fusion between influenza virus and target membranes is mediated by the viral glycoprotein hemagglutinin (HA). Replacement of the transmembrane domain of HA with a glycosylphosphatidylinositol (GPI) membrane anchor allows lipid mixing but not the establishment of cytoplasmic continuity. This observation led to the proposal that the fusion mechanism passes through an intermediate stage corresponding to hemifusion between outer monolayers. We have used confocal fluorescence microscopy to study the movement of probes for specific bilayer leaflets of erythrocytes fusing with HA-expressing cells. N-Rh-PE and NBD-PC were used for specific labeling of the outer and inner membrane leaflet, respectively. In the case of GPI-HA-induced fusion, different behaviors of lipid transfer were observed, which include 1) exclusive movement of N-Rh-PE (hemifusion), 2) preferential movement of N-Rh-PE relative to NBD-PC, and 3) equal movement of both lipid analogs. The relative population of these intermediate states was dependent on the time after application of a low pH trigger for fusion. At early time points, hemifusion was more common and full redistribution of both bilayers was rare, whereas later full redistribution of both probes was frequently observed. In contrast to wild-type HA, the latter was not accompanied by mixing of the cytoplasmic marker Lucifer Yellow. We conclude that 1) the GPI-HA-mediated hemifusion intermediate is meta-stable and 2) expansion of an aqueous fusion pore requires the transmembrane and/or cytoplasmic domain of HA.     

A point mutation in the transmembrane domain of the hemagglutinin of influenza virus stabilizes a hemifusion intermediate that can transit to fusion

A hemagglutinin (HA) of influenza virus having a single semiconserved Gly residue within the transmembrane domain mutated to Leu (G520L) was expressed on cells; these cells were bound to red blood cells. By decreasing pH at 23 degrees C rather than 37 degrees C, an intermediate with properties expected of hemifusion just as the membranes are about to transit to full fusion was captured. As evidence: 1) increasing temperature to 37 degrees C at neutral pH allowed fusion to proceed; 2) after achieving the intermediate, the two membranes did not separate from each other after proteolytic cleavage of G520L because cells treated with proteinase K could not fuse upon temperature increase but could fuse upon the addition of chlorpromazine; and 3) at the point of the intermediate, adding exogenous lipids known to promote or inhibit the creation of hemifusion did not significantly alter the lipid dye spread that occurred upon increasing temperature, implying that at the intermediate, contacting membrane leaflets had already merged. A stable intermediate of hemifusion that could transit to fusion was also generated for wild-type HA, but pH had to be reduced at the significantly lower temperature of 4 degrees C. The fusion pores generated by G520L did not enlarge, whereas those induced by wild-type HA did. The finding that a state of transitional hemifusion can be readily obtained via a point mutation without the need for unusually low temperature supports the hypothesis that hemifusion occurs before pore formation.     

Control of membrane fusion mechanism by lipid composition: predictions from ensemble molecular dynamics

Membrane fusion is critical to biological processes such as viral infection, endocrine hormone secretion, and neurotransmission, yet the precise mechanistic details of the fusion process remain unknown. Current experimental and computational model systems approximate the complex physiological membrane environment for fusion using one or a few protein and lipid species. Here, we report results of a computational model system for fusion in which the ratio of lipid components was systematically varied, using thousands of simulations of up to a microsecond in length to predict the effects of lipid composition on both fusion kinetics and mechanism. In our simulations, increased phosphatidylcholine content in vesicles causes increased activation energies for formation of the initial stalk-like intermediate for fusion and of hemifusion intermediates, in accordance with previous continuum-mechanics theoretical treatments. We also use our large simulation dataset to quantitatively compare the mechanism by which vesicles fuse at different lipid compositions, showing a significant difference in fusion kinetics and mechanism at different compositions simulated. As physiological membranes have different compositions in the inner and outer leaflets, we examine the effect of such asymmetry, as well as the effect of membrane curvature on fusion. These predicted effects of lipid composition on fusion mechanism both underscore the way in which experimental model system construction may affect the observed mechanism of fusion and illustrate a potential mechanism for cellular regulation of the fusion process by altering membrane composition.     

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.     

GPI-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes

Under fusogenic conditions, fluorescent dye redistributed from the outer monolayer leaflet of red blood cells (RBCs) to cells expressing glycophosphatidylinositol-anchored influenza virus hemagglutinin (GPI- HA) without transfer of aqueous dye. This suggests that hemifusion, but not full fusion, occurred (Kemble, G. W., T. Danieli, and J. M. White. 1994. Cell. 76:383-391). We extended the evidence for hemifusion by labeling the inner monolayer leaflets of RBCs with FM4-64 and observing that these inner leaflets did not become continuous with GPI-HA- expressing cells. The region of hemifusion-separated aqueous contents, the hemifusion diaphragm, appeared to be extended and was long-lived. But when RBCs hemifused to GPI-HA-expressing cells were osmotically swollen, some diaphragms were disrupted, and spread of both inner leaflet and aqueous dyes was observed. This was characteristic of full fusion: inner leaflet and aqueous probes spread to cells expressing wild-type HA (wt-HA). By simultaneous video fluorescence microscopy and time-resolved electrical admittance measurements, we rigorously demonstrated that GPI-HA-expressing cells hemifuse to planar bilayer membranes: lipid continuity was established without formation of fusion pores. The hemifusion area became large. In contrast, for cells expressing wt-HA, before lipid dye spread, fusion pores were always observed, establishing that full fusion occurred. We present an elastic coupling model in which the ectodomain of wt-HA induces hemifusion and the transmembrane domain, absent in the GPI-HA-expressing cells, mediates full fusion.     

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.     

Lipid Mixing and Content Release in Single-Vesicle, SNARE-Driven Fusion Assay with 1-5 ms Resolution

A single-vesicle, fluorescence-based, SNARE-driven fusion assay enables simultaneous measurement of lipid mixing and content release with 5 ms/frame, or even 1 ms/frame, time resolution. The v-SNARE vesicles, labeled with lipid and content markers of different color, dock and fuse with a planar t-SNARE bilayer supported on glass. A narrow (<5 ms duration), intense spike of calcein fluorescence due to content release and dequenching coincides with inner-leaflet lipid mixing within 10 ms. The spike provides more sensitive detection of productive hemifusion events than do lipid labels alone. Consequently, many fast events previously thought to be prompt, full fusion events are now reclassified as productive hemifusion. Both full fusion and hemifusion occur with a time constant of 5-10 ms. At 60% phosphatidylethanolamine lipid composition, productive and dead-end hemifusion account for 65% of all fusion events. However, quantitative analysis shows that calcein is released into the space above the bilayer (vesicle bursting), rather than the thin aqueous space between the bilayer and glass. Evidently, at the instant of inner-leaflet mixing, flattening of the vesicle increases the internal pressure beyond the bursting point. This may be related to in vivo observations suggesting that membrane lysis often competes with membrane fusion.