Membrane lysis during biological membrane fusion: collateral damage by misregulated fusion machines

In the canonical model of membrane fusion, the integrity of the fusing membranes is never compromised, preserving the identity of fusing compartments. However, recent molecular simulations provided evidence for a pathway to fusion in which holes in the membrane evolve into a fusion pore. Additionally, two biological membrane fusion models—yeast cell mating and in vitro vacuole fusion—have shown that modifying the composition or altering the relative expression levels of membrane fusion complexes can result in membrane lysis. The convergence of these findings showing membrane integrity loss during biological membrane fusion suggests new mechanistic models for membrane fusion and the role of membrane fusion complexes.     

Participation of two fusion peptides in measles virus-induced membrane fusion: emerging similarity with other paramyxoviruses

Paramyxoviruses penetrate into their host cells by fusing their membranes with the plasma membrane. The hydrophobic N terminus of their F1 protein, termed the 'fusion peptide', is thought to be responsible for this process. Recently, an additional internal fusion peptide, homologous in sequence to the N-terminal fusion peptide of HIV-1, was identified in the Sendai virus F1 protein. Here, we investigated whether the presence of an additional internal fusion peptide is a general feature of paramyxoviridae. To this end, we synthesized and structurally and functionally characterized three peptides: (i) MV-197, which corresponds to an internal segment of the F1 protein of the measles virus (amino acids 197-225), homologous in location but not in sequence to the internal fusion peptide of the Sendai virus, (ii) Mu-MV-197, a randomized version of MV-197, and (iii) the 33 amino acid N-terminal fusion peptide of the measles virus. Remarkably, only MV-197 was highly fusogenic toward large unilamellar vesicles composed of either zwitterionic (phosphatidylcholine or phosphatidylcholine/sphingomyelin/cholesterol, a composition similar to that of human cell membranes) or negatively charged phospholipids. Binding experiments, circular dichroism spectroscopy in phospholipid membranes, and homo energy-transfer studies with fluorescently labeled peptides revealed that MV-197 adopts a predominant alpha-helical structure and shares properties similar to those reported for known fusion peptides. These results suggest that the presence of two fusion peptides in the F1 protein is a general feature of paramyxoviruses.     

Membrane transport: deciphering fusion

Membrane fusion events that occur in yeast have been reconstituted with a minimal set of SNARE protein components. This system has been exploited to establish the syntax underlying specificity of intracellular fusion events from yeast to mammals.     

Membrane fusion

Membrane fusion, one of the most fundamental processes in life, occurs when two separate lipid membranes merge into a single continuous bilayer. Fusion reactions share common features, but are catalyzed by diverse proteins. These proteins mediate the initial recognition of the membranes that are destined for fusion and pull the membranes close together to destabilize the lipid/water interface and to initiate mixing of the lipids. A single fusion protein may do everything or assemblies of protein complexes may be required for intracellular fusion reactions to guarantee rigorous regulation in space and time. Cellular fusion machines are adapted to fit the needs of different reactions but operate by similar principles in order to achieve merging of the bilayers.     

Membrane fusion

The process of membrane fusion in the case of lipid bilayers, as well as induced by influenza virus is reviewed shortly. The methods of studying fusion kinetics in pure lipid and lipid-protein systems are described. The main theories of molecular fusion machines are presented. Open questions and unsolved problems are discussed in details. In conclusion, possible ways to solve the remaining problems are suggested.     

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.     

Membrane topologies of neuronal SNARE folding intermediates

Assembly of the SNARE complex is essential for neurotransmitter release at synapses. Target plasma membrane SNAREs (t-SNAREs) syntaxin 1A and SNAP-25 form the t-SNARE complex that serves as an intermediate toward final SNARE assembly with vesicle-associated SNARE (v-SNARE). Membrane topologies of syntaxin 1A and the t-SNARE complex were investigated using site-directed spin labeling EPR. EPR analysis revealed that the basic region at the membrane-water interface is unstructured but inserted into the membrane. Such membrane insertion leaves no gap between the t-SNARE core and the membrane. Yet the lack of structure could provide the flexibility necessary for the t-SNARE core. Further, the insertion of the basic interfacial region into the membrane may have profound implications for the mechanism of SNARE-induced membrane fusion.     

Membrane trafficking: three steps to fusion

Membrane fusion involves the action of members of the SNARE protein family as well as Sec1/Munc18 (SM) proteins, which have been found to interact with SNAREs in three distinct ways. Recent work has established that Munc18-1 directly stimulates fusion and possibly uses all three modes of SNARE interaction.     

Membrane transport: Take your fusion partners

Recent studies of how vesicles are targeted to fuse with specific membranes inside cells highlight a role for extended coiled-coil proteins in tethering partner membranes prior to formation of the 'SNARE complex' that mediates the fusion reaction. The tethering protein is recruited to membranes by a Rab family GTPase     

Membrane fusion: caught in a trap

SNAREs are small coiled-coil proteins required for specific membrane fusion events in eukaryotic cells. Recent evidence points to the existence of an inhibitory class of SNAREs, i-SNAREs, which prevent incorrect fusions from occurring, adding a further layer of regulation to the process of membrane docking and fusion.     

Membrane fusion: structure snared at last

The structure of the core of the neuronal 'SNARE complex', involved in neurotransmitter release, has been determined recently. Its topological similarity to viral fusion proteins suggests how the SNARE complex might facilitate membrane fusion.     

Membrane fusion: stalk model revisited.

Membrane fusion is believed to proceed via intermediate structures called stalks. Mathematical analysis of the stalk provided the elastic energy involved in this structure and predicted the possible evolution of the overall process, but the energies predicted by the original model were suspiciously high. This was due to an erroneous assumption, i.e., that the stalk has a figure of revolution of a circular arc. Here we abandon this assumption and calculate the correct shape of the stalk. We find that it can be made completely stress free and, hence, its energy, instead of being positive and high can become negative, thus facilitating the fusion process. Based on our new calculations, the energies of hemifusion, of complete fusion, and of the pore in a bilayer were analyzed. Implications for membrane fusion and lipid phase transitions are discussed.     

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.     

Lipid bilayer vesicle fusion: intermediates captured by high-speed microfluorescence spectroscopy

The fusion of lipid bilayers can be visualized under the fluorescence microscope, but the process is very fast and requires special techniques for its study. It is reported here that vesicle fusion is susceptible to analysis by microspectrofluorometry and that for the first time, the entire fusion process has been captured. In the case of giant (>10- micro m diameter) bilayer vesicles having a high density of opposite charge, fusion proceeds through stages of adhesion, flattening, hemifusion, elimination of the intervening septum, and uptake of excess membrane to generate a spherical product very rapidly. These investigations became possible with a fluorescence microscope that was modified for recording of images simultaneously with the collection of fluorescence emission spectra from many (>100) positions along the fusion axis. Positively-charged vesicles, composed of O-ethylphosphatidylcholine and dioleoylphosphatidylcholine, were labeled with a carbocyanine fluorophore. Negatively-charged vesicles, composed of dioleoylphosphatidylglycerol and dioleoylphosphatidylcholine, were labeled with a rhodamine fluorophore that is a resonance energy transfer acceptor from the carbocyanine fluorophore. An electrophoretic chamber allowed selection of pairs of vesicles to be brought into contact and examined. Spectral changes along the axis of fusion were captured at high speed (a few ms/frame) by operating a sensitive digital camera in the virtual-chip mode, a software/hardware procedure that permits rapid readout of selected regions of interest and by pixel binning along the spectral direction. Simultaneously, color images were collected at video rates (30 frame/s). Comparison of the spectra and images revealed that vesicle fusion typically passes through a hemifusion stage and that the time from vesicle contact to fusion is <10 ms. Fluorescence spectra are well suited to rapid collection in the virtual-chip mode because spectra (in contrast to images) are accurately characterized with a relatively small number of points and interfering signals can be removed by judicious choice of barrier filters. The system should be especially well-suited to phenomena exhibiting rapid fluorescence change along an axis; under optimal conditions, it is possible to obtain sets of spectra (wavelength range of approximately 150 nm) at >100 positions along a line at rates >1000 frames/s with a spectral resolution of approximately 10 nm and spatial resolution at the limit of the light microscope ( approximately 0.2 micro m).     

HIV-1 fusion peptide decreases bending energy and promotes curved fusion intermediates

A crucial step in human immunodeficiency virus (HIV) infection is fusion between the viral envelope and the T-cell membrane, which must involve intermediate membrane states with high curvature. Our main result from diffuse x-ray scattering is that the bending modulus K(C) is greatly reduced upon addition of the HIV fusion peptide FP-23 to lipid bilayers. A smaller bending modulus reduces the free energy barriers required to achieve and pass through the highly curved intermediate states and thereby facilitates fusion and HIV infection. The reduction in K(C) is by a factor of 13 for the thicker, stiffer 1,2-sn-dierucoylphosphatidylcholine bilayers and by a factor of 3 for 1,2-sn-dioleoylphosphatidylcholine bilayers. The reduction in K(C) decays exponentially with concentration of FP-23, and the 1/e concentration is <1 mol % peptide/lipid, which is well within the physiological range for a fusion site. A secondary result is, when FP-23 is added to the samples which consist of stacks of membranes, that the distance between membranes increases and eventually becomes infinite at full hydration (unbinding); we attribute this both to electrostatic repulsion of the positively charged arginine in the FP-23 and to an increase in the repulsive fluctuation interaction brought about by the smaller K(C). Although this latter interaction works against membrane fusion, our results show that the energy that it requires of the fusion protein machinery to bring the HIV envelope membrane and the target T-cell membrane into close contact is negligible.