Pictures of the prologue to neurotransmitter release

Membrane fusion and fission determine compartmentalization in eukaryotic cells and uptake or secretion of various molecular species. For example, a very rapid, tightly regulated, Ca²⁺-triggered fusion event releases neurotransmitters into the synaptic cleft (1). A long history of identifying and characterizing its essential components has allowed in vitro reconstitution, as modeled by fusion of two liposomes: one bearing the components of the fusion machinery associated with a synaptic vesicle and the other, those associated with the plasma membrane (2). Gipson et al., in a paper in PNAS entitled “Morphologies of synaptic protein membrane fusion interfaces,” visualize steps in the fusion process directly, by applying recent advances in electron cryotomography (cryo-ET) (3). The authors find that productive fusion proceeds through point contacts between membranes created by a small number (probably just two) of fusion-protein complexes.

Fusion of Lipid Bilayers

Lipid bilayers repel each other through a so-called “hydration force” when they approach each other more closely than about 15–20 Å (4). To overcome this barrier, fusion requires a counteracting force, either from a bulk effect, such as pressure by polymer exclusion, or from a localized fusion facilitator, such as the presynaptic fusion complexes Gipson et al. (3) describe. Computational studies have suggested that formation of a localized contact would minimize the hydration force by minimizing the area of closely apposed membranes, and hence reduce the kinetic barrier, leading to a “hemifusion stalk” as a likely fusion intermediate (5). The cryo-ET images of Gipson et al. (3) are in accord with those proposals.

The fusogens for synaptic-vesicle fusion are the “neuronal SNAREs”: synaptobrevin-2 (also known as VAMP2), anchored in the membrane of the synaptic vesicle; syntaxin-1, anchored in the plasma membrane; and SNAP-25, associated with the plasma membrane by palmitoylated cysteine residues. The Ca²⁺ sensor, synaptotagmin-1, anchored in the vesicle, completes a minimal, Ca²⁺-triggered fusion system, but addition of two other regulatory factors (complexin and Munc13) change both the speed and yield of the response and, as shown in by Gipson et al. (3), the range of morphologies present.

Like medical tomography, cryo-ET reconstitutes a 3D image of a single entity from a large number of projected views, related by tilting about a fixed axis (6). (In medical tomography, the X-ray beam rotates about the subject; in EM tomography, the stage bearing the sample tilts in a fixed beam.) Because electron damage severely limits the exposure, each tilt image is very noisy, and the achievable resolution is substantially more modest than can be reached with very large numbers of identical molecular objects. One way to enhance the signal-to-noise in cryo-ET is to use phase-contrast imaging, now made relatively practical by the Volta phase plates that Danev et al. [coauthors of the Gipson et al. (3) paper] have shown to be effective (7, 8). Biological samples are essentially pure phase objects; at perfect focus there is no detectable image. In the absence of a phase plate—the electron-beam equivalent of a phase plate for an optical phase-contrast microscope—the standard method for obtaining an interpretable image is careful underfocusing, with subsequent computational correction to restore uniform contrast. The phase plate retains important information lost by the underfocusing approach, while avoiding potential computational problems that result from variation in defocus across a titled sample.

Gipson et al. (3) describe the contacts they see as “point contacts”—with a thin bridge of density (interpreted as the assembled protein fusion machinery) between the liposomes bearing vesicle proteins (synaptobrevin-2 and synaptotagmin) and those bearing plasma-membrane proteins (syntaxin-1 and SNAP-25)—and “long contacts,” with a laterally more extended region of density between the two liposomes. Addition of both regulatory proteins restricted the contacts largely to the former class and a spacing of ∼100 Å between the centers of the two bilayers (i.e., >50 Å between their apposed surfaces). Moreover, after addition to Ca²⁺, which induces fusion on a millisecond timescale when these proteins are present, nearly all of the point contacts had disappeared, leaving only a few long contacts and “hemifusion diaphragms,” both apparently nonproductive states. Gipson et al. interpret these results to mean that the point contacts represent the physiologically functional state and that complexin and Munc13 ensure fidelity and yield, by modulating SNARE-complex geometry and conformational change. Moreover, having found good evidence that the point contacts are productive intermediates, the authors estimate, from the volume of the density bridge and the total mass of the components, that two complete, Ca²⁺-triggered complexes are probably sufficient to generate a productive fusion event.

SNAREs and Viral Fusion Proteins

When the Ca²⁺ concentration reaches a threshold, the transition from the point contacts seen by Gipson et al. (3) to opening of a fusion pore takes a few milliseconds (2). The fusion of viral membranes with a membrane of a host cell—essential for entry and infection of enveloped viruses—has molecular features very similar to those outlined for the SNARE-mediated process, but the delay time from triggering to fusion is substantially longer. The reason for this difference is instructive.

In both viral and SNARE-mediated fusion, the molecular transition that forces the apposed membrane across the hydration-force barrier is a zippering-like rearrangement of the fusion complex that brings together parts anchored in one membrane (e.g., the synaptobrevin transmembrane anchor) with parts anchored in the other (e.g., the syntaxin transmembrane anchor and the SNAP-25 palmitoyl chains) (9, 10). In the case of the SNAREs, the zippering results in a four-strand, α-helical coiled-coil, with the transmembrane anchors together at one end (11). Structural and kinetic studies have shown that complexin and synaptotagmin trap the complex in a partly zippered configuration and that release of this restraint when synaptotagmin binds Ca²⁺ produces very rapid fusion (2, 12). The direct cryo-ET visualization now accomplished shows that when both complexin and Munc13 are present, the correct number of primed complexes are preassembled about a point contact and that no slow molecular steps intervene between Ca²⁺ triggering and complete zippering. In the point contacts described by Gipson et al. (3), the two lipid bilayers are farther apart than the critical hydration-force separation; free energy released by completing the formation of the SNARE coiled-coils is evidently enough to drive local extrusions of the two membranes across that barrier.

In the best-studied examples of viral membrane fusion, the active complex is not preassembled. The triggering event in those examples is proton binding (reduced pH in an endosome), but several triggered fusion proteins must come together to generate a fusion event. Single-particle kinetic analysis of three different enveloped viruses has shown that when exposed to low pH, each of the relatively large number of fusion proteins in the contact zone between virus and target membrane can undergo a conformational change that inserts a hydrophobic “fusion peptide” or “fusion loop” into the target bilayer (13–16). Each such transition creates a molecular bridge between the two membranes, functionally analogous to the partially zippered SNARE complex trapped by complexin and syaptotagmin. Although the transition for a single fusion protein—such as influenza virus hemagglutinin—can occur on a time scale of seconds or less, contraction of the bridge, by the zippering that induces fusion, cannot ensue until there is a critical number of bridging complexes (between two and four, for different viruses) around a single point. The same number of bridges, but with not-yet-extended, nonbridging fusion proteins between them, is insufficient. As long as the largest number of adjacent bridging proteins in the contact zone is below the requirement for drawing the membranes together, the resistance of the membranes to deformation and the hydration-force barrier prevent the transition from proceeding further. When that critical number of adjacent bridges has been reached, anywhere in the contact zone, their zippering (coordinated by their common insertion in the two membranes) liberates enough free energy to overcome the resistance, and fusion proceeds rapidly. The delay of tens of seconds that intervenes between proton binding and hemifusion-stalk formation depends on the probability that the critical number of neighboring bridges will have formed by any given time.

Cryo-ET images of influenza virus fusing with liposomes show intermediate structures that conform to the above description (17), which derives from studies of single-particle fusion kinetics (13). Less than a minute after lowering the pH of the virus-liposome mixture, there are thin, straight bridges across a gap of ∼100 Å between virus and liposome. In many images, the lateral extent of the contact zone includes several such bridges, but at later time points, close approach or continuity of the two bilayers, in the form of either a fusion pore or a putative hemifusion stalk, is always at a localized point contact. As in studies of SNARE-mediated fusion, direct visualization by cryo-ET has therefore substantiated and extended the less direct inferences from single-molecule kinetics and high-resolution structures of the protein machinery. Visualizing the rapid transition from a Ca²⁺ triggered complex (or a critical accumulation of bridging viral-protein intermediates) to a hemifusion stalk—the event that all these proteins “catalyze”—is clearly a formidable next challenge.