Hormones and neurotransmitters are released through fluctuating exocytotic fusion pores that can flicker open and shut multiple times. Cargo release and vesicle recycling depend on the fate of the pore, which may reseal or dilate irreversibly. Pore nucleation requires zippering between vesicle-associated v-SNAREs and target membrane t-SNAREs, but the mechanisms governing the subsequent pore dilation are not understood. Here, we probed the dilation of single fusion pores using v-SNARE-reconstituted ~23-nm-diameter discoidal nanolipoprotein particles (vNLPs) as fusion partners with cells ectopically expressing cognate, 'flipped' t-SNAREs. Pore nucleation required a minimum of two v-SNAREs per NLP face, and further increases in v-SNARE copy numbers did not affect nucleation rate. By contrast, the probability of pore dilation increased with increasing v-SNARE copies and was far from saturating at 15 v-SNARE copies per face, the NLP capacity. Our experimental and computational results suggest that SNARE availability may be pivotal in determining whether neurotransmitters or hormones are released through a transient ('kiss and run') or an irreversibly dilating pore (full fusion).DOI: http://dx.doi.org/10.7554/eLife.22964.001

The initial, nanometer-sized connection between the plasma membrane and a hormone- or neurotransmitter-filled vesicle –the fusion pore– can flicker open and closed repeatedly before dilating or resealing irreversibly. Pore dynamics determine release and vesicle recycling kinetics, but pore properties are poorly known because biochemically defined single-pore assays are lacking. We isolated single flickering pores connecting v-SNARE-reconstituted nanodiscs to cells ectopically expressing cognate, “flipped” t-SNAREs. Conductance through single, voltage-clamped fusion pores directly reported sub-millisecond pore dynamics. Pore currents fluctuated, transiently returned to baseline multiple times, and disappeared ~6 s after initial opening, as if the fusion pore fluctuated in size, flickered, and resealed. We found that interactions between v- and t-SNARE transmembrane domains (TMDs) promote, but are not essential for pore nucleation. Surprisingly, TMD modifications designed to disrupt v- and t-SNARE TMD zippering prolonged pore lifetimes dramatically. We propose that the post-fusion geometry of the proteins contribute to pore stability.

In neurotransmission synaptotagmin-1 tethers synaptic vesicles to the presynaptic plasma membrane by binding to acidic membrane lipids and SNAREs and promotes rapid SNARE-mediated fusion upon Ca 2+ triggering. However, recent studies suggested that upon membrane contact synaptotagmin may not only bind in trans to the target membrane but also in cis to its own membrane. Using a sensitive membrane tethering assay we have now dissected the structural requirements and concentration ranges for Ca 2+ -dependent and -independent cis-binding and trans-tethering in the presence and absence of acidic phospholipids and SNAREs. Using variants of membrane-anchored synaptotagmin in which the Ca 2+ -binding sites in the C2 domains and a basic cluster involved in membrane binding were disrupted we show that Ca 2+ -dependent cis-binding prevents trans-interactions if the cis-membrane contains 12-20% anionic phospholipids. Similarly, no trans-interactions were observable using soluble C2AB-domain fragments at comparable concentrations. At saturating concentrations, however, tethering was observed with soluble C2AB domains, probably due to crowding on the vesicle surface and competition for binding sites. We conclude that trans-interactions of synaptotagmin considered to be essential for its function are controlled by a delicate balance between cis-and trans-binding, which may play an important modulatory role in synaptic transmission. . This increase is sensed by synaptotagmin-1, a 65-kDa protein anchored to synaptic vesicles (1, 2). Synaptotagmin-1 then triggers fusion of the synaptic vesicles with the plasma membrane resulting in release of neurotransmitter. Fusion itself is mediated by the vesicular R-SNARE synaptobrevin-2 and the plasma membrane Q-SNAREs SNAP-25 and syntaxin-1A. These SNAREs assemble in trans between the membranes and form a tight coiled-coil complex which overcomes the energy barrier of membrane fusion. Synaptotagmin-1 consists of an N-terminal transmembrane helix connected by a long (61-residue) unstructured linker to two C2 domains, called C2A and C2B. The C2A and C2B domains bind three and two Ca 2+ ions, respectively (3, 4). They also bind to both individual Q-SNAREs and assembled SNARE complexes (1, 5-7) and to anionic membranes (3,(8)(9)(10)(11)(12)(13)(14)(15)(16)). Both of these interactions are modulated by Ca 2+ and have been implicated in the mechanism of synaptotagmin-1 action (1, 2). In addition, synaptotagmin-1 possesses a polybasic stretch in the C2B domain that is structurally separated from the calcium-binding domain and that mediates calcium-independent binding to acidic phospholipids, particularly phosphatidylinositol-4,5-bisphosphate (PiP 2 ) (8, 9, 15-17).Despite intense research over the past two decades, it is still unclear by which molecular mechanism synaptotagmin-1 is capable of accelerating exocytosis by more than four orders of magnitude (18). Two types of models are presently discussed that are not necessarily exclusive. The first proposes a direct action of synaptotagmin-1 on th...

Background: Soluble N-ethylmaleimide-sensitive factor attachment protein ␣ (␣-SNAP) regulates the pre-fusion step as well as SNARE disassembly. Results: ␣-SNAP on its own interferes with SNARE zippering and inhibits chromaffin granule fusion, but not synaptic vesicle fusion. Conclusion: Retardation of SNARE zippering by ␣-SNAP results in the partial SNARE zippering. Significance: This is the first direct evidence showing the partial SNARE zippering in the physiological context.

Exocytosis underlies release of neurotransmitters and hormones. Electrophysiological and electrochemical measurements from live cells have shown that the initial fusion pore is small (~1 nm diameter) and can repeatedly flicker before either dilating fully, or closing permanently. The fraction of flickering pores and flicker characteristics vary with stimulation strength, regulating the

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The AP1 complex, which is one of four structurally related protein complexes, forms a bridge between the clathrin coat and membrane components (cargo) during the formation of clathrincoated vesicles at the trans-Golgi network.is greater than 100. Most of the evidence is confined to qualitative approaches such as pull-downs and coimmunoprecipitations, which are notorious for yielding false positives with 'sticky' proteins. Quantitative and/or structural data about these presumed SNARE-targetprotein complexes are therefore largely missing. To validate these interactions, detailed structural information and a rigorous assessment of their in vivo relevance are required.Plasma-membrane SNAREs are not uniformly distributed in the membrane, but are clustered in nano domains, the stability of which depends on cholesterol [51][52][53] . The homomeric association of SNARE transmembrane domains has been reported, and this might contribute to cluster formation 54 . Secretory vesicles selectively dock and fuse at such clusters 51 . It remains to be seen whether cluster formation is a hallmark of all SNAREs or is a speciality of plasma membranes and other membranes that are rich in steroid lipids. Clustering achieves high local SNARE concentrations that might result in more efficient fusion. Acceptor complexes: intermediates in the fusion pathway?How does the assembly of four unstructured SNAREs into a SNARE complex proceed? Detailed studies on exocytic S. cerevisiae and neuronal SNARE complexes in vitro have shown that assembly proceeds through a defined and partially helical Qabc intermediate [55][56][57] , the formation of which is rate limiting. Although it is not yet known whether other SNAREs form such intermediate acceptor complexes, it is probable that they represent a key step in the fusion pathway of all SNAREs -that is, it is likely that assembly is an ordered, sequential reaction rather than a random collision of four different SNARE motifs. Only when an acceptor scaffold is available in which the N-terminal ends of the SNARE motifs are structured is the final SNARE able to bind with biologically relevant kinetics and nucleate the zippering reaction.Acceptor complexes are highly reactive and are therefore difficult to characterize. For example, in vitro, the neuronal acceptor complex readily recruits a second Qa-SNARE, which results in a 'dead-end' Qaabc complex.

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