The Ca2+-sensor synaptotagmin-1 that triggers neuronal exocytosis binds to negatively charged membrane lipids (mainly phosphatidylserine (PtdSer) and phosphoinositides (PtdIns)) but the molecular details of this process are not fully understood. Using quantitative thermodynamic, kinetic and structural methods, we show that synaptotagmin-1 (from Rattus norvegicus and expressed in Escherichia coli) binds to PtdIns(4,5)P2 via a polybasic lysine patch in the C2B domain, which may promote the priming or docking of synaptic vesicles. Ca2+ neutralizes the negative charges of the Ca2+-binding sites, resulting in the penetration of synaptotagmin-1 into the membrane, via binding of PtdSer, and an increase in the affinity of the polybasic lysine patch to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). These Ca2+-induced events decrease the dissociation rate of synaptotagmin-1 membrane binding while the association rate remains unchanged. We conclude that both membrane penetration and the increased residence time of synaptotagmin-1 at the plasma membrane are crucial for triggering exocytotic membrane fusion.DOI: http://dx.doi.org/10.7554/eLife.15886.001

In dendritic cells, the NADPH oxidase 2 complex (NOX2) is recruited to the phagosomal membrane during antigen uptake. NOX2 produces reactive oxygen species (ROS) in the lumen of the phagosome that kill ingested pathogens, delay antigen breakdown and alter the peptide repertoire for presentation to T cells. How the integral membrane component of NOX2, cytochrome b558 (which comprises CYBB and CYBA), traffics to phagosomes is incompletely understood. In this study, we show in dendritic cells derived from human blood-isolated monocytes that cytochrome b558 is initially recruited to the phagosome from the plasma membrane during phagosome formation. Cytochrome b558 also traffics from a lysosomal pool to phagosomes and this is required to replenish oxidatively damaged NOX2. We identified syntaxin-7, SNAP23 and VAMP8 as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediating this process. Our data describe a key mechanism of how dendritic cells sustain ROS production after antigen uptake that is required to initiate T cell responses.

Legionella pneumophila, the Gram-negative pathogen causing Legionnaires’ disease, infects host cells by hijacking endocytic pathways and forming a Legionella-containing vacuole (LCV) in which the bacteria replicate. To promote LCV expansion and prevent lysosomal targeting, effector proteins are translocated into the host cell where they alter membrane traffic. Here we show that three of these effectors [LegC2 (Legionella eukaryotic-like gene C2)/YlfB (yeast lethal factor B), LegC3, and LegC7/YlfA] functionally mimic glutamine (Q)-SNARE proteins. In infected cells, the three proteins selectively form complexes with the endosomal arginine (R)-SNARE vesicle-associated membrane protein 4 (VAMP4). When reconstituted in proteoliposomes, these proteins avidly fuse with liposomes containing VAMP4, resulting in a stable complex with properties resembling canonical SNARE complexes. Intriguingly, however, the LegC/SNARE hybrid complex cannot be disassembled by N-ethylmaleimide-sensitive factor. We conclude that LegCs use SNARE mimicry to divert VAMP4-containing vesicles for fusion with the LCV, thus promoting its expansion. In addition, the LegC/VAMP4 complex avoids the host’s disassembly machinery, thus effectively trapping VAMP4 in an inactive state.

The large gap in time scales between membrane fusion occurring in biological systems during neurotransmitter release and fusion observed between model membranes has provoked speculations over a large number of possible factors that might explain this discrepancy. One possible reason is an elevated lateral membrane tension present in the presynaptic membrane. We investigated the tension-dependency of fusion using model membranes equipped with a minimal fusion machinery consisting of syntaxin 1, synaptobrevin and SNAP 25. Two different strategies were realized; one based on supported bilayers and the other one employing sessile giant liposomes. In the first approach, isolated patches of planar bilayers derived from giant unilamellar vesicles containing syntaxin 1 and preassembled SNAP 25 (ΔN-complex) were deposited on a dilatable PDMS sheet. In a second approach, lateral membrane tension was controlled through the adhesion of intact giant unilamellar vesicles on a functionalized surface. In both approaches fusion efficiency increases considerably with lateral tension and we identified a threshold tension of 3.4 mN m−1, at which the number of fusion events is increased substantially.

Fusion of lipid bilayers is usually prevented by large energy barriers arising from removal of the hydration shell, formation of highly curved structures, and, eventually, fusion pore widening. Here, we measured the force-dependent lifetime of fusion intermediates using membrane-coated silica spheres attached to cantilevers of an atomicforce microscope. Analysis of time traces obtained from force-clamp experiments allowed us to unequivocally assign steps in deflection of the cantilever to membrane states during the SNARE-mediated fusion with solid-supported lipid bilayers. Force-dependent lifetime distributions of the various intermediate fusion states allowed us to propose the likelihood of different fusion pathways and to assess the main free energy barrier, which was found to be related to passing of the hydration barrier and splaying of lipids to eventually enter either the fully fused state or a long-lived hemifusion intermediate. The results were compared with SNARE mutants that arrest adjacent bilayers in the docked state and membranes in the absence of SNAREs but presence of PEG or calcium. Only with the WT SNARE construct was appreciable merging of both bilayers observed.embrane fusion plays a pivotal role in many fundamental biological processes, comprising viral infection, cell-cell fusion during fertilization, tissue formation, and intracellular transport during exo-and endocytosis (1). Among the best-studied biological examples is the Ca 2+ -regulated fusion of synaptic vesicles with the presynaptic plasma membrane in neurons and chromaffin cells (2, 3). Fusion in these cells is catalyzed by concerted action of SNARE proteins through formation of a tetrameric coiled-coil complex that releases a sufficient amount of free energy to lower the barriers for membrane merging. One major energy barrier is associated with the strong repulsive hydration forces when two smooth bilayers approach each other. Other energy-costly contributions originate from lipid splaying as the initiation of stalk formation, expansion of the stalk structure driven by the strong curvature associated with membrane destabilization, hemifusion diaphragm expansion, and, finally, pore formation (4-8). The free energy associated with complex formation and folding of SNAREs was reported to be ∼35 k B T, close to the energy required to create the highly curved transition structures (40-50 k B T) (9, 10). Albeit a part of this free energy might be dissipated as heat, this coincidence of matching energy is also supported by experimental studies showing that only very few SNARE complexes are sufficient to induce fusion in artificial systems (5,6,(11)(12)(13). Although precise data for thermodynamics and kinetics of SNARE zippering and unzippering are now available (11,14,15), few studies address how the energy landscape of membrane fusion is shaped by participation of SNAREs. were among the first to measure fusion events in the absence of proteins as a function of external force using a surface force apparatus, and Moy and coworkers (19) m...