Discrimination between docking and fusion of liposomes reconstituted with neuronal SNARE-proteins using FCS (original) (raw)
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SNARE-Driven, 25-Millisecond Vesicle Fusion In Vitro
Biophysical Journal, 2005
Docking and fusion of single proteoliposomes reconstituted with full-length v-SNAREs (synaptobrevin) into planar lipid bilayers containing binary t-SNAREs (anchored syntaxin associated with SNAP25) was observed in real time by wide-field fluorescence microscopy. This enabled separate measurement of the docking rate k dock and the unimolecular fusion rate k fus. On low t-SNARE-density bilayers at 37°C, docking is efficient: k dock ¼ 2.2 3 10 7 M ÿ1 s ÿ1 , ;40% of the estimated diffusion limited rate. Full vesicle fusion is observed as a prompt increase in fluorescence intensity from labeled lipids, immediately followed by outward radial diffusion (D lipid ¼ 0.6 mm 2 s ÿ1); ;80% of the docked vesicles fuse promptly as a homogeneous subpopulation with k fus ¼ 40 6 15 s ÿ1 (t fus ¼ 25 ms). This is 10 3-10 4 times faster than previous in vitro fusion assays. Complete lipid mixing occurs in ,15 ms. Both the v-SNARE and the t-SNARE are necessary for efficient docking and fast fusion, but Ca 21 is not. Docking and fusion were quantitatively similar on syntaxin-only bilayers lacking SNAP25. At present, in vitro fusion driven by SNARE complexes alone remains ;40 times slower than the fastest, submillisecond presynaptic vesicle population response.
Determinants of liposome fusion mediated by synaptic SNARE proteins
Proceedings of the National Academy of Sciences, 2004
Preparation of Recombinant Proteins. The following constructs were described (all from rat): SNAP-25a (all cysteines replaced by serines; ref. 16), SNAP-25 C-terminal fragment (amino acid positions 120-206) (17), SNAP-25 N-terminal fragment (amino acids 1-180) (18), endobrevin N-terminal fragment (amino acids 1-76) (16), and tomosyn C-terminal fragment (amino acids 1051-1116) (19). Synaptobrevin 2 (amino acids 1-116; ref. 20) was subcloned into pET15 (Novagen) via the NdeI͞XhoI sites.
Single-Molecule Studies of the Neuronal SNARE Fusion Machinery
Annual Review of Biochemistry, 2009
SNAREs are essential components of the machinery for Ca2+-triggered fusion of synaptic vesicles with the plasma membrane, resulting in neurotransmitter release into the synaptic cleft. Although much is known about their biophysical and structural properties and their interactions with accessory proteins such as the Ca2+ sensor synaptotagmin, their precise role in membrane fusion remains an enigma. Ensemble studies of liposomes with reconstituted SNAREs have demonstrated that SNAREs and accessory proteins can trigger lipid mixing/fusion, but the inability to study individual fusion events has precluded molecular insights into the fusion process. Thus, this field is ripe for studies with single-molecule methodology. In this review, we discuss applications of single-molecule approaches to observe reconstituted SNAREs, their complexes, associated proteins, and their effect on biological membranes. Some of the findings are provocative, such as the possibility of parallel and antiparallel...
Proceedings of the National Academy of Sciences, 1999
Membrane bilayer fusion has been shown to be mediated by v-and t-SNAREs initially present in separate populations of liposomes and to occur with high efficiency at a physiologically meaningful rate. Lipid mixing was demonstrated to involve both the inner and the outer leaflets of the membrane bilayer. Here, we use a fusion assay that relies on duplex formation of oligonucleotides introduced in separate liposome populations and report that SNARE proteins suffice to mediate complete membrane fusion accompanied by mixing of luminal content. We also find that SNARE-mediated membrane fusion does not compromise the integrity of liposomes.
Single SNARE-Mediated Vesicle Fusion Observed In Vitro by Polarized TIRFM
Biophysical Journal, 2010
Single-vesicle fusion assays in vitro are useful tools for examining mechanisms of membrane fusion at the molecular level mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). This approach allows the experimentalist to define the lipid and protein composition of the two fusing membranes and perform experiments under highly controlled conditions. In previous experiments, in which we reconstituted a SNARE acceptor complex into supported membranes and observed the docking and fusion of fluorescently labeled synaptobrevin proteoliposomes by total internal reflection fluorescence microscopy with millisecond time resolution, we were able to determine the optimal number of SNARE complexes needed for fast fusion. Here, we utilize this assay in combination with polarized total internal reflection fluorescence microscopy to investigate topology changes that vesicles undergo after the onset of fusion. The theory that describes the fluorescence intensity during the transformation of a single vesicle from a spherical particle to a flat membrane patch is developed and confirmed by experiments with three different fluorescent probes. Our results show that on average, the fusing vesicles flatten and merge into the planar membrane within 8 ms after fusion starts.
Biophysical Journal, 2010
The influence of the lipid environment on docking and fusion of synaptobrevin 2 (Syb2) vesicles with target SNARE complex membranes was examined in a planar supported membrane fusion assay with high time-resolution. Previously, we showed that approximately eight SNARE complexes are required to fuse phosphatidylcholine (PC) and cholesterol model membranes in~20 ms. Here we present experiments, in which phosphatidylserine (PS) and phosphatidylethanolamine (PE) were added to mixtures of PC/cholesterol in different proportions in the Syb2 vesicle membranes only or in both the supported bilayers and the Syb2 vesicles. We found that PS and PE both reduce the probability of fusion and that this reduction is fully accounted for by the lipid composition in the vesicle membrane. However, the docking efficiency increases when the PE content in the vesicle (and target membrane) is increased from 0 to 30%. The fraction of fast-activating SNARE complexes decreases with increasing PE content. As few as three SNARE complexes are sufficient to support membrane fusion when at least 5% PS and 10% PE are present in both membranes or 5% and 30% PE are present in the vesicle membrane only. Despite the smaller number of required SNAREs, the SNARE activation and fusion rates are almost as fast as previously reported in reconstituted PC/cholesterol bilayers, i.e., of 10 and~20 ms, respectively.
Frontiers in molecular neuroscience, 2017
Synaptic vesicle membrane fusion, the process by which neurotransmitter gets released at the presynaptic membrane is mediated by a complex interplay between proteins and lipids. The realization that the lipid bilayer is not just a passive environment where other molecular players like SNARE proteins act, but is itself actively involved in the process, makes the development of biochemical and biophysical assays particularly challenging. We summarize in vitro assays that use planar supported membranes and fluorescence microscopy to address some of the open questions regarding the molecular mechanisms of SNARE-mediated membrane fusion. Most of the assays discussed in this mini-review were developed in our lab over the last 15 years. We emphasize the sample requirements that we found are important for the successful application of these methods.
Membrane Fusion Induced by Neuronal SNAREs Transits through Hemifusion
Journal of Biological Chemistry, 2005
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.
Rapid Fusion of Synaptic Vesicles with Reconstituted Target SNARE Membranes
Biophysical Journal, 2013
Neurotransmitter release at neuronal synapses occurs on a timescale of 1 ms or less. Reconstitution of vesicle fusion from purified synaptic proteins and lipids has played a major role in elucidating the synaptic exocytotic fusion machinery with ever increasing detail. However, one limitation of most reconstitution approaches has been the relatively slow rate of fusion that can be produced in these systems. In a related study, a notable exception is an approach measuring fusion of single reconstituted vesicles bearing the vesicle fusion protein synaptobrevin with supported planar membranes harboring the presynaptic plasma membrane proteins syntaxin and SNAP-25. Fusion times of~20 ms were achieved in this system. Despite this advance, an important question with reconstituted systems is how well they mimic physiological systems they are supposed to reproduce. In this work, we demonstrate that purified synaptic vesicles from rat brain fuse with acceptor-SNARE containing planar bilayers equally fast as equivalent reconstituted vesicles and that their fusion efficiency is increased by divalent cations. Calcium boosts fusion through a combined general electrostatic and synaptotagmin-specific mechanism.
SNARE-Mediated Lipid Mixing Depends on the Physical State of the Vesicles
Biophysical Journal, 2006
Reconstitution experiments have suggested that N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins constitute a minimal membrane fusion machinery but have yielded contradictory results, and it is unclear whether the mechanism of membrane merger is related to the stalk mechanism that underlies physiological membrane fusion. Here we show that reconstitution of solubilized neuronal SNAREs into preformed 100 nm liposomes (direct method) yields proteoliposomes with more homogeneous sizes and protein densities than the standard reconstitution method involving detergent cosolubilization of proteins and lipids. Standard reconstitutions yield slow but efficient lipid mixing at high protein densities and variable amounts of lipid mixing at moderate protein densities. However, the larger, more homogenous proteoliposomes prepared by the direct method yield almost no lipid mixing at moderate protein densities. These results suggest that the lipid mixing observed for standard reconstitutions is dominated by the physical state of the membrane, perhaps due to populations of small vesicles (or micelles) with high protein densities and curvature stress created upon reconstitution. Accordingly, changing membrane spontaneous curvature by adding lysophospholipids inhibits the lipid mixing observed for standard reconstitutions. Our data indicate that the lipid mixing caused by high SNARE densities and/or curvature stress occurs by a stalk mechanism resembling the mechanism of fusion between biological membranes, but the neuronal SNAREs are largely unable to induce lipid mixing at physiological protein densities and limited curvature stress.