Marta K. Domanska

Single vesicle millisecond fusion kinetics reveals number of SNARE complexes optimal for fast SNARE-mediated membrane fusion.

SNAREs mediate membrane fusion in intracellular vesicle traffic and neuronal exocytosis. Reconstitution of membrane fusion in vitro proved that SNAREs constitute the minimal fusion machinery. However, the slow fusion rates observed in these systems are incompatible with those required in neurotransmission. Here we present a single vesicle fusion assay that records individual SNARE-mediated fusion events with millisecond time resolution. Docking and fusion of reconstituted synaptobrevin vesicles to target SNARE complex-containing planar membranes are distinguished by total internal reflection fluorescence microscopy as separate events. Docking and fusion are SNAP-25-dependent, require no Ca2+, and are efficient at room temperature. Analysis of the stochastic data with sequential and parallel multi-particle activation models reveals six to nine fast-activating steps. Of all the tested models, the kinetic model consisting of eight parallel reaction rates statistically fits the data best. This might be interpreted by fusion sites consisting of eight SNARE complexes that each activate in a single rate-limiting step in 8 ms.

Docking and Fast Fusion of Synaptobrevin Vesicles Depends on the Lipid Compositions of the Vesicle and the Acceptor SNARE Complex-Containing Target Membrane

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., ~10 and ~20 ms, respectively [corrected].

Rapid fusion of synaptic vesicles with reconstituted target SNARE membranes.

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.

Multiphasic effects of cholesterol on influenza fusion kinetics reflect multiple mechanistic roles.

The envelope lipid composition of influenza virus differs from that of the cellular plasma membrane from which it buds. Viruses also appear to fuse preferentially to specific membrane compartments, suggesting that the lipid environment may influence permissiveness for fusion. Here, we investigated the influence of the membrane environment on fusion, focusing on cholesterol composition. Strikingly, manipulating cholesterol levels in the viral membrane had different effects on fusion kinetics compared with analogous changes to the target membrane. Increasing cholesterol content in target vesicles increased lipid- and contents-mixing rates. Moderate cholesterol depletion from the viral membrane sped fusion rates, whereas severe depletion slowed the process. The pleiotropic effects of cholesterol include alterations in both membrane-bending moduli and lateral organization. Because influenza virions have demonstrated cholesterol-dependent lateral organization, to separate these effects, we deliberately selected a target vesicle composition that does not support lateral heterogeneity. We therefore postulate that the monotonic response of fusion kinetics to target membrane cholesterol reflects bending and curvature effects, whereas the multiphasic response to viral cholesterol levels reflects the combined effects of lateral organization and material properties.

Fast Topology Changes During SNARE-Mediated Vesicle Fusion Observed in Supported Membranes by Polarized Tirfm

In vitro reconstitution experiments have played an essential role in a large body of research on SNARE-mediated membrane fusion. Recently, new single vesicle assays have been developed to gain more detailed insight into the kinetics of vesicle docking and fusion. Previously, we used supported membranes in combination with total internal reflection microscopy (TIRFM) to record the docking and fusion of synaptobrevin containing vesicles with 4 ms time resolution. Depending on the lipid conditions, we found that between 3 and 8 SNARE complexes are needed for fast fusion in this system. Here we utilize polarized TIRFM to investigate topology changes that the docked 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 the three fluorescent probes Rh-DOPE, NBD-DPPE and BODIPY-PC. Our results show that, on average, the fusing vesicles flatten and merge into the planar membrane within 8 ms after fusion starts.

Fast Single Vesicle SNARE-Mediated Membrane Fusion Assay in Planar Supported Bilayers Reveals Details About Fusion Mechanism

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane fusion in neuronal exocytosis and intracellular membrane trafficking. It is widely accepted that the zippering interaction between syntaxin1a, SNAP25 and synaptobrevin2 brings plasma- and vesicle membrane together and leads to their merger. Membrane fusion has been extensively studied by liposome and single vesicle-planar membrane fusion assays. However, both approaches have been criticized because of slow fusion rates incompatible with cellular rates or lack of physiological specificity, respectively.Here we present a fluorescence-basedsingle vesicle-planar bilayer fusion assay with millisecond time resolution and an improved reconstitution procedure that preserves the native topology and mobility of the SNAREs. The acceptor-SNARE complex, composed of syntaxin1a (SyxH3), SNAP25 and a short synaptobrevin2 peptide (Syb49-96), was reconstituted into planar bilayers by a combined Langmuir-Blodgett - vesicle fusion technique. Docking and fusion of single Rh-DOPE labeled Syb vesicles to the supported acceptor-SNARE membranes were observed by total internal reflection fluorescence microscopy. No docking or fusion was observed in protein-free control membranes. Vesicle SNARE docking was dependent on the acceptor-SNARE complex density in the membranes. Moreover, docking was SNAP25-dependent, and subsequent fusion did not require Ca2+ and was efficient at ambient temperature. A detailed kinetic analysis of >1000 single fusion events revealed that each fusion reaction consists of 6 to 9 activating steps with 8 steps fitting the data best. This could be interpreted by fusion sites consisting of 8 SNARE complexes that each activate in a single rate-limiting step in 8 ms. We find that different lipid compositions of the supported and the vesicle membrane have a relatively minor influence on docking, but modulate the fusion efficiency and fusion kinetics more dramatically.Supported by NIH grant GM072694