Single Vesicle Millisecond Fusion Kinetics Reveals Number of SNARE Complexes Optimal for Fast SNARE-mediated Membrane Fusion

Abstract

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 Ca 2؉ , 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 ratelimiting step in 8 ms.Neurotransmitter release in synaptic transmission by fusion of synaptic vesicles with the presynaptic membrane is tightly regulated and is probably the fastest membrane fusion event in mammalian cells. Synaptic vesicles are primed and docked to the plasma membrane but do not fuse until triggered by an influx of Ca 2ϩ from opened Ca 2ϩ channels. After electrical stimulation, neurotransmitter release is observed in less than 1 ms (1-3). The neuronal fusion and disassembly machinery is composed of the soluble N-ethylmaleimide-sensitive factor, N-ethylmaleimide-sensitive factor attachment proteins (SNAPs), 2 and the SNAP receptors (SNAREs) syntaxin1a (Syx1a), SNAP-25, and synaptobrevin2 (Syb). Proteins such as the Ca 2ϩ sensor synaptotagmin, complexin, Sec1/Munc18 homologs, Munc13, and synaptophysin are involved in regulating the fusion process, and Rab GTPases function as upstream tethering factors (4 -6). SNARE proteins, which assemble during fusion with equimolar stoichiometry into a parallel four-helix coiled-coil structure with their C termini oriented toward their respective membranes (7-9), play the most essential role in this machinery (10 -12). Energy released from a proposed N 3 C folding process pulls the two membranes together, deforms them, and eventually fuses them in a process that is mechanistically still poorly understood. SNARE-mediated fusion between target (t)-SNARE (Syx1a and SNAP-25) and vesicle (v)-SNARE (Syb) liposomes has been reconstituted in vitro (12). This and many subsequent similar studies were initially criticized because the reaction was very slow (minutes to hours). Adding a C-terminal fragment (residues 49 -96) of Syb to the Syx1a/SNAP-25 heterodimer, resulting in a ternary acceptor-SNARE complex, increased the rate of fusion with Syb liposomes by more than an order of magnitude, presumably by preventing the formation of a nonproductive 2:1 Syx1a⅐SNAP-25 complex (13)...

Assembly and Comparison of Plasma Membrane SNARE Acceptor Complexes

“…To separate the docking and fusion steps of the overall fusion reaction, we reconstituted the different SNARE acceptor complexes into planar-supported bilayers. As expected from previous studies, the DN complex showed a much higher docking efficiency than the syntaxin:SNAP-25(CHAPS) complex (4,5), while the syntaxin:SNAP-25(DPC) complex purified in DPC docked synaptobrevin-2 vesicles docked almost as efficiently as the DN complex (Fig. 1 b; Fig.…”

“…1 d). As described in Domanska et al (5), the initial fast component of the fusion kinetics was examined and fit with a parallel-activation mixed fusion-site model. Similar numbers of activation steps (~7 5 1) were found for each complex, and the fast rates of fusion ranged between 60 and 150 s À1 for all investigated complexes (5).…”

“…Traditionally, syntaxin-1a is purified in bOG (octyl-b-D-glucoside) (2,(11)(12)(13)(14) or sodium cholate (3,4,9), and allowed to assemble into an acceptor complex with soluble SNAP-25a during coexpression in Escherichia coli or postexpression in cholate, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), or bOG. Depending on input stoichiometries and other conditions, this results, to different degrees, in the formation of a 2:1 syntaxin-1a:SNAP-25a complex that does not readily bind to synaptobrevin-2 (4,5). We found that the oligomeric state of syntaxin-1a is dependent on the purification and assembly detergent and that an exclusively monomeric syntaxin-1a can be produced in DPC (dodecylphosphocholine) (15).…”

“…Full-length synaptobrevin-2 readily binds this complex, replacing the shorter peptide. Upon using this acceptor complex in single liposome and single vesicle assays, average fusion times of 20 ms and faster have been observed (5,6).…”

Variable cooperativity in SNARE-mediated membrane fusion

“…Although some studies have left open the possibility that the number of SNARE complexes that cooperate during fusion is variable (11,12), much attention has been given to the notion of a preferred number of SNARE complexes, with estimates varying from a single SNARE complex (13) to 15 (14), although more recent estimates vary between two and eight (12,(15)(16)(17)(18). Unfortunately, this large disparity in results has not been appropriately explained, and it remains unclear whether the differences are a result of inherent properties of the particular set of SNAREs involved or rather originate from the biophysical characteristics of the fusing vesicles.…”