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 rate-limiting step in 8 ms.
Highlights
We present a single vesicle-supported membrane fusion assay based on lipid mixing with millisecond time resolution that reveals the kinetics of SNAP receptors (SNAREs)-mediated membrane fusion
In supplemental Movies S1 and S2, we show by single particle tracking that the stabilized 1:1:1 acceptor SNARE complexes used in the current study are laterally mobile in POPC bilayers without and with cholesterol
Numerous previous studies established that SNAREs constitute the minimal machinery required for intracellular membrane fusion, an important caveat concerned the speed of fusion, which, at least in synaptic exocytosis, is 5 orders of magnitude faster in cells than in even the fastest reconstituted biochemical systems
Summary
Protein Expression and Purification—SNARE proteins from Rattus norvegicus cloned in pET28a vector were expressed in BL21(DE3) Escherichia coli and purified as described previously [9, 17]. SNARE Reconstitution into Proteoliposomes—Single SNAREs and binary and ternary acceptor SNARE complexes were reconstituted into vesicles by rapid dilution of micellar protein/ lipid/detergent mixtures followed by dialysis as described previously [18]. Single Vesicle Docking and Fusion Assay—Supported bilayers containing acceptor SNARE complex (or other proteins as described) were perfused with 3 ml of 0.6 M Syb116 vesicles containing 1 mol % lissamine-rhodamine-B-dioleoylphosphatidylcholine (Rh-DOPE) mixed with 3.3 M protein-free vesicles in fusion buffer on the microscope stage (concentration refers to total lipid). Three to five such series were usually collected from each supported membrane preparation This fast image acquisition period was followed by ϳ30 min of single image acquisitions every 30 s with 20-ms exposure times to measure additional vesicle docking in bulk mode. Where D∞ is the total concentration of active docking sites, and kon is the docking rate
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