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. 2011 May 4;100(9):2141-50.
doi: 10.1016/j.bpj.2011.03.015.

Docking, not fusion, as the rate-limiting step in a SNARE-driven vesicle fusion assay

Elizabeth A Smith  1 , James C Weisshaar
Affiliations

Docking, not fusion, as the rate-limiting step in a SNARE-driven vesicle fusion assay

Elizabeth A Smith et al. Biophys J. .

Abstract

In vitro vesicle fusion assays that monitor lipid mixing between t-SNARE and v-SNARE vesicles in bulk solution exhibit remarkably slow fusion on the nonphysiological timescale of tens of minutes to several hours. Here, single-vesicle, fluorescence resonance energy transfer-based assays cleanly separate docking and fusion steps for individual vesicle pairs containing full-length SNAREs. Docking is extremely inefficient and is the rate-limiting step. Of importance, the docking and fusion kinetics are comparable in the two assays (one with v-SNARE vesicles tethered to a surface and the other with v-SNARE vesicles free in solution). Addition of the V(C) peptide synaptobrevin-2 (syb(57-92)) increases the docking efficiency by a factor of ∼30, but docking remains rate-limiting. In the presence of V(C) peptide, the fusion step occurs on a timescale of ∼10 s. In previous experiments involving bulk fusion assays in which the addition of synaptotagmin/Ca(2+), Munc-18, or complexin accelerated the observed lipid-mixing rate, the enhancement may have arisen from the docking step rather than the fusion step.

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Figures

Figure 1
Figure 1
Two-step model of SNARE-driven vesicle-vesicle docking and fusion. The t-SNARE vesicles (T) dock (second-order rate constant kdock) and fuse (first-order rate constant kfus) with v-SNARE vesicles (V). The single-vesicle assay resolves these two steps. Docked but unfused pairs of vesicles differ from docked and fused pairs in the measured FRET efficiency from green labels in the t-SNARE vesicles to red labels in the v-SNARE vesicles.
Figure 2
Figure 2
Alternating laser excitation of tethered vesicles with two-color imaging. Example images from the tethered vesicle assay after incubation of tethered v-SNARE vesicles with t-SNARE vesicles for 140 min. (a) On excitation at 633 nm, DiD fluorescence from each tethered v-SNARE vesicle gives rise to puncta in the red emission channel, enabling location of each tethered v-SNARE vesicle. Boxes mark positions of tethered v-SNARE vesicles in both the red and green images. No green emission appears. (b) Subsequent excitation at 514 nm locates green t-SNARE vesicles (DiI green emission), most of which are nonspecifically bound and do not colocalize with the v-SNARE vesicle locations. For those t-SNARE vesicles that colocalize with v-SNARE vesicles, fusion is detected as a greatly enhanced brightening of the red emission on 514 nm excitation. Several examples of docked and fused vesicle pairs are circled. Scale bar = 3.0 μm.
Figure 3
Figure 3
FRET efficiency histograms versus reaction time. Sparsely tethered v-SNARE vesicles were incubated with a 10 nM solution of t-SNARE vesicles in the (a) absence and (b) presence of 5 μM VC peptide. After the specified reaction time, excess (undocked) t-SNARE vesicles were rinsed away. Histograms of DiI to DiD FRET efficiency E (Eq. 1) are presented only for the subset of v-SNARE vesicles that were colocalized with a t-SNARE vesicle. Normalization accounts for variations in the total number of v-SNARE vesicles observed for each sample. Reaction times as shown. The strong peaks at E = 0 are due to false colocalization events (see Supporting Material).
Figure 4
Figure 4
Docking and fusion versus time in tethered-vesicle assay. Percentage of tethered v-SNARE vesicles that docked but did not fuse with a t-SNARE vesicle (gray data points) or docked and fused with a t-SNARE vesicle (black data points) at each reaction time, as determined by the absolute FRET efficiency. The t-SNARE vesicle concentration was 10 nM. (a) Without VC peptide. (c) In the presence of 5 μM VC peptide. Docked but unfused curves are corrected for false colocalizations between a tethered v-SNARE vesicle and a t-SNARE vesicle (see SupportingMaterial). The total docking curves in panels b and d are the sum of the docked but unfused plus the docked and fused curves in panels a and c. Docked but unfused curves are corrected for false colocalizations between a v-SNARE vesicle and a t-SNARE vesicle (see Supporting Material). The dotted lines represent the linear fits used to determine kdock,teth.
Figure 5
Figure 5
Histogram of τfus for single-vesicle fusion events with VC peptide. The histogram shows the dwell time in the docked but unfused state for 36 fusion events observed in real time with the tethered vesicle assay in the presence of VC peptide at 5 μM. Data were obtained with 3 s time resolution. See Fig. S9 for six examples of real-time FRET traces. Movie S1 shows an example of a single fusion event.
Figure 6
Figure 6
Docking and fusion versus time in bulk assay. The percentage of free v-SNARE vesicles that docked but did not fuse with a free t-SNARE vesicle (gray data points) or docked and fused with a free t-SNARE vesicle (black data points) at each reaction time is shown, as determined by a vesicle pair's absolute FRET efficiency. Mixtures were 10 nM t-SNARE vesicles and 5 nM v-SNARE vesicles without VC peptide (a and b) and in the presence of 5 μM VC peptide (c and d). The total docking curves in panels b and d are the sum of the docked and fused plus the docked but unfused curves in panels a and c. Docked but unfused curves are corrected for false colocalizations between a v-SNARE vesicle and a t-SNARE vesicle (see Supporting Material). The dotted lines represent the linear fits to the total docking curves that were used to determine kdock,bulk.
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