Dynamics and number of trans-SNARE complexes determine nascent fusion pore properties

Abstract

The fusion pore is the first crucial intermediate formed during exocytosis, yet little is known about the mechanisms that determine the size and kinetic properties of these transient structures. Here, we reduced the number of available SNAREs (proteins that mediate vesicle fusion) in neurons and observed changes in transmitter release that are suggestive of alterations in fusion pores. To investigate these changes, we employed reconstituted fusion assays using nanodiscs to trap pores in their initial open state. Optical measurements revealed that increasing the number of SNARE complexes enhanced the rate of release from single pores and enabled the escape of larger cargoes. To determine whether this effect was due to changes in nascent pore size or to changes in stability, we developed an approach that uses nanodiscs and planar lipid bilayer electrophysiology to afford microsecond resolution at the single event level. Both pore size and stability were affected by SNARE copy number. Increasing the number of vesicle (v)-SNAREs per nanodisc from three to five caused a twofold increase in pore size and decreased the rate of pore closure by more than three orders of magnitude. Moreover, pairing of v-SNAREs and target (t)-SNAREs to form trans-SNARE complexes was highly dynamic: flickering nascent pores closed upon addition of a v-SNARE fragment, revealing that the fully assembled, stable SNARE complex does not form at this stage of exocytosis. Finally, a deletion at the base of the SNARE complex, which mimics the action of botulinum neurotoxin A, markedly reduced fusion pore stability. In summary, trans-SNARE complexes are dynamic, and the number of SNAREs recruited to drive fusion determines fundamental properties of individual pores.

Extended Data Figure 1. Viral expression of cd-syb2

a, cDNA encoding the cytosolic domain of synaptobrevin 2 (cd-syb2, residues 1-95) was cloned into a FUGW transfer vector modified to have a synapsin promoter and to co-express soluble eGFP via an IRES sequence; eGFP serves as a marker for infection efficiency. For control experiments, eGFP alone was expressed. Both constructs were packaged into lentivirus for expression in neuronal cultures. b, Representative images of cells stained for a neuronal marker (MAP2, magenta) and GFP (green). Images were adjusted for brightness and contrast for the sake of presentation. Both preparations used for Fig. 1g were examined and had similar GFP expression levels and coverage across cells. The scale bar (50 μm) applies to all nine images shown. c, Quantification of the ICC demonstrating that both cd-syb2 and control viruses achieved a nearly 100% infection rate. Percent infected refers to the number of visually identified MAP2-positive somas (i.e. neurons) that were also positive for GFP. Three fields of view were quantified for each condition. d, Representative traces (left) and quantification (right) demonstrating that cd-syb2 was expressed at levels sufficient to inhibit evoked IPSCs triggered by field stimulation (p = 0.032, two-tailed t-test; n = 10 neurons for each condition, using 2 litters of mice, 3 coverslips per condition). Data are presented as mean ± s.e.m.. * denotes p < 0.05.

Extended Data Figure 2. Binding of different maltodextrins to the maltose sensor, determination of pore sizes and the relative fraction of open pores, and characterization of the single vesicle fusion assay

a, Fluorescence emission spectra of the maltose sensor in the absence or presence of the indicated maltodextrin (top). Equilibrium titration of maltodextrin binding to the maltose sensor. The data were fitted with a single site binding equation, using Prism 6 (GraphPad software, lnc.), to determine the dissociation constants. n = 3 independent experiments. Data are presented as mean ± s.d. (bottom). b, Kinetics of maltodextrin binding to the maltose sensor using stopped-flow (top). The observed rate constants (K_obsd) were plotted against maltodextrin concentration. The data were fitted with linear functions, yielding the off and on-rates for binding of each maltodextrin to the maltose sensor, as follows: 3 ± 1 S⁻¹ and 0.58 ± 0.03 μM⁻¹S⁻¹ (maltose), 14 ± 1 S⁻¹ and 6.7 ± 0.3 μM⁻¹S⁻¹ (maltotriose), 29 ± 9 S⁻¹ and 7.3 ± 0.2 μM⁻¹S⁻¹ (maltoheptaose), respectively (bottom). n = 3 independent experiments. Data are presented as mean ± s.d.. c, The lengths of the three principal axes of each sugar were averaged during 10 ns simulations (left). Representative snapshots of the sugars from the simulations. Error bars indicate s.d. from 1000 snapshots taken every 10 ps during the simulation (right). Data are presented as mean ± s.e.m.. d, Pore sizes were determined from the maltodextrin flux assays shown in Fig. 2c, as described in Methods. n = 3 independent experiments. e, Representative traces of dithionite quenching experiments using ND3 and ND8. Dithionite was added at the indicated time points, during on-going fusion reactions, to determine the degree of protection of NBD. The degree of protection was plotted against the incubation time, as shown in Fig. 2d. Similar results were obtained in three independent trials. We note that quenching by dithionite is much faster than cargo release (e.g. Fig. 2b). This is because the kinetics of most of the dithionite quenching that was observed was not a reflection of its influx via fusion pores, as >50% of the NBD-PE is on the outer leaflet. It is likely that dithionite can readily enter even small, flickering fusion pores, such as those formed by ND3, because it is smaller (174.11 Da) than the smallest maltodextrin used in this study (maltose; 360.31 Da). Also, the dithionite is present at high concentrations (5 mM). f, Plot of fusion probability observed using the indicated NDs; the black bars indicate experiments in which t-SNARE SUVs were pre-incubated with cd-syb2 to prevent trans-SNARE pairing. Data are presented as mean ± s.d.. g, Histograms of the fluorescence intensities of the tethered t-SNARE vesicles. n values were 54, 51, and 53 traces respectively, obtained from four independent trials under each condition.

Extended Data Figure 3. Characterization of the ND-BLM system: effect of t-SNARE density and detection of multiple pores

a–b, Fusion pores were formed using ND5 and BLMs bearing different t-SNARE densities. t-SNARE density was varied by using SUVs that harbored 100 (SUV_{t-SNARE (100)}), 200 (SUV_{t-SNARE (200)}) or 400 (SUV_{t-SNARE (400)}) copies of the SNAP-25B/syntaxin1A heterodimer per liposome. Since SUV_{t-SNARE (200)} and SUV_{t-SNARE (400)} resulted in fusion pores with similar sizes and kinetics properties, SUV_{t-SNARE (200)} was used for all other experiments in this study. n = 5 for SUV_{t-SNARE (100)} and 20 for (SUV_{t-SNARE (200)}); n = 5 for SUV_{t-SNARE (400)}. The representative traces (a) correspond to data points demarcated with red arrows in current versus t-SNARE copy number plot (b). Data are presented as mean ± s.e.m.. c, Estimation of the t-SNARE density in the BLMs used to form fusion pores. Typical recording showing that multiple t-SNARE SUVs, bearing a single gramicidin pore, fuse with the planar lipid bilayer. d, Histogram of the number of gramicidin pores formed, as shown in panel c, from 21 trials.Histogram of the number of gramicidin pores formed (n = 21). e, Multiple pores sometimes form in the ND-BLM assay. Example of a recording (SUV_{t-SNARE (200)} and ND5) in which a second pore appeared (top). Current histograms of the recording in the upper panel are shown (bottom). Similar results were obtained in fifteen independent trials.

Extended Data Figure 4. ND-BLM fusion pore properties at various time points

a, Typical recording of a fusion pore formed using ND5; this pore eventually closed after ~100 min. Similar results were obtained in eleven independent trials. b, Stability of fusion pores in the ND-BLM assay. Current histograms of ND-BLM assays using SUV_{t-SNARE (200)} and ND0, ND3, ND5, or ND7 at different time points in the recordings. There were no significant differences at the beginning, middle, or end of a recording session, so fusion pores are stable. The baseline was also stable over course of all recordings reported in this study. n = 14 for ND3; n = 20 for ND5 and ND7. For clarity, the closed state is shown in black and the open state is shown in red. In the case of ND0 and ND3, a cyan box is included to mark the appearance of open pores in ND3.

Extended Data Figure 5. Fusion pores formed using 50 nm NDs often dilate

a–c, Current histograms (left) and representative traces (right) of dilating fusion pores formed using ND3 (a, n = 7), ND5 (b, n = 9) and ND7 (c, n = 10). d, Fraction of time that pores are open. Since fusion pores often dilated, we analyzed the currents during an early phase of their initial open state (0.5 s after pore formation). Increasing the copy number of SNAREs per nanodisc resulted in larger pores (a–c) that spent more time in the open state (before they dilated; d). Data are presented as mean ± s.e.m.. e, A subpopulation of fusion pores formed using 50 nm NDs fail to dilate. Representative traces (left), current (middle) and open dwell time (right) histograms of non-dilating fusion pores (observed in 5 out of 14 trials) formed using 50 nm ND5. These pores exhibit well-defined open and closed states. There is some degree of heterogeneity regarding the v-SNARE copy number per ND (Fig. 3c and ref. 9). The non-dilating pores likely arise from NDs with the lower v-SNARE densities, consistent with a model in which SNARE density drives dilation.

Extended Data Figure 6. Characterization of fusion pores formed by yeast SNAREs

a, Illustration of pores formed using the yeast SNARE complex comprising Sso1p, the appropriate fragment of Sec9c (residues 401-651), and Snc2p. b, Typical recordings of fusion pores formed using ND0, ND3, ND5, and ND7. c–d) open dwell time histogram (c) and a scatter plot of the currents (d) that result from fusion pores formed using ND3 (n = 10), ND5 (n= 14) and ND7 (n = 14). ANOVA p<0.001; linear trend post-hoc p<0.001. Red arrows in panel d indicate the representative pores shown in panel c. Data are presented as mean ± s.e.m.. There is significant increase in pore size and stability as the v-SNARE copy number is increased. The rate constants for pore closure are reported in Extended Data Table 3. *** indicates p<0.001.

Extended Data Figure 7. Closure of fusion pores caused by cd-syb2

a, Fusion pores were first formed using ND5; cd-syb2 was then added at the indicated concentrations. Partial closure of fusion pores was sometimes observed after addition of cd-syb2, as shown in the representative current trace. b, Current histogram of all data from the 4 out of 11 trials in which partial closure was observed. In the remaining trials, these sub-conductance states were not observed (Fig. 4a). c, Representative recording (top) and current histogram (bottom) of a pore (ΔΨ = −50 mV) formed using ND7, before and after addition of cd-syb2 at the indicated concentrations. d, Fraction of closed pores formed using ND5 and ND7 in the presence of 20.25 μM cd-syb2. Data are presented as mean ± s.e.m..

Extended Data Figure 8. BSA, cd-syb2_4A and a C-terminal truncation of SNAP-25B have limited effects on fusion pore current

Representative traces (top) and current histograms (bottom) of fusion pores before (left) and after (right) addition of BSA (a) or cd-syb2_4A (b). These reagents had no effect on the magnitude of the currents, but cd-syb2_4A causes increases in flickering behavior, likely due to weak residual t-SNARE binding activity; this effect was limited, as the same concentration of cd-syb2 completely closed pores under the same conditions. n = 6 for both BSA and cd-syb2_4A. c, Fusion pore current is unaffected in SNAP-25B_1-197. n = 20 for SNAP-25B (WT) and n = 5 for SNAP-25B_1-197; Data are presented as mean ± s.e.m..

Figure 1. Reducing v- or t-SNARE availability alters the shape of mEPSCs

a, Cleavage of syb2 by TeNT. b–c, Representative traces (b) and (c) quantification of mEPSC frequency after treatment with TeNT (Ctrl: 0.81 ± 0.28 Hz [0.18, 1.44], n = 10; 100 pM TeNT: 0.10 ± 0.02 Hz [0.04, 0.17], n = 10; p < 0.001, Kruskal-Wallis test; p = 0.001 for Ctrl vs 100 pM, Dunn’s multiple comparison post-hoc test). Inset in panel (c): immunoblot of syb2, β-actin and synaptotagmin 1 (syt1) in control and TeNT treated neurons. Similar results were obtained in three independent trials. d, Averaged mEPSC traces after treatment with TeNT (left); amplitudes (Ctrl: −27 ± 3 pA [−20, −33], n = 10 neurons; 100 pM TeNT: −19 ± 2 pA [−15, −22], n = 10 neurons; p = 0.021, two-tailed t-test) and 10–90% rise times are plotted on the right (Ctrl: 1.2 ± 0.2 milliseconds (ms) [0.9, 1.6 ms], n = 10 neurons; 100 pM TeNT: 1.8 ± 0.2 ms [1.3, 2.2], n = 10 neurons; p = 0.020, two-tailed t-test). e, Averaged traces (left) and quantification (right) of γ-DGG mediated inhibition of mEPSCs. (Ctrl: 8 ± 3% reduction in amplitude [1, 16], n = 7 neurons; 100 pM TeNT: 23 ± 3% inhibition [17, 31], n = 6 neurons; p = 0.005, two-tailed Mann-Whitney test). a–e, Experiments were performed using one coverslip from each of 3 independent litters of mice. f, Cd-syb2 occupies t-SNAREs to inhibit fusion. g, Averaged mEPSC traces (left); frequencies, amplitudes, and rise times are plotted on the right (Ctrl: 1.6 ± 0.3 Hz [1.0, 2.1] / −17 ± 1 pA [−15, −20] / 1.1 ± 0.1 ms [0.9, 1.3], n = 19; cd-syb2: 0.9 ± 0.2 Hz [0.6, 1.2] / −13 ± 1 pA [−12, −15] / 1.4 ± 0.1 ms [1.2, 1.7], n = 21; p = 0.049 -freq., two-tailed Mann-Whitney test; p = 0.017 -amp., Welch’s two-tailed t-test; p = 0.021 -rise, Welch’s two-tailed t-test). f–g, experiments were performed using 2 litters, 2 coverslips per litter. * denotes p < 0.05 and ** denotes p < 0.01. All data are presented as mean ± s.e.m.. [95% C.I.].

Figure 2. Reconstituted fusion assays reveal changes in cargo efflux rates as a function of syb2 copy number

a, Ensemble fusion assay using NDs and SUVs b, Release time courses of different maltodextrins, using ND6. c, Maltodextrin release efficiency versus the syb2 copy number per ND. d, Dithionite quenching of NBD labeled lipid revealed that the number of open pores was the same for ND3 and ND8. The values plotted in panels (b–d) represent mean ± s.d. (n = 3 independent experiments). e, Illustration of single-vesicle fusion assays. f, Representative trace showing SRB efflux through a single fusion pore (left); expanded time scale, fitted with a single exponential (red) function, is shown on the right. Similar results were obtained in four independent experiments. g, SRB efflux rates using ND3, ND5, and ND7; n values were 54, 51, and 53 respectively, obtained from four independent trials under each condition.

Figure 3. Properties of single fusion pores measured via planar lipid bilayer electrophysiology

a, Illustration of the ND-BLM assay. b, Traces of single pores at ΔΨ = −50 mV for ND0, ND3, ND5, and ND7 are shown. Closed (C) and open (O) states are indicated, along with the respective currents. c, Pore currents obtained using the indicated ND; red arrows indicate representative pores shown in panel (b). ND3: −4 ± 1 pA [−3, −5 pA], ND5: −11 ± 1 pA [−9, −13 pA], ND7: −18 ± 2 pA [−15, −21 pA]; p < 0.001, Kruskal-Wallis test; p < 0.001 ND3 vs. ND5, p < 0.001 ND3 vs. ND7, p = 0.036 ND5 vs. ND7, Dunn’s multiple comparison post-hoc tests. Data in (c) are presented as mean ± s.e.m.. [95% C.I.]. d, Current (I, in pA) versus voltage (V, in millivolt) relationships for pores formed using the indicated ND. Data are presented as mean ± s.e.m.. e, Open dwell time histograms of pores. n = 14, 20 and 20 independent BLMs for ND3, ND5, and ND7 respectively, and 5 different sets of NDs of each type were used. *** indicates p<0.001 and * indicates p = 0.05.

Figure 4. Trans-SNARE complexes are dynamic

a–b, Representative recording (a) and current histogram (b) of a pore formed using ND5, before and after addition of cd-syb2 at the indicated concentrations. c, Fraction of time that pores are fully closed, plotted as a function of [cd-syb2]. n = 11 independent BLMs, and 5 different sets of NDs. Data are presented as mean ± s.e.m.. d, Illustration of dynamic trans-SNARE pairing, as evidenced by cd-syb2 (purple) mediated destabilization of open pores. e, ND-BLM assays were performed using ND5 and the indicated SNAP-25B mutants; open dwell time histograms are shown. SNAP-25B_1-197 gave rise to partial conductances, denoted P. n = 5 independent BLMs for each mutant, using 3 different sets of NDs.