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- Multiple intermediates in SNARE-induced membrane fusion

mcith_zpq0510645070001.jpg Figure 1 Single-liposome fluorescence assay of SNARE-mediated membrane fusion. (a) Schematics of the assay. The v-SNARE liposomes containing membrane fluorescent acceptors are tethered on a PEG-coated quartz slide, and Sso1pHT-reconstituted liposomes doped with membrane fluorescent donors are introduced together with Sec9c to induce fusion. The mixing of donor and acceptor dyes caused by fusion between the cognate liposomes leads to increase in E, which is being monitored by wide-field total internal reflection (TIR) microscopy. (b) Negative staining electron micrograph of the Sso1pHT-reconstituted liposomes (1200 EX; JEOL, Tokyo, Japan). (Scale bar: 100 nm.) (cf) Final E distribution of the products of SNARE-driven single-liposome fusion (after 30 min of reaction) in the absence of Sec9c (c) and at the Sec9c/Sso1pHT ratio of 2:1 (d and e) and 1:1 (f). The fusion reactions of c, d, and f were induced on surface (as illustrated in a), whereas in e, fusion was induced in bulk solution and the products were subsequently immobilized on the quartz surface for observation. We notice that after 30 min of reaction the fraction of the population that remains at the docked state varies significantly sample by sample.

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mcith_zpq0510645070002.jpg Figure 2 Multiple intermediate states of SNARE-induced fusion. (af) Single-liposome fusion time traces; full fusion events with no intermediates (classified as α class, a), one intermediate state (β1 class, b and c), two intermediate states (β2 class, d and e), and three intermediate states (β3 class, f). (Upper) Shown is the fluorescence intensity time traces of the donor (ID, green) and the acceptor (IA, red) channels. (Lower) Shown is the corresponding FRET efficiency (blue) where the intermediate states are marked with black bars. In b, docking of a t-SNARE liposome is followed by a gradual FRET increase (between the two arrows), which culminates with the first intermediate state. (gi) FRET histograms of the first intermediate state for the subclasses β1, β2, and β3 (g). The narrow distribution is not disturbed when traces obtained at the different Sec9c/Sso1pHT ratios of 2:1 (h) and 1:1 (i) are separately considered. The histograms are fitted with the Gaussian distributions, and the center (Ec) and the standard deviation (σ) of the Gaussians are shown. (j) Schematic illustration of a typical single-liposome fusion time trace. (k) Pathway of SNARE-driven membrane fusion. For the class β, docking of a t-SNARE liposome and close contacting between two liposomes (state D) are followed by an obligatory intermediate state, the hemifusion state (state H). The number of premature closings of the fusion pore (F′, F″, and so on) between the hemifusion state and the full fusion state (state F) then determines the subclass such as β1, β2, and β3. Class α evolves from the D to F state without discernible intermediate states. In contrast, the FRET increase of class γ is too gradual for intermediates to be identified, which could be caused by many pore-flickering phenomena.

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mcith_zpq0510645070003.jpg Figure 3 Kinetic analysis of single-liposome fusion time traces. In this analysis, only the class β (β2 and β3 in the case of the second intermediate) obtained at the Sec9c/Sso1pHT ratio of 2:1 was used. Shown are cumulative dwell time histograms of docked (a), first intermediate (b), and second intermediate (c) states. The docked state is defined as an intermediate plateau with the largest E < 0.25. With the first-order kinetics assumed, the dwell time histograms are fitted by using two exponentials, A1(1 − expt/t1) + A2(1 − expt/t2) (red curve). (Insets) The dwell times (t1, t2) and the corresponding number of traces (A1, A2) are shown.

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mcith_zpq0510645070004.jpg Figure 4 Kiss-and-run-like fusion events. Shortly after the fusion-pore opening, which leads to complete lipid mixing, both the donor and acceptor fluorescence signals decrease without appreciable changes in the FRET efficiency for the class β (a and c) and the class α (b and d). Such an effect is consonant with the detachment of what remains of the t-SNARE liposome. (Upper) Shown are the changes in the donor (green) and the acceptor (red) fluorescence intensities. (Lower) The corresponding changes in the FRET efficiency (blue) are shown.

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