Abstract
Membrane fusion and fission are vital to eukaryotes’ life1–5. For three decades, it has been proposed that fusion is mediated by fusion between proximal leaflets of two bilayers (hemi-fusion) that produces a hemi-fused structure, followed by fusion between distal leaflets, whereas fission is via hemi-fission, which also produces a hemi-fused structure, followed by full fission1, 4, 6–10. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion/hemi-fission in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed2, 11–15. Using confocal and super-resolution STED microscopy, we observed the hemi-fused Ω-shaped structure for the first time in live cells, neuroendocrine chromaffin cells and pancreatic β-cells. This structure was generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, its transition to full fusion or fission was determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and was surprisingly slow (seconds to tens of seconds) in a significant fraction of the events. These results provide key missing evidence over the past three decades proving the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion/fission, as fusion and fission mechanisms compete to determine its transition to fusion or fission.
Chromaffin cells were transfected with EGFP-tagged phospholipase Cδ1 PH domain (PH-EGFP), bathed with cell-impermeable Atto 655 (A655), and stimulated with 1 s depolarization (from −80 to +10 mV, depol1s), which induced calcium currents (ICa) and capacitance (Cm) changes reflecting exo- and endocytosis (Fig. 1a). PH-EGFP binds specifically to PtdIns(4,5)P2 (PIP2)16, a phospholipid located at the cytoplasm-facing (PMcyto), but not extracellular facing leaflet (PMextra) of the plasma membrane (PM) or the cytoplasm-facing or lumen-facing leaflet of the vesicular membrane (VMcyto or VMlumen)17. Accordingly, PH-EGFP labels PMcyto in chromaffin cells18 (see also Fig. 1b, n =16; Extended Data Figure 1a–b). Diffusion of PIP2-bound PH-EGFP from PMcyto to VMcyto may indicate PMcyto-VMcyto fusion, whereas A655 labels opened Ω-profiles19.
Confocal imaging of PH-EGFP and A655 (PH-EGFP/A655) at XY plane with a fixed Z plane ~100 nm above cell-bottom (XY/Zfix imaging, Extended Data Fig. 1c–d) revealed A655 spots (25.1 ± 3.9 per cell, 16 cells) induced by depol1s19, 98% of which were accompanied by PH-EGFP fluorescence (FPH) increase (Fig. 1c). FPH rise τ (417 ± 31 ms) was slower than A655 fluorescence (F655) increase (122 ± 16 ms, n = 69 spots randomly selected, Fig. 1d). A655 spots without FPH increase was due to Ω-profile shrinking and merging faster than FPH rise19 (Extended Data Fig. 1e).
At our imaging time resolution of 33–100 ms, FPH increased at the same onset as F655 increase (termed same-onset spots, Fig. 1d, Supplementary video 1, 224 spots), or 0.1–26 s (8.0 ± 1.5 s, 40 spots) earlier (termed PH-earlier spots, Fig. 1e, Extended Data Fig. 1f, Supplementary video 2). We also observed PH-EGFP spots without F655 increase, but with size and FPH similar to same-onset or PH-earlier spots (termed PH-only spots, Fig. 1f, Supplementary video 3, 137 spots, 16 cells, Extended Data Fig. 1g). Since PH-EGFP and A655 report PMcyto-VMcyto fusion and full-fusion, respectively, depol1s-induced same-onset (54 ± 6%), PH-earlier (14 ± 5%), and PH-only spots (32 ± 5%, 16 cells, Fig. 1g, Extended Data Table 1) may correspond to full-fusion, hemi-fusion then full-fusion, and hemi-fusion, respectively. This conclusion was further strengthened by the following six sets of evidence.
First, when PH-EGFP was replaced with GFP tagged to Lyn kinase’s myristoylation and palmitoylation sequence (PM-GFP) that is targeted to PMcyto20 (Extended Data Fig. 2a), PM-GFP/A655 imaging revealed same-onset, PM-GFP-earlier, and PM-GFP-only spots analogous to PH-EGFP/A655 imaging results (Fig. 1h–i, Extended Data Table 1). Similarly, replacing PH-EGFP with CAAX-EGFP, a motif targeted to PMcyto via its cysteine residue isoprenylation21 (Extended Data Fig. 2b), revealed same-onset, CAAX-EGFP-earlier, and CAAX-EGFP-only events (Extended Data Fig. 2c–e). Thus, PH-EGFP-reported hemi-fusion (PMcyto-VMcyto fusion) is independent of PIP2.
Second, FPH increase was not due to lipid transport from nearby vesicles or endoplasmic reticulum (ER), because 1) lipid transport (minutes)22 is much slower than FPH increase (Fig. 1d–f), 2) PH-EGFP (or PH-mCherry or PH-YFP) labelled only PM, but not vesicles (Fig. 1b, Extended Data Fig. 3a–b) or ER (Extended Data Fig. 3c–d), and 3) FPH increase was independent of ER location (Extended Data Fig. 3e–f).
Third, in cells transfected with PH-mCherry and pH-sensitive VAMP2-pHluorin, depol1s induced VAMP2-pHluorin spots with PH-mCherry fluorescence (FPH-mCh) increase at the same onset (Same-onset, 52 spots, 72 ± 6%, n=11 cells, Fig. 1j) or 6.7 ± 1.4 s earlier (PH-earlier, 26 spots, 28 ± 6%, Fig. 1k), analogous to PH-EGFP/A655 imaging results. Since VAMP2-pHluorin fluorescence (FVAMP2) increase is due to proton (molecular weight: 1 Dalton, hydrated proton: ~0.1 nm) and OH− (molecular weight: 17 Dalton) exchange via an open pore23, PH-earlier spots (before FVAMP2 increase) could not permeate the smallest molecules. Thus, PH-earlier events were not due to an opened pore too small to permeate A655 (molecular weight: 528 Dalton), but reflected hemi-fusion followed by full-fusion. Although PH-mCherry-only spots were also observed, it could be due to lack of VAMP2-pHluorin expression. However, infrequently, FPH-mCh increased and decreased without FVAMP2 changes, and then increased to the previous level with FVAMP2 increase (Fig. 1l, n = 11 spots), suggesting sequential transition of hemi-fusion, fission (FPH-mCh decay, described later), and full-fusion.
Fourth, stimulated emission depletion (STED) imaging of PH-EGFP and Alexa 532 (A532) in the XZ plane with a fixed Y plane (XZ/Yfix scanning; Extended Data Fig. 1d) directly revealed depol1s-induced same-onset (53%, Fig. 2a), PH-earlier (12%, Fig. 2b) and PH-only Ω-profiles (34%, Fig. 2c; total Ω-profile number: 108, 35 cells). The Ω-profile diameter at the fusion onset (318 ± 28 nm, n = 63 Ω-profiles with clear edges) was similar to granule diameter (~300 nm)24. Ω-profile PH-EGFP fluorescence was often brighter than PM (Fig. 2a–c). Simulation suggests that this is due to neighbouring fluorescence contribution from the Ω-profile in the Z-axis direction (Extended Data Fig. 4). Scanning every ~50 ms showed that FPH increase near the Ω-profile base (near PM) was earlier than the top (far from PM) by 111 ± 9 ms (n = 11 Ω-profiles, Fig. 2d), indicating PIP2 diffusion from the PM to Ω-profile.
Fifth, we applied mCLING-Atto 488 (membrane-binding fluorophore-cysteine-lysine-palmitoyl group, mCLING-A488) to the bath, which inserted into and thus labelled PMextra25. mCLING fluorescence increased in nearly all A655 spots (not shown), suggesting that mCLING labels fully fused Ω-profiles. STED XZ imaging of mCLING-A488/PH-mCherry revealed same-onset Ω-profiles (labelled with mCLING and PH-mCherry, Fig. 2e) and PH-only Ω-profiles (labelled with only PH-mCherry, n = 16, Fig. 2f, g). Some PH-only Ω-profiles exhibited an open neck (n = 5, Fig. 2g), consistent with a hemi-fused diaphragm. By repeating XZ scanning every 100 nm along the Y-axis for 1–2 µm (XZ/Ystack scanning), we reconstructed XY-plane images showing a smaller ring across the Ω-profile neck and a larger ring across the Ω-profile center (Fig. 2g). 5 out of 16 PH-only Ω-profiles showed a detectable open neck of 70–130 nm (Fig. 2g). These results directly demonstrate the hemi-fused Ω-profile by differential labelling of PMcyto and PMextra.
Sixth, during high potassium application, which induced PH-only and close-fusion events (n = 8 cells, Extended Data Fig. 5a–b), electron microscopic examination of ~800 granules close to the PM (< 30 nm, Extended Data Fig. 5c) revealed 28 granules (~3.5%) in tight contact with PM (Extended Data Fig. 5d). Electron tomography revealed no PMcyto–VMcyto connection in 6 tight-contacts (Extended Data Fig. 5e), potential PMcyto–VMcyto connection in 7 tight-contacts (Extended Data Fig. 5f), and evident PMcyto–VMcyto connection in 5 tight-contacts (Fig. 2h, Extended Data Fig. 5g) similar to hemi-fusion candidates reported in cells26, 27. These results support live-cell detection of the hemi-fused structure.
Next, we studied fission during fusion pore closure (Fig. 3a–i). Full-fusion-generated Ω-profiles may maintain an open pore (stay fusion), close the pore (close fusion) or shrink to merge with PM (Ω-shrink fusion)19. With strong A655 excitation, A655 spots persisted during stay fusion (Fig. 3a, n = 33 spots), dimmed while maintaining a constant size after pore closure (close fusion) that prevented bleached A655 exchange with extracellular fluorescent A655 (Fig. 3b–c), and dimmed rapidly while spot size reduced during Ω-shrink fusion19 (Extended Data Fig. 1e). At confocal cell-bottom PH-EGFP/A655 image setting, depol1s induced close-fusion (5.2 ± 1.0 spots per cell, n = 10 cells) where F655 decayed while FPH remained unchanged for > 40 s (24 spots, 46%, Fig. 3b) or decayed 4.9 ± 1.0 s after pore closure (F655 decay onset) with a τ of 5.5 ± 1.0 s (28 spots, 54%, Fig. 3c, 3i). Similar FPH patterns were observed when pore closure was detected by VAMP2-pHluorin imaging, where FVAMP2 increase and decrease reflect pore opening and closure/re-acidification, respectively (Extended Data Fig. 6). They were also observed with STED XZ/Yfix imaging of PH-EGFP/A532 (strong A532 excitation), where A532 fluorescence (F532) dimming reflected pore closure (Fig. 3d–f; stay-fusion, 30 spots; close-fusion with no FPH decay, 10 spots; close-fusion with delayed FPH dimming, 8 spots).
FPH decay after F532 or F655 dimming was not due to vesicle movement, because as FPH decayed, the PH-EGFP-labelled Ω-profile dimmed, but did not move (Fig. 3f, n = 8 spots), and A655 spots remained unchanged if excited minimally to avoid bleaching (Extended Data Fig. 7a, 22 spots). While FPH decayed, the fluorescence of a PI(4)P probe increased (Extended Data Fig. 7b, 13 spots), suggesting PIP2 conversion to PI(4)P, which may explain why PIP2 is not detected in resting vesicles (Fig. 1b, Extended Data Fig. 3a)18.
FPH decay must reflect PMcyto-VMcyto fission that prevents PH-EGFP diffusion from PMcyto to VMcyto. The interval between F655 decay (or F532 decay, pore closure) and FPH decay may reflect the lifetime of the hemi-fused Ω-profile generated by hemi-fission (PMextra-VMlumen fission). To test this, we applied a high laser power (100%, 1 s) 5 s after depol1s to photo-bleach PH-EGFP in a small area. Bleached PH-EGFP recovered during stay-fusion, due to fluorescent PH-EGFP diffusion through the PMcyto-VMcyto connection (Fig. 3g, n = 22 spots, 11 cells). Bleached PH-EGFP recovered after close-fusion with no FPH decay in 28 out of 37 spots (76%, 11 cells, Fig. 3h), suggesting a PMcyto-VMcyto connection after pore closure, i.e., hemi-fission. FPH did not recover in 9 close-fusion spots, suggesting PMcyto-VMcyto fission after pore closure, i.e., full-fission. Full-fission in these spots likely occurred during or right before bleaching, because FPH did not recover in two close-fusion spots, where FPH started to decrease right before bleaching (Extended Data Fig. 7c). Thus, the interval between F655 and FPH dimming reflected hemi-fission intermediate lifetime (Fig. 3i).
Occasionally (4 out of 28 spots), the hemi-fission intermediate returned to full-fusion status, reflected as F655 decay (pore closure) and then increase while FPH remained elevated and could recover if being bleached (Fig. 3j). Not only the hemi-fission intermediate (Fig. 3c, f), but also the hemi-fusion intermediate could proceed to full-fission, reflected as FPH decay after dwelling for 8.6 ± 1.1 s in 80 out of 137 PH-only spots (n=16 cell, Fig. 3k, see also Fig. 1l).
Summarized in Fig. 4a, hemi-fused Ω-profiles were generated by hemi-fusion (Fig. 1f, 2c, 2f–g) or hemi-fission (Fig. 3b, e, h), and proceeded to full-fusion (Fig. 1e, 1k, 2b, 3j; Extended Data Fig. 1f) or full-fission (Fig. 3c, f, k) in chromaffin cells. Similar transitions were observed with PH-EGFP/A655 imaging in rat pancreatic β cells (24 INS-1 cells, 179 fusion spots, Extended Data Fig. 8), suggesting wider applications of our observations.
The schematics in Fig. 4a suggest that hemi-fission (PMextra-VMlumen fission) counteracts hemi-to-full-fusion transition. Consistent with this suggestion, as close-fusion or ICa that triggers close-fusion19 increased, PH-only events reflecting hemi-fusion (not hemi-fission) increased, whereas full-fusion (including same-onset and PH-earlier events) percentage decreased (Fig. 4b, Extended Data Table 1), likely due to increased hemi-fission that counteracts hemi-to-full-fusion transition. Inhibition of dynamin by dynasore, overexpressed dynamin dominant-negative mutant Dynamin 1-K44A, or dynamin 1 and 2 knockdown (Extended Data Fig. 9a–b) substantially reduced depol1s-induced close-fusion and PH-only percentage without affecting ICa, but increased same-onset percentage (Fig. 4c–e, Extended Data Fig. 9c–e, Extended Data Table 1). Thus, inhibition of dynamin-dependent close-fusion may reduce PH-only events, but increase full-fusion percentage (Fig. 4f, Extended Data Fig. 9f) by inhibiting hemi-fission that counteracts hemi-to-full-fusion transition. Dynamin 1-EGFP puncta were observed at PM (Extended Data Fig. 9g, Fig. 4g) and co-localized with 11 out of 16 depol1s-induced A532 spots at the Ω-profile pore region (Fig. 4g, 10 cells), suggesting that dynamin is physically available before fusion to counteract hemi-to-full-fusion transition. Insufficient dynamin 1-EGFP expression and endogenous dynamin might explain the lack of dynamin-EGFP in remaining spots.
In summary, by developing STED- and confocal-based methods, we resolved PMcyto-VMcyto and PMextra-VMlumen fusion/fission in live chromaffin and pancreatic β cells, and discovered the hemi-fused Ω-profile as the structural pathway to fusion and fission. The hemi-fused Ω-profile generated by hemi-fusion or hemi-fission is a key structure controlling fusion and fission. It proceeds to full-fusion or full-fission depending on the net outcome of competition between fusion and calcium/dynamin-dependent fission mechanisms (Fig. 4a).
Lacking direct live-cell evidence, whether fusion is mediated via forming a protein-lined pore2, 11–13, 15 or through hemi-fusion1, 6–8 has been intensely debated. The hemi-fusion hypothesis remains to be proved with evidence showing sequential transition from intact vesicle to hemi-fusion and then to full-fusion. The present work reveals this sequential transition in live cells and thus proves the hemi-fusion hypothesis. Studies interpreting fusion pore regulation by SNARE proteins as SNARE-lined pore in PC12 and chromaffin cells2, 11–15 may be re-interpreted under the hemi-fusion framework to gain new insight.
Hemi-fused vesicles were proposed as release-ready vesicles in cortical synapses28 (but see Ref. 29) and sea urchin eggs30, whereas reconstituted vesicle-vesicle fusion starts from vesicle contact to either full-fusion8, possibly via hemi-fusion too transient to detect, or to hemi-fused diaphragms that do not or are highly reluctantly to proceed to full-fusion7, 8. We observed not only transitions (same-onset or PH-only events) analogous to those observed in reconstituted vesicle-vesicle fusion, but also many other dynamic reversible transitions, including from intact vesicle to a hemi-fused structure for 0.1–26 s and then to full-fusion or back to intact vesicle, and from fully-fused Ω-profile to a hemi-fused structure for various times and then to intact vesicle or back to fully fused Ω-profile (Fig. 4a). These differences may reflect differential protein and lipid composition in cells and reconstituted systems.
Hemi-fission was proposed based on simulation and conductance measurements from artificially pulled lipid nanotube3, 4, 9. Here we provided the first evidence showing hemi-fission as the pathway to fission during fusion pore closure in live cells. Unexpectedly, the hemi-fission intermediate had a long lifetime, ranging from <1 s to > 40 s, before proceeding to full-fission or full-fusion (Fig. 3). Fission in live cells is therefore composed of two kinetically distinguishable steps, hemi-fission (PMextra-VMlumen fission) and full-fission (PMcyto-VMcyto fission).
We found surprisingly that dynamin-dependent fission mechanisms compete with fusion mechanisms at the hemi-fused state before full-fusion occurs to counteract the hemi-to-full-fusion transition. Such a competition is sometimes observed in real time; the sequential observation of full-fusion, hemi-fission, and back to full-fusion (Fig. 3j) may explain the widely observed capacitance flickers, repeated fusion pore opening and closure5, 24. Regulation of fission mechanisms may thus regulate fusion efficiency.
Hemi-fusion and hemi-fission were studied mostly in reconstituted membranes different from cells in molecular composition and geometry. To what extent these studies apply to cells is unclear. Our work provides the foundation and techniques to further study hemi-fusion and hemi-fission in live cells.
Methods
Cell culture
We prepared primary chromaffin cell culture as described previously19. In brief, fresh adult (21 – 27 months old) bovine adrenal glands (from a local abattoir), were immersed in pre-chilled Lock’s buffer on ice containing: NaCl, 145 mM; KCl, 5.4 mM; Na2HPO4, 2.2 mM; NaH2PO4, 0.9 mM; glucose, 5.6 mM; HEPES, 10 mM (pH 7.3, adjusted with NaOH). Glands were perfused with Lock’s buffer, then infused with Lock's buffer containing collagenase P (1.5 mg/ml, Roche), trypsin inhibitor (0.325 mg/ml, Sigma) and bovine serum albumin (5 mg/ml, Sigma), and incubated at 37°C for 20 min. The digested medulla was minced in Lock’s buffer, and filtered through a 100 µm nylon mesh. The filtrate was centrifuged (48 ×g, 5 min), re-suspended in Lock’s buffer and re-centrifuged until the supernatant was clear. Final cell pellet was re-suspended in pre-warmed DMEM medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and plated onto poly-L-lysine (0.005 % w/v, Sigma) and laminin (4 µg/ml, Sigma) coated glass coverslips. The cells were incubated at 37°C with 9% CO2 and used within 1 week. Before plating, some cells were transfected by electroporation using Basic Primary Neurons Nucleofector Kit (Lonza), according to the manufacturer’s protocol. The INS-1 cell line was purchased from AddexBio (San Diago, USA).
Electrophysiology
At room temperature (22 – 24°C), whole-cell voltage-clamp and capacitance recordings were performed with an EPC-10 amplifier together with the software lock-in amplifier (PULSE, HEKA, Lambrecht, Germany)19, 31. The holding potential was −80 mV. The frequency of the sinusoidal stimulus was 1000 – 1500 Hz with a peak-to-peak voltage ≤ 50 mV. The bath solution contained 125 mM NaCl, 10 mM glucose, 10 mM HEPES, 5 mM CaCl2, 1 mM MgCl2, 4.5 mM KCl, 0.001 mM TTX and 20 mM TEA (pH 7.3, adjusted with NaOH). The pipette (2 – 5 MΩ) solution contained 130 mM Cs-glutamate, 0.5 mM Cs-EGTA, 12 mM NaCl, 30 mM HEPES, 1 mM MgCl2, 2 mM ATP, and 0.5 mM GTP, pH 7.2 adjusted with CsOH. These solutions pharmacologically isolated calcium currents.
Setup and parameters for confocal and STED imaging
With an inverted confocal microscope (Nikon A1R, 60× oil objective, numerical aperture: 1.4), A655 (68 µM in bath, Sigma) and PH-EGFP were excited by a Diode laser at 640 nm (maximum power: 40 mW) and an Argon laser at 488 nm (maximum power: 50 mW), respectively. Unless mentioned otherwise, the 640 nm laser was set at 16% of the maximum power, whereas 488 nm laser was set at 1.5%. A655 and PH-EGFP fluorescence were collected with a photomultiplier at 650 – 720 nm and 500 – 550 nm, respectively. Both excitation and fluorescence collection were done simultaneously. VAMP2-pHluorin was imaged with the same setting as for PH-EGFP imaging. When PH-mCherry was used, it was excited with 561 nm laser (maximal power: 50 mW) at 3% maximal power. Confocal images were collected every 33 ms at 50 nm per pixel at the cell bottom (70–160 µm2).
STED images were acquired with Leica TCS SP8 STED 3× microscope that is equipped with a 100 × 1.4 NA HC PL APO CS2 oil immersion objective and operated with the LAS-AX imaging software. Excitation was with a tunable white light laser and emission was detected with hybrid (HyD) detectors. In time gated STED mode, PH-EGFP and A532 were sequentially excited with a tunable white light laser at 470 and 532 nm, respectively. The STED beam was generated by a 592 nm depletion beam. Similarly, mCLING-A488 and PH-mCherry were sequentially imaged, in this case excitation laser was set to 488 and 570 nm, respectively, STED beam was generated by a 660 nm depletion beam. All STED images were acquired in the XZ plane at the cell bottom that was attached to the coverslip, where a cross-sectional view of the cell and thereby fused vesicles could be observed. The STED images of PH-EGFP and A532 was acquired every 50–280 ms at 16 nm per pixel, in an XZ area of 19.4 µm×1.2 µm, with fixed Y plane. The probability of observing a fusion event induced by depol1s during XZ scanning (with a fix y-axis location) varied from 0 to 0.38 per cell depending on the number of fusion events induced by depol1s in the scanned region. To reconstruct the structure of fused vesicles, STED XZ images were also acquired with Y stacks at 100 nm interval for a total length of 1–2 µm. All STED images were deconvolved using Huygens software (Scientific Volume Imaging).
Image analysis
Confocal images were analyzed using NIS-Elements AR (Nikon). STED images were analyzed with ImageJ. The fluorescence intensity from an area covering the fluorescence spot was measured at every image frame. For images shown in figures, 5–15 frames were averaged. The full-width-half-maximum (WH) was measured from intensity profiles of 1 – 4 lines across the spot center.
For plot of fluorescence intensity, such as FPH and F655, we normalized the fluorescence intensity to the baseline in all figures. For calculation of the onset of spot F655 and FPH increase, we fit the 10–30% rising phase of F655 or FPH, and extrapolated the fit to the baseline. The time point at which the fit line crosses the baseline is the onset. Our sampling time was 33 ms in most experiments, but 100 ms for experiments where PH-EGFP was bleached. Thus, our time resolution was 33 – 100 ms. With this time resolution, spots with delay of F655 rise within 33 ms from the FPH rise onset could not be identified, which may lead to an underestimation of PH-EGFP spots with a delayed F655 rise. Similar analysis was applied to measure the onset of FPH-mCherry and FVMAP2 rise.
When A655 (strong excitation: 16% maximal power) and PH-EGFP (weak excitation: 1.5% maximal power) were imaged, fusion pore closure was identified if after A655 spot appeared, the spot F655 decayed to baseline with a time constant more than 2 s (~2–5 s) while the spot WH did not change. Another form of fusion, the Ω-shrink fusion, in which the fusion-generated Ω-profile shrinks until undetectable, could also cause F655 decay to baseline19. However, this decay is different from the decay caused by Ω-profile pore closure in three aspects. First, the decay time constant of nearly all Ω-shrink events is < 1.7 s19. Thus, a decay time constant of > 2s is a safe criteria for identifying fusion pore closure19. Second, Ω-shrink fusion is accompanied by a reduction of WH, whereas Ω-profile closure is not19. The WH can be measured from either A655 spot or PH-EGFP spot, the latter of which is often easier to measure because FPH often did not decrease in parallel with F655 during pore closure. Third, F655 and FPH decayed approximately in parallel during Ω-shrink fusion that reduces the size of Ω-profile till undetectable, but usually did not decay in parallel during fusion pore closure. After pore closure, there is often a delay of FPH decay due to the long lifetime of the hemi-fused structure generated by hemi-fission (Fig. 3i). A combination of these three criteria allowed us to clearly identify fusion pore closure events, as has been recently characterized extensively and confirmed with several other independent methods19.
Representative images were shown with 5–15 frame averages. Since the STED Z-resolution (~150 nm) is worse than the X-axis resolution (~65 nm), we measured the vesicle diameter on the X-axis direction for images obtained with STED XZ scanning. The vesicle at the Z-axis direction may appear elongated due to a lower Z-axis resolution.
Electron microscopy
Bovine chromaffin cells were stimulated for 2–10 minutes using a solution containing 30 mM KCl 115 mM NaCl, 10 mM glucose, 10 mM HEPES, 2 mM CaCl2, and 1 mM MgCl2 (pH 7.3, adjusted with NaOH). The cells were immediately fixed with 2% paraformaldehyde, 2% glutaraldehyde, and 1% tannic acid or 1% acrolein and 1% tannic acid for 30 min at room temperature. The results obtained from both fixation methods were undistinguishable. The cells were stained with 1% OsO4 for 1 hour, en bloc stained with 0.25–0.5% uranyl acetate in acetate buffer at pH 5.0 for 1 h at 4°C, and gradually dehydrated with increasingly pure solutions of ethanol (50%, 70%, 90%, and then 100%) before infiltration with ethanol as a diluent and embedding with Embed-812, then cured over night at 50°C and then at 60°C for 36 hours. The embedded samples were sectioned on an ultramicrotome with thicknesses of 50–70 nm. The resulting sections were placed on 400 mesh carbon coated copper grids.
Single images were collected at up to 73,000X magnification on a JEOL JEM-200CX, 120kV electron microscope with an AMT XR-100 CCD. Tilt-series were collected with SerialEM32 at 15,000X magnification on a JEOL 2100, 200kV electron microscope, with an Orius 832 CCD. Tilt-series were acquired at tilt increments of 1° from −70° to +70°. Tomographic reconstruction was performed using the IMOD software suite33 using patch-tracking or 10 nm gold fiducials for alignment, with slices of 1 nm thickness examined for hemi-fusion structures.
Data selection and statistics
The data within the first 2 min after whole-cell break-in were used to avoid whole-cell endocytosis rundown19, 34. The statistical test used is t test (two-sided) and ANOVA test. The data were expressed as mean ± s.e.m. Sample size was chosen based on our previous experience. The present work did not involve cell authentication, mycoplasma testing, randomization or blinding.
Extended Data
Extended Data Table 1. Mean ± s.d. values in main figures.
a. | |||||||
---|---|---|---|---|---|---|---|
Same-onset (%) |
PH-earlier (%) |
PH-only (%) |
Close fusion (%) |
Cell # |
Animal # |
||
Fig 1g | 53.8 ± 23.0 | 13.9 ± 19.0 | 32.3 ± 20.8 | 16 | 5 | ||
Fig. 1i | 60.4 ± 14.2 | 14.7 ± 12.2 | 24.9 ± 9.8 | 7 | 5 | ||
Fig. 4c | Ctrl | 54.6 ± 16.7 | 15.8 ± 16.9 | 29.7 ± 16.7 | 60.9 ± 15.0 | 19 | 6 |
Dynasore | 81.4 ± 18.8 (P< 0.0001) |
12.2 ± 12.2 (P = 0.8201) |
6.4 ± 8.2 (P< 0.0001) |
17.9 ± 12.1 (P< 0.0001) |
23 | 7 | |
Fig. 4d | Vector | 57.4 ± 23.5 | 12.9 ± 11.1 | 29.7 ± 18.1 | 58.4 ± 12.9 | 21 | 5 |
DynI-K44A | 91.4 ± 14.7 (P< 0.0001) |
6.1 ± 9.6 (P = 0.3440) |
2.5 ± 6.7 (P< 0.0001) |
13.4 ± 10.0 (P< 0.0001) |
23 | 6 | |
Fig. 4e | si-Ctrl | 52.2 ± 15.7 | 13.5 ± 8.7 | 34.2 ± 12.2 | 60.6 ± 11.7 | 24 | 6 |
si-Dyn | 78.1 ± 14.9 (P< 0.0001) |
10.6 ± 9.7 (P = 0.7872) |
11.3 ± 10.6 (P< 0.0001) |
20.0 ± 18.8 (P< 0.0001) |
24 | 7 |
b. | ||||
---|---|---|---|---|
Group 1 | Group 2 | Group 3 | Group 4 | |
ICa (pA) | 204.0 ± 52.0 | 298.6 ± 34.4 | 517.5 ± 81.2 | 829.0 ± 41.6 |
PH-only (%) | 13.8 ± 19.1 | 26.7 ± 21.3 | 39.4 ± 10.9 | 41.6 ± 10.0 |
Full fusion (%) | 83.2 ± 19.1 | 73.3 ± 21.3 | 60.6 ± 10.9 | 58.4 ± 10.0 |
Cell # | 5 | 7 | 6 | 6 |
Supplementary Material
Acknowledgments
We thank Drs. Tamas Balla, Gero Miesenböck, and Dong-Sheng Wang for providing construct containing GFP-fused PH domain of PLCδ1, VAMP2-pHluorin construct, and dynamin siRNA, respectively. We thank Dr. Carolyn Smith for technical support of STED microscopy. We thank Susan Cheng and Virginia Crocker of NINDS EM facility for their EM technical support. This work was supported by the National Institute of Neurological Disorders and Stroke Intramural Research Program (ZIA NS003009-13 and ZIA NS003105-08) and National Institute on Deafness and other Communication Disorders (NIDCD) Intramural Research Program (Z01-DC000002 and NIDCD Advanced Imaging Core ZIC DC000081).
Footnotes
Author contributions. W.D.Z, E.H., W.S. performed and analysed most experiments, P.J.W. and H.C.C. initiated STED imaging of PH-EGFP, E.S.K., S.A.V. and B.K. performed electron microscopic works. L.G.W. designed experiments and wrote the manuscript with helps from all authors. W.D.Z. and E.H. participated in designing experiments and writing the manuscript.
The authors declare no competing financial interest.
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