1. The Presynaptic v-ATPase Reversibly Disassembles and Thereby Modulates Exocytosis but Is Not Part of the Fusion Machinery 
Anna Bodzęta, Martin Kahms, Jürgen Klingauf 
Cell Reports 
Volume 20, Issue 6, Pages (August 2017)
DOI: /j.celrep
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2. Cell Reports 2017 20, 1348-1359DOI: (10.1016/j.celrep.2017.07.040)
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3. Figure 1
Fluorescently Tagged v-ATPase Subunits Incorporate into v-ATPases at Presynaptic Boutons
(A) Scheme of v-ATPase structure. Subunits used in this study are labeled.
(B) Confocal images of hippocampal neurons expressing GFP-tagged v-ATPase subunits. Scale bar, 10 μm.
(C) Co-localization of TdTom-V1B2 and the presynaptic marker Syb2-pHl. Scale bar, 10 μm.
(D) Average fluorescence transients of Syb2-pHl in response to 200 APs at 20 Hz from boutons co-expressing TdTom-V1B2 (n = 12 coverslips, 50–300 boutons each).
(E and F) Pseudocolor images of V0a1-GFP and GFP-V1B2 distributions along the axon at rest and during stimulation. Scale bar, 2 μm. Normalized average signal of fluorescence at boutons and adjacent axonal regions in response to stimulation for (E) V0a1-GFP and (F) GFP-V1B2 (n = 17 and 16 boutons respectively).
Error bars represent SEM. See also Figures S1 and S2.
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4. Figure 2
Presynaptic Boutons Feature a PM Pool of v-ATPases at Rest that Participates in SV Recycling
(A) Fluorescence signal from neurons expressing V0c-pHl at rest, during stimulation, during an acidic pulse to quench the surface pool, and during superfusion with NH4Cl to unmask the internalized fraction. Scale bar, 10 μm.
(B) Normalized average fluorescence transients of Syp-pHl and V0c-pHl (n = 6–16 coverslips, 50–200 boutons each).
(C and D) Half time (τ1/2) of recovery (C) and plasma membrane pool (D) of the pHluorin-based reporters. ∗p < 0.05 (unpaired t test).
(E) Fluorescence signal of V0c-pHl before and after selective photobleaching of the PM pool and after stimulation (50 APs at 20 Hz). Scale bar, 5 μm.
(F) Fluorescence transients of V0c-pHl in response to 50 APs at 20 Hz before and after selective photobleaching (n = 18 coverslips, 50–250 boutons each).
(G) Confocal images of V0c-pHl transfected neurons upon live labeling of the PM pool of V0c-pHl with an anti-GFP Alexa647 nanobody (NB anti-GFP-A647) and subsequent co-staining with Bassoon (Alexa568-conjugated secondary antibody, anti-Bassoon-A568). Scale bars, 2 μm.
(H) Images of neurons co-expressing V0a1-TdTom and YFP-RIM3. Scale bar, 10 μm.
(I) Fluorescence transients of the pH-dependent YFP-RIM3 signal in response to a stimulation train of 900 APs at 20 Hz under control conditions and in presence of the v-ATPase blocker Baf (1 μM, 2 min; n = 14 coverslips for control, 10 coverslips for Baf, 50–200 boutons each). Experiments were performed in bicarbonate buffer gassed with 5% CO2.
Error bars represent SEM. See also Figure S3.
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5. Figure 3 FRAP Analysis Reveals Transient v-ATPase Assembly
(A) Exemplary images from a FRAP time series in neurons expressing GFP-V1B2 under silenced conditions (0 mM Ca2+, 1 μM TTX). The dotted circle highlights the bleached bouton. Scale bar, 1 μm.
(B) 3D plot of the integrated fluorescence signal from (A).
(C) Normalized fluorescence recovery of GFP and GFP-tagged v-ATPase subunits under silenced conditions (n = 40–75 boutons).
(D) Ratios of GFP-V1B2 and V1C1-GFP fluorescence intensities in boutons over those in axons (enrichment factors, Ef) before and after the FRAP experiment (n = 25 boutons for V1B2, 18 boutons for V1C1). Enrichment factors were corrected for apparent enrichment due to larger bouton volume using the measured ratios of GFP-transfected neurons.
(E) Neurons expressing Mitotrap-YFP and TdTom-FKBP-V1B2 before and after application of rapamycin (rap; 500 nM, 2 min). Scale bar, 10 μm.
(F) Intensity line profiles along axons highlighted in (E) before and after application of rap. Almost complete re-routing of TdTom-FKPB-V1B2 to mitochondria within 2 min was observed.
(G) Recovery curves of GFP and GFP-tagged v-ATPase subunits like in (C) but normalized to the fluorescence signal after 180 s (n = 40–60 boutons).
(H) Quantification of half times of fluorescence recovery (τ1/2) of GFP and GFP-tagged v-ATPase subunits. All FRAP experiments were performed at 37°C. ∗∗p < (unpaired t test). Error bars represent SEM.
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6. Figure 4
The Affinity of the V1 Domain to the V0 Membrane Sector Depends on the Lumenal pH of SVs
(A) FRAP recovery curves of GFP-V1B2 under silenced conditions (0 mM Ca2+, 1 μM TTX) under depolarizing conditions (45 mM K+) and under silenced conditions in the presence of 50 mM NH4Cl or the protonophore FCCP (1 μM; n = 40–95 boutons).
(B) FRAP recovery curves of GFP-V1B2 under silenced conditions and under silenced conditions in presence of Baf (1 μM) and SaliPhe (1 μM; n = 40–130 boutons).
(C) Normalized fluorescence recovery curves of V1C1-GFP under silenced, depolarizing conditions and under depolarizing conditions in presence of SaliPhe (n = boutons). All FRAP experiments were performed at 37°C.
Error bars represent SEM. See also Figure S4.
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7. Figure 5
Artificially Induced v-ATPase Assembly Induces Use- and Time-Dependent Depression of SV Release
(A) Average fluorescence transients of V0c-pHl in response to 50 APs at 20 Hz for control and in the presence of SaliPhe (1 μM, 2 min) measured at the same boutons. Traces were normalized to the fluorescence signal during NH4Cl superfusion at the end of the experiment (not depicted).
(B and C) V0c-pHl (B) and Syb2-pHl (C) exocytosis amplitudes in response to 50 APs at 20 Hz slightly decreased after treatment with SaliPhe, while after application of Baf (1 μM, 2 min), exocytosis amplitudes slightly increased. Black squares represent mean values; gray diamonds represent real exocytosis amplitudes estimated by linear back-extrapolation of the post-stimulus endocytosis rate.
(D) Average fluorescence transients of V0c-pHl like in (A), but for of 200 APs at 20 Hz.
(E and F) V0c-pHl (E) and Syb2-pHl (F) exocytosis amplitudes in response to 200 APs at 20 Hz decreased upon treatment with SaliPhe, but not with Baf.
(G) Average V0c-pHl fluorescence transients for two consecutive stimuli (50 APs and 200 APs at 20 Hz) with a 2 s interval.
(H) V0c-pHl release amplitudes from (G).
(I) Average V0c-pHl fluorescence transients in response to multiple spaced stimuli (4 × 50 APs at 20 Hz with 60 s interval) in presence of SaliPhe and Baf (n = 5 coverslips for SaliPhe, 9 coverslips for Baf, each 50–300 boutons). Traces were normalized to the amplitude of the first response.
(J) Quantification of exocytosis amplitudes from (I).
Error bars represent SEM. See also Figure S5.
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8. Figure 6
The V0 and V1 Domains of v-ATPase Disperse Differently upon Stimulation
(A) Images of hippocampal neurons co-expressing V0a1-GFP and TdTom-V1B2 at rest and during stimulation. Scale bar, 5 μm.
(B) Kymographs showing the redistribution of V0a1-GFP and TdTom-V1B2 from bouton (b) to neighboring axonal regions (a) during stimulation. Scale bar, 2 μm. Time bar, 2 s.
(C and D) Exemplary time courses of V0a1-GFP and TdTom-V1B2 dispersion into axonal regions in response to 100 APs at 50 Hz for control (C) and in the presence of SaliPhe (D) and corresponding fits with logistic functions.
(E) Quantification of differences in τ95 (time points of 95% dispersion amplitude) for activity-dependent dispersion of v-ATPase subunits into axonal regions (n = 14 coverslips, 70 boutons for V0a1-GFP/TdTom-V1B2; n = 11 coverslips, 80 boutons for V0a1-GFP/V0a1-TdTom; and n = 5 coverslips, 37 boutons for V0a1-GFP/V1B2-TdTom in the presence of SaliPhe). Δτ95 was calculated as the difference between the τ95 values of the GFP-tagged and the TdTom-tagged subunit obtained from the same bouton. Boxes represent 25th/75th percentiles, solid lines represent mean, dashed lines represent median, and whiskers represent minima/maxima. ∗p < 0.02 (unpaired t test).
See also Figure S6.
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9. Figure 7
A Minimal Reaction-Diffusion Model Reproduces Experimental FRAP Data
(A) Schematic of discretization in space for numerical simulation of 1D diffusion and reaction. Veff: effective presynaptic cytosolic volume; gray region: bouton grid elements with diffusion sink representing homogeneously distributed immobile V0 sectors.
(B) Kymographs of experimental and simulated FRAP data for V1 diffusion-reaction under silenced conditions.
(C) Normalized FRAP curves of GFP-V1B2 under silenced, depolarizing conditions and under silenced conditions in the presence of NH4Cl and fit with the “kinetic” numerical model by a Levenberg-Marquardt algorithm with koff and bound fraction Fbound as free parameters (Table S1).
(D) Recovery curves of GFP-V1B2 like in (C) but normalized to the fluorescence signal before photobleaching and fit with the “amplitude” numerical model. Both numerical models differ in axon length and thus the extent of diffusional exchange.
(E) Model of v-ATPase assembly/disassembly coupled to the SV exo/endocytosis cycle. (1) v-ATPases of acidified and fully NT-loaded SVs disassemble into three parts: the V0 membrane sector, the cytosolic core V1 domain, and V1C1. (2) During exocytosis, SVs devoid of V1 preferentially fuse with the PM. (3) Upon exposure of the lumenal part of V0 to the neutral extracellular pH, V1 is recruited to form an assembled holoenzyme at the presynaptic PM. SVs with fully assembled v-ATPases are endocytosed. (4) v-ATPase pumps protons to generate the proton-electrochemical gradient for NT uptake. When the final intravesicular pH is established, v-ATPase disassembles and SVs gain full fusion competence. The exact role of V1C1 during this assembly cycle remains elusive.
See also Figure S7 and Table S1.
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