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Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca2+ channel–vesicle coupling

Nature Neuroscience volume 19, pages 1311–1320 (2016)Cite this article

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Abstract

Brain function relies on fast and precisely timed synaptic vesicle (SV) release at active zones (AZs). Efficacy of SV release depends on distance from SV to Ca2+ channel, but molecular mechanisms controlling this are unknown. Here we found that distances can be defined by targeting two unc-13 (Unc13) isoforms to presynaptic AZ subdomains. Super-resolution and intravital imaging of developing Drosophila melanogaster glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent AZs by the scaffolding proteins Syd-1 and Liprin-α, and Unc13A was positioned by Bruchpilot and Rim-binding protein complexes at maturing AZs. Unc13B localized 120 nm away from Ca2+ channels, whereas Unc13A localized only 70 nm away and was responsible for docking SVs at this distance. Unc13Anull mutants suffered from inefficient, delayed and EGTA-supersensitive release. Mathematical modeling suggested that synapses normally operate via two independent release pathways differentially positioned by either isoform. We identified isoform-specific Unc13-AZ scaffold interactions regulating SV-Ca2+-channel topology whose developmental tightening optimizes synaptic transmission.

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Figure 1: Generating mutants of Unc13A and Unc13B.
Figure 2: In vivo analysis of Unc13A and Unc13B accumulation.
Figure 3: Two different scaffold complexes organize the AZ localization of Unc13 isoforms.
Figure 4: Unc13A is essential for fast, efficient synaptic transmission.
Figure 5: Two-color STED microscopy analysis of Unc13A and Unc13B organization at Drosophila NMJ synapses.
Figure 6: Unc13A and Unc13B localized in distinct distances from the presynaptic Ca2+ channels.
Figure 7: Unc13A increased extracellular Ca2+ sensitivity, release probability and mediated tight SV to Ca2+ channel coupling.

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Acknowledgements

This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) to S.J.S. (Exc 257, TP A3 and A6 SFB 958, TP B9/SFB665; TP09/SFB740), and J.H.D., M.C.W. and B.L. (TP A6/SFB958). A.M.W. was supported by a DFG Emmy Noether grant. M.A.B. was supported by a PhD fellowship from the graduate school GRK 1123 funded by the DFG and by the SFB 740. A.T.G. was supported by a NeuroCure PhD fellowship within the International Graduate Program Medical Neurosciences funded by the DFG (Exc 257). We thank M. Brünner and A. Stawrakakis for excellent technical assistance, S. Sweeney for help with the EMS screen and T. Matkovic for help with cloning.

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Author notes

  1. Mathias A Böhme, Christina Beis, Suneel Reddy-Alla and Eric Reynolds: These authors contributed equally to this work.

  2. Alexander M Walter and Stephan J Sigrist: These authors jointly directed this work.

Authors and Affiliations

  1. Institute for Biology/Genetics, Freie Universität Berlin, Berlin, Germany

    Mathias A Böhme, Christina Beis, Suneel Reddy-Alla, Eric Reynolds, Malou M Mampell, Janine Lützkendorf, Dominique Dufour Bergeron, Husam Babikir & Stephan J Sigrist

  2. NeuroCure, Cluster of Excellence, Charité Universitätsmedizin, Berlin, Germany

    Mathias A Böhme, Andreas T Grasskamp & Stephan J Sigrist

  3. Molecular and Theoretical Neuroscience, Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany

    Andreas T Grasskamp & Alexander M Walter

  4. Institute of Chemistry and Biochemisty/Structural Biochemistry, Freie Universität Berlin, Berlin, Germany

    Jan H Driller, Markus C Wahl & Bernhard Loll

  5. Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Fabian Göttfert & Stefan W Hell

  6. Plymouth University Peninsula School of Medicine, Plymouth University, Plymouth, UK

    Iain M Robinson

  7. Department of Genetics, University of Cambridge, Cambridge, UK

    Cahir J O'Kane

  8. Institute of Pharmaceutical Sciences, Pharmaceutical Chemistry, University of Graz, Graz, Austria

    Ulrich Stelzl

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  1. Mathias A Böhme
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Contributions

M.A.B., S.R.-A., A.M.W. and S.J.S. conceived the project. M.A.B. and M.M.M. performed all confocal and in vivo imaging experiments and analyzed the data. M.A.B. performed STED experiments and analyzed the data. C.B., S.R.-A. and E.R. performed all electrophysiological experiments, and C.B. and E.R. analyzed the data. J.L., U.S. and J.H.D. performed and analyzed Y2H experiments. M.C.W. and B.L. contributed to the Y2H experiments and analysis. C.B. and D.D.B. performed HPF experiments and C.B. analyzed the data. A.T.G. performed modeling and simulation. H.B. and M.M.M. created antibodies and isoform-specific mutants. I.M.R. and C.J.O. performed the EMS screen. F.G. and S.W.H. developed and built the STED microscope. M.A.B., A.M.W. and S.J.S. wrote the paper with input from all coauthors.

Corresponding authors

Correspondence to Alexander M Walter or Stephan J Sigrist.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Liprin-ɑ/Syd-1 scaffold complexes organize the AZ localization of Unc13B

(a,b) Muscle 4 NMJs of segments A2-A4 from 3rd instar larvae of the displayed genotypes labelled with the antibodies (ABs) indicated. (c) Mean BRP intensity measured over the whole NMJ was unchanged in Syd-1null and Liprin-αnull in comparison to the Wild type (Wild type (n=13 NMJs from 4 larvae) vs. Syd-1null (n=11 NMJs from 4 larvae) vs. Liprin-αnull (n=12 NMJs from 4 larvae): p >0.05 for Wild type vs Syd-1null; p >0.05 for Wild type vs. Liprin-αnull; p >0.05 for Syd-1null vs. Liprin-αnull; p=0.2883 (F(2,33)=1.29)). (d) BRP spots per µm² NMJ were slightly reduced in Syd-1null and significantly reduced in Liprin-αnull in comparison to the Wild type (Wild type (n=13 NMJs from 4 larvae) vs. Syd-1null (n=11 NMJs from 4 larvae) vs. Liprin-αnull (n=12 NMJs from 4 larvae): p >0.05 for Wild type vs Syd-1null; p ≤0.01 for Wild type vs. Liprin-αnull; p >0.05 for Syd-1null vs. Liprin-αnull; p=0.0108 (F(2,33)=5.207)). (e) Mean Unc13A intensity measured over the whole NMJ was unchanged in Syd-1null and Liprin-αnull in comparison to the Wild type (Wild type (n=13 NMJs from 4 larvae) vs. Syd-1null (n=11 NMJs from 4 larvae) vs. Liprin-αnull (n=12 NMJs from 4 larvae): p >0.05 for Wild type vs Syd-1null; p >0.05 for Wild type vs. Liprin-α null; p >0.05 for Syd-1null vs. Liprin-αnull; p=0.2105 (F(2,33)=1.63)). (f) Unc13A spots per µm² NMJ were slightly reduced in Syd-1null and Liprin-αnull in comparison to the Wild type (Wild type (n=13 NMJs from 4 larvae) vs. Syd-1null (n=11 NMJs from 4 larvae) vs. Liprin-αnull (n=12 NMJs from 4 larvae): p ≤0.05 for Wild type vs Syd-1null; p >0.05 for Wild type vs. Liprin-αnull; p >0.05 for Syd-1null vs. Liprin-αnull; p=0.0278 (F(2,33)=4.00)). (g,h) Muscle 4 NMJs of segments A2-A4 from 3rd instar larvae of the displayed genotypes labelled with the ABs indicated. (i) Mean Unc13B intensity measured over the whole NMJ was slightly reduced in Syd-1null but severely reduced in Liprin-αnull in comparison to the Wild type (Wild type (n=13 NMJs from 5 larvae) vs. Syd-1null (n=11 NMJs from 5 larvae) vs. Liprin-αnull (n=15 NMJs from 5 larvae): p >0.05 for Wild type vs Syd-1null; p ≤0.001 for Wild type vs. Liprin-αnull; p ≤0.01 for Syd-1null vs. Liprin-αnull; p <0.0001 (F(2,36)=15.13)). (j) Unc13B spots per µm² NMJ were significantly reduced in Syd-1null but severely reduced in Liprin-αnull in comparison to the Wild type (Wild type (n=13 NMJs from 5 larvae) vs. Syd-1null (n=11 NMJs from 5 larvae) vs. Liprin-αnull (n=15 NMJs from 5 larvae): p ≤0.01 for Wild type vs Syd-1null; p ≤0.001 for Wild type vs. Liprin-αnull; p ≤0.001 for Syd-1null vs. Liprin-αnull; p <0.0001 (F(2,36)=42.93)). Number and p values are listed in Supplementary Table 1. Statistics for c-f, i, j: nonparametric one-way analysis of variance (ANOVA) test, followed by a Turkey’s multiple comparison test. All panels show mean ± SEM; *, p ≤0.05; **, p ≤0.01; ***, p ≤0.001; ns, not significant, p >0.05. Scale bars: 5 μm.

Supplementary Figure 2 Unc13B interacts with Syd-1/Liprin-α; Unc13A interacts with BRP/RBP

(a) Schematic representation of Unc13B N-terminus including three fragments (1-3) that were used in the Y2H screen; Liprin-α domain structure containing three C-terminal SAM domains (I-III from the N terminus); Syd-1 domain structure containing an N-terminal PDZ domain, a C2 and a Rho-GAP domain. The corresponding fragments of each protein used in the Y2H screen are indicated. A central N-terminal fragment of Unc13B interacted with an N-terminal part of Liprin-α. Both very N-terminal fragments of Unc13B interacted with a central stretch of Syd-1 located in-between PDZ- and C2-domain. (b) Schematic representation of Unc13A N-terminus including three fragments (1-3) that were used in the Y2H screen. The RBP-binding PxxP motif is indicated; RBP domain structure containing three SH3 domains (I-III from the N terminus) and three Fibronectin 3 (FN3) domains; BRP domain structure containing several coiled-coil (CC) domains. The corresponding fragments of each protein used in the Y2H screen are indicated. The most N-terminal fragment of Unc13A (including the RBP binding PxxP motif) interacted with both C-terminal fragments of RBP including the SH3-domains II and III, and with an N-terminal part of BRP.

Supplementary Figure 3 BRP/RBP scaffold complexes organize the AZ-localization of Unc13A

(a-c) Muscle 4 NMJs of segments A2-A4 from 3rd instar larvae of the displayed genotypes labelled with the ABs indicated. BRP as well as Unc13A were severely decreased upon motoneuronal downregulation of BRP or in Rbpnull mutants. (d,e) BRP as well as Unc13A intensity were severely decreased upon motoneuronal downregulation of BRP or in Rbpnull mutants with the strongest downregulation upon Brp knockdown in Rbpnull (BRP intensity: Ctrl (n=14 NMJs from 5 larvae) vs. BrpRNAi (n=15 NMJs from 5 larvae) vs. Rbpnull (n=15 NMJs from 5 larvae) vs. BrpRNAi;RBPnull (n=13 NMJs from 5 larvae): p ≤0.001 for Ctrl vs BrpRNAi; p ≤0.001 for Ctrl vs. Rbpnull; p ≤0.001 for Ctrl vs. BrpRNAi;RBPnull; p ≤0.05 for BrpRNAi vs. Rbpnull; p >0.05 for BrpRNAi vs. BrpRNAi;RBPnull; p ≤0.05 for Rbpnull vs. BrpRNAi;RBPnull; p <0.0001 (F(3,53)=31.96)); Unc13A intensity: Ctrl (n=14 NMJs from 5 larvae) vs. BrpRNAi (n=15 NMJs from 5 larvae) vs. Rbpnull (n=15 NMJs from 5 larvae) vs. BrpRNAi;RBPnull (n=13 NMJs from 5 larvae): p ≤0.001 for Ctrl vs BrpRNAi; p ≤0.001 for Ctrl vs. Rbpnull; p ≤0.001 for Ctrl vs. BrpRNAi;RBPnull; p >0.05 for BrpRNAi vs. Rbpnull; p >0.05 for BrpRNAi vs. BrpRNAi;RBPnull; p >0.05 for Rbpnull vs. BrpRNAi;RBPnull; p <0.0001 (F(3,53)=30.07)). (f) Unc13A spots per µm² NMJ were decreased upon motoneuronal downregulation of BRP or in Rbpnull mutants with the strongest downregulation upon Brp knockdown in Rbpnull (Ctrl (n=14 NMJs from 5 larvae) vs. BrpRNAi (n=15 NMJs from 5 larvae) vs. Rbpnull (n=15 NMJs from 5 larvae) vs. BrpRNAi;RBPnull (n=13 NMJs from 5 larvae): p ≤0.001 for Ctrl vs BrpRNAi; p ≤0.001 for Ctrl vs. Rbpnull; p ≤0.001 for Ctrl vs. BrpRNAi;RBPnull; p ≤0.01 for BrpRNAi vs. Rbpnull; p >0.05 for BrpRNAi vs. BrpRNAi;RBPnull; p ≤0.001 for Rbpnull vs. BrpRNAi;RBPnull; p <0.0001 (F(3,53)=63.28)). (g-i) Muscle 4 NMJs of segments A2-A4 from 3rd instar larvae of the genotypes indicated labelled with the ABs indicated. BRP but not Unc13B were severely decreased upon motoneuronal downregulation of BRP as well as in Rbpnull mutants. (j) Mean Unc13B intensity measured over the whole NMJ was not affected upon downregulation of BRP or in Rbpnull mutants or even upon Brp knockdown in Rbpnull (Ctrl (n=12 NMJs from 5 larvae) vs. BrpRNAi (n=11 NMJs from 5 larvae) vs. Rbpnull (n=13 NMJs from 5 larvae) vs. BrpRNAi;RBPnull (n=10 NMJs from 5 larvae): p >0.05 for Ctrl vs BrpRNAi; p >0.05 for Ctrl vs. Rbpnull; p >0.05 for Ctrl vs. BrpRNAi;RBPnull; p >0.05 for BrpRNAi vs. Rbpnull; p >0.05 for BrpRNAi vs. BrpRNAi;RBPnull; p >0.05 for Rbpnull vs. BrpRNAi;RBPnull; p=0.3491 (F(3,42)=1.127)). (k) Unc13B spots per µm² NMJ were unchanged upon downregulation of BRP or in Rbpnull mutants or even upon Brp knockdown in Rbpnull (Ctrl (n=12 NMJs from 5 larvae) vs. BrpRNAi (n=11 NMJs from 5 larvae) vs. Rbpnull (n=13 NMJs from 5 larvae) vs. BrpRNAi;RBPnull (n=10 NMJs from 5 larvae): p >0.05 for Ctrl vs BrpRNAi; p >0.05 for Ctrl vs. Rbpnull; p >0.05 for Ctrl vs. BrpRNAi;RBPnull; p >0.05 for BrpRNAi vs. Rbpnull; p >0.05 for BrpRNAi vs. BrpRNAi;RBPnull; p >0.05 for Rbpnull vs. BrpRNAi;RBPnull; p=0.6570 (F(3,42)=0.5408)). Number and p values are listed in Supplementary Table 1. Statistics for d-f, j,k: nonparametric one-way analysis of variance (ANOVA) test, followed by a Turkey’s multiple comparison test. All panels show mean ± SEM; *, p ≤0.05; **, p ≤0.01; ***, p ≤0.001; ns, not significant, p >0.05. Scale bars: 5 μm.

Supplementary Figure 4 TEVC analysis of Unc13A and Unc13B mutant terminals

(a,b) The time-to-peak (time difference between stimulation pulse to the afferent nerve and the eEJC minimum) and eEJC decay, which is the time constant τ resulting from a single exponential fit in the range from 60% of the eEJC amplitude back to baseline, both are similar in Ctrl (black) and Unc13Bnull (blue) (time to peak: Ctrl (n=12 NMJs from 12 larvae) vs Unc13Bnull (n=12 NMJs from 12 larvae), p=0.1333 (t(22)=1.559); eEJC decay: Ctrl (n=12 NMJs from 12 larvae) vs Unc13Bnull (n=11 NMJs from 11 larvae), p=0.2413 (t(21)=1.206)). (c) The time to peak is significantly prolonged in Unc13Anull mutant synapses (Wild type (n=12 NMJs from 12 larvae) vs Unc13Anull (n=12 NMJs from 12 larvae), p=0.0162 (t(22)=2.605)). (d) The eEJC decay is similar in Wild type and Unc13Anull (Wild type (n=12 NMJs from 12 larvae) vs Unc13Anull (n=9 NMJs from 9 larvae), p=0.2136 (U=36)). (e) The mEJC rise time is unaltered in Unc13Bnull mutant synapses compared to Ctrl (Ctrl (n=14 NMJs from 10 larvae) vs Unc13Bnull (n=7 NMJs from 5 larvae), p=0.7652 (U=44.5)). (f) In Unc13Bnull the mEJC decay is significantly increased compared to Ctrl (Ctrl (n=14 NMJs from 10 larvae) vs Unc13Bnull (n=7 NMJs from 5 larvae), p=0.0480 (U=22.00)). (g,h) mEJC kinetics do not differ between Wild type and Unc13Anull (mEJC rise time: Wild type (n=12 NMJs from 6 larvae) vs Unc13Anull (n=11 NMJs from 6 larvae), p=0.1914 (t(21)=1.350); mEJC decay: Wild type (n=12 NMJs from 6 larvae) vs Unc13Anull (n=11 NMJs from 6 larvae), p=0.2546 (t(21)=1.171)). All recordings were performed in the presence of 1.5 mM extracellular Ca2+. Number and p values are listed in Supplementary Table 1. Statistics: Student's t-test besides panels d,e,f where a Mann Whitney U-test was performed. All panels show mean ± SEM; *, p ≤0.05; **, p ≤0.01; ***, p ≤0.001; ns, not significant, p >0.05.

Supplementary Figure 5 Ca2+ sensitivity and release probability is altered upon loss of Unc13A but not -B

(a-f) Plot of eEJC amplitude as a function of extracellular Ca2+ concentrations [Ca2+]ext fitted with Hill equations to determine the values for slope and KD. A clear shift can be observed in (a) Unc13Anull mutant synapses (red) compared to Wild type (black), whereas in (d) there is no change upon loss of Unc13Bnull (blue) compared to Ctrl (black) (a: Wild type (n=12 NMJs from 12 larvae per Ca2+ concentration) vs Unc13Anull (n=10 NMJs from 10 larvae per Ca2+ concentration): 0.75 mM [Ca2+]ext: p=0.0092 (U=20); 1.5 mM [Ca2+]ext: p <0.0001 (U=0); 3 mM [Ca2+]ext: p=0.0005 (U=7); 6 mM [Ca2+]ext: p=0.0272 (U=26); 10 mM [Ca2+]ext: p=0.0062 (U=18)); d: Ctrl (n=12 NMJs from 12 larvae per Ca2+ concentration) vs Unc13Bnull (n=12 NMJs from 12 larvae per Ca2+ concentration): 0.75 mM [Ca2+]ext: p=0.1971 (t(22)=1.330); 1.5 mM [Ca2+]ext: p=0.2652 (t(22)=1.143); 3 mM [Ca2+]ext: p=0.9269 (t(22)=0.09278); 6 mM [Ca2+]ext: p=0.5181 (t(22)=0.6569); 10 mM [Ca2+]ext: p=0.6284 (t(22)=0.4908)). The values for I/Imax can be found in Supplementary Table 1. (b) Ca2+-dependence of release analysis revealed an increased Ca2+ requirement (KD, obtained from fitting with the Hill function) in Unc13Anull mutant synapses (Wild type (n=12 NMJs from 12 larvae) vs Unc13Anull (n=10 NMJs from 10 larvae), p=0.0004 (U=6)). (c) The apparent Ca2+ cooperativity of release (slope, obtained from fitting with the Hill function) is not different in Unc13Anull relative to Wild type (Wild type (n=12 NMJs from 12 larvae) vs Unc13Anull (n=10 NMJs from 10 larvae), p=0.6682 (U=53)). (e,f) The Ca2+-dependence and Ca2+-cooperativity of release are both unaltered upon loss of Unc13B (KD: Ctrl (n=12 NMJs from 12 larvae) vs Unc13Bnull (n=12 NMJs from 12 larvae), p=0.9566 (t(22)=0.05502); slope: Ctrl (n=12 NMJs from 12 larvae) vs Unc13Bnull (n=12 NMJs from 12 larvae), p=0.1574 (t(22)=1.464)). (g) Sample traces of paired pulse stimulation for Ctrl (black) and Unc13Bnull (blue) at 10 ms ISI show no differences between genotypes. (h) The paired pulse ratios were not significantly changed in Unc13Bnull at 10 ms ISI, in all Ca2+ concentrations (Ctrl (n=12 NMJs from 12 larvae per Ca2+ concentration) vs Unc13Bnull (n=12 NMJs from 12 larvae per Ca2+ concentration): 0.75 mM [Ca2+]ext: p=0.1971 (t(22)=1.33); 1.5 mM [Ca2+]ext: p=0.1678 (t(22)=1.426); 3 mM [Ca2+]ext: p=0.474 (t(22)=0.7284); 6 mM [Ca2+]ext: p=0.3726 (t(22)=0.9102); 10 mM [Ca2+]ext: p=0.2602 (t(22)=1.156)). Values can be found in Supplementary Table 1. (i) Unc13Anull (gold: control with DMSO, red: with EGTA-AM/DMSO) shows faster and stronger inhibition of eEJC amplitudes after addition of 200 µM EGTA-AM to the extra-cellular solution compared to Wild type (black: control with DMSO, blue: with EGTA-AM/DMSO). Amplitudes are normalized to average eEJC amplitudes obtained during 1 min of baseline recording prior to the addition of EGTA-AM/DMSO or DMSO, each with Pluronic F-127. Synaptic transmission was stimulated by single action potentials every 10 s. Experiments were performed in the presence of 2.5 mM extracellular Ca2+. Values can be found in Supplementary Table 1. (j) The time constant of the inhibition caused by EGTA-AM application was determined by fitting a single exponential decay function to 100 peak amplitude values after addition of EGTA-AM in individual cells. This revealed a significantly faster inhibition in Unc13Anull compared to Wild type animals (Wild type + EGTA (n=10 NMJs from 10 larvae) vs Unc13Anull + EGTA (n=10 NMJs from 10 larvae), p=0.0012 (t(18)=3.835)). (k) The asymptotic inhibition is captured in the exponential fit as the plateau value which was significantly decreased in Unc13Anull in comparison to Wild type (Wild type + EGTA (n=10 NMJs from 10 larvae) vs Unc13Anull + EGTA (n=10 NMJs from 10 larvae), p=0.016 (t(18)=2.6508)). (l) 30 min incubation with the fast Ca2+-buffer Bapta-AM reduced eEJC amplitudes in both genotypes to a similar extent. Sample traces for Wild type (black with DMSO, blue with Bapta-AM/DMSO) and Unc13Anull (gold with DMSO, red with Bapta-AM/DMSO) exhibit similar Bapta-sensitivity for both genotypes. For clarity, the stimulation artefact was removed and replaced by a straight line. (m,n) The significant reduction of the eEJC amplitude after 30 min Bapta-AM incubation is similar in Wild type (m) and Unc13Anull (n) compared to DMSO incubated cells (m: Wild type + DMSO (n=15 NMJs from 9 larvae) vs Wild type + Bapta (n=14 NMJs from 9 larvae), p <0.0001 (t(27)=12.59); n: Unc13Anull + DMSO (n=14 NMJs from 10 larvae) vs Unc13Anull + Bapta (n=14 NMJs from 8 larvae), p=0.0004 (t(26)=4.095)). Values can be found in Supplementary Table 1. (o) The Bapta sensitivity is calculated as the ratio of eEJC amplitude size in the presence of Bapta-AM/DMSO to the eEJC amplitude size in the presence of DMSO. The Bapta-sensitivity does not differ between Wild type and Unc13Anull (Bapta sensitivity: Wild type (n=14 NMJs from 9 larvae) vs Unc13Anull (n=14 NMJs from 8 larvae), p=0.304 (t(26)=1.049)). Values can be found in Supplementary Table 1. Statistics: Student's t-test except for panels (a-c) where a Mann-Whitney U-test was performed. All panels show mean ± SEM; *, p ≤0.05; **, p ≤0.01; ***, p ≤0.001; ns, not significant, p >0.05.

Supplementary Figure 6 Increased Ca2+ channel abundance at Unc13Anull mutant AZs

(a) Two-color STED images of multiple AZs from 3rd instar larvae of the displayed genotypes labelled with the indicated ABs. BRP rings were larger in Unc13Anull. (b) BRP ring diameters were increased in Unc13Anull in comparison to the Wild type (Wild type (n=9 NMJs from 3 larvae) vs Unc13Anull (n=12 NMJs from 3 larvae), p=0.0001 (U=0)). (c) Two-color STED images of multiple AZs from 3rd instar larvae of the displayed genotypes labelled with the indicated ABs. BRP ring structure appeared normal in Unc13Bnull (d) BRP ring diameters were unchanged in Unc13Bnull in comparison to Ctrl (Ctrl (n=8 NMJs from 3 larvae) vs Unc13Bnull (n=8 NMJs from 3 larvae), p=0.9591 (U=31)). (e) Muscle 4 NMJs of segments A2-A4 from 3rd instar larvae of the displayed genotypes labelled with the ABs indicated. (f) Cac-GFP spot sizes were increased in Unc13Anull but not Unc13Bnull in comparison to Ctrl (Ctrl (n=19 NMJs from 5 larvae) vs. Unc13Anull (n=15 NMJs from 5 larvae) vs. Unc13Bnull (n=21 NMJs from 5 larvae): p >0.05 for Ctrl vs Unc13Bnull; p ≤0.001 for Ctrl vs. Unc13Anull; p ≤0.001 for Unc13Bnull vs. Unc13Anull; p <0.0001 (F(2,52)=54.12)). (g) Mean Cac-GFP intensity measured over the whole NMJ was similar in Unc13Anull, Unc13Bnull and Ctrl (Ctrl (n=19 NMJs from 5 larvae) vs. Unc13Anull (n=15 NMJs from 5 larvae) vs. Unc13Bnull (n=21 NMJs from 5 larvae): p >0.05 for Ctrl vs Unc13Bnull; p >0.05 for Ctrl vs. Unc13Anull; p >0.05 for Unc13Bnull vs. Unc13Anull; p=0.166 (F(2,52)=1.855)). Number and p values are listed in Supplementary Table 1. Statistics: Mann-Whitney U-test, except for f and g where a nonparametric one-way analysis of variance (ANOVA) test, followed by a Turkey’s multiple comparison test was performed. All panels show mean ± SEM; *, p ≤0.05; **, p ≤0.01; ***, p ≤0.001; ns, not significant, p >0.05. Scale bar: (a,c) 500 nm; (e) 5 μm.

Supplementary Figure 7 Sketch of de novo synapse formation

During the process of AZ assembly, clusters of Syd-1 and Liprin-α undergo rounds of assembly and disassembly at the presynaptic membrane. Unc13B localizes to sites of de novo synapse formation via the Syd-1/Liprin-α scaffold. At nascent synapses, this induces a loose SV-Ca2+ channel coupling. Later during the AZ maturation process, Unc13A localizes to more mature synapses via a second, central RBP/BRP scaffold that concentrates Unc13A at the center of the AZ. Unc13A facilitates a close localization of SVs to the presynaptic Ca2+ channels and therefore maintains a tight stimulus/secretion coupling.

Supplementary Figure 8 Allosteric five-site binding model of Ca2+-driven exocytosis

Reaction scheme (derived from the ‘allosteric model’; Lou et al., 2005. Nature. 435:497-501) depicts the sequential binding of up to five Ca2+ ions to a single vesicle (RCa0-5).

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Böhme, M., Beis, C., Reddy-Alla, S. et al. Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca2+ channel–vesicle coupling. Nat Neurosci 19, 1311–1320 (2016). https://doi.org/10.1038/nn.4364

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