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. 2019 Mar 6;10:1085. doi: 10.1038/s41467-019-08977-6

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — Unc13A and its N-terminus are critical for rapid PHP and AZ-remodeling. a Confocal images of muscle 4 NMJs of abdominal segment 2–5 from 3rd instar larvae at wild-type (left) and unc13A^Null (right) NMJs labelled with the indicated antibodies without (control; black) and with 10 min PhTx (+PhTx; blue) treatment. b Quantification of percentage change of synaptic BRP, RBP and Cac AZ-levels in wild-type (blue) and unc13A^Null (red) upon PhTx-treatment compared to the same measurement in the absence of PhTx for each genotype (dashed grey line indicates 100%/no change). c Representative traces of eEPSP (evoked) and mEPSP (spont.) in wild-type and unc13A^Null animals without (Ctrl; black or grey) and with 10 min PhTx (+PhTx; blue or light red) treatment. d Quantifications of percentage change of mEPSP amplitude, eEPSP amplitude and quantal content (QC) in PhTx-treated wild-type (blue) and unc13A^Null (light red) cells compared to the same measurement obtained without PhTx for each genotype. Traces for (c) were replotted from Fig. 1. e Left: Full-length Unc13A construct used in rescue experiments of unc13^Null animals. Functional domains for AZ localization, Calmodulin- (CAM), lipid-binding (C1, C2B, C2C) and the MUN domain relevant for SV release are shown. Right: Schematic of Unc13A construct lacking the N-terminal localization sequence (C-term-GFP rescue). f–i Same as in (a–d) for cells re-expressing Unc13A-GFP (blue) or C-term-GFP (light red) in the unc13^Null background. See also Supplementary Figure 8. See also Supplementary Figure 10 for non-normalized values. Source data as exact normalized and raw values, detailed statistics including sample sizes and P values are provided in the Source Data file. Scale bars: a, f 5 µm; c, h eEPSP: 25 ms, 5 mV; mEPSP: 50 ms, 1 mV. Statistics: Student’s unpaired T-test was used for comparisons in ((d) mEPSP, eEPSP change), ((i) mEPSP change) and Mann–Whitney U test for all other comparisons. *P ≤ 0.05; **P ≤ 0.01; n.s., not significant, P > 0.05. All panels show mean ± s.e.m.