Munc13 supports fusogenicity of non-docked vesicles at synapses with disrupted active zones
- Department of Neurobiology, Harvard Medical School, United States
- Department of Neuroscience, University of Copenhagen, Denmark
- Department of Molecular Neurobiology, Max Planck Institute for Multidisciplinary Sciences, Germany
Figures
Figure 1 with 3 supplements
Action potential-evoked neurotransmitter release and Munc13 active zone levels after ablation of RIM + ELKS.
(A) Strategy for deletion of RIM1, RIM2, ELKS1, and ELKS2 in cultured hippocampal neurons. Neurons of mice with floxed alleles for all four genes were infected with Cre-expressing lentiviruses (to …
Figure 1—figure supplement 1
Assessment of Munc13-1 levels with STED microscopy in Munc13 knockout synapses.
(A) Strategy for deletion of Munc13-1, Munc13-2, and Munc13-3 in cultured hippocampal neurons (cKOM). Cultured hippocampal neurons of mice with floxed alleles for Munc13-1 (Banerjee et al., 2022) …
Figure 1—figure supplement 2
Action potential-evoked neurotransmitter release after ablation of Munc13.
(A, B) Sample traces (A) and quantification (B) of excitatory postsynaptic currents (EPSCs) evoked by focal electrical stimulation in controlM and cKOM neurons, 16 cells/3 cultures each. (C–E) …
Figure 1—figure supplement 3
Assessment of Munc13-1 levels with confocal microscopy and Western blot in RIM + ELKS or Munc13 knockout neurons.
(A, B) Sample confocal images (A) and quantification of Munc13-1 levels (B) at synapses of controlR+E and cKOR+E neurons. Confocal scans were taken from the same areas that were analyzed by STED …
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Figure 1—figure supplement 3—source data 1
Western blots for Figure 1—figure supplement 3.
(A) Original scans (1 s and 30 s) of Western blots shown in Figure 1—figure supplement 3C. (B) Raw grayscale scans (left) and brightness- and contrast-adjusted scans (right) of Western blots shown in Figure 1—figure supplement 3C. (C) Original scans (5 s and 1 min) of Western blots shown in Figure 1—figure supplement 3F. (D) Raw grayscale scans (left) and brightness- and contrast-adjusted scans (right) of Western blots shown in Figure 1—figure supplement 3F.
- https://cdn.elifesciences.org/articles/79077/elife-79077-fig1-figsupp3-data1-v2.zip
Figure 2 with 2 supplements
Simultaneous deletion of RIM, ELKS, and Munc13 does not disrupt synapse formation.
(A) Strategy for simultaneous deletion of RIM1, RIM2, ELKS1, ELKS2, Munc13-1, and Munc13-2 in cultured hippocampal neurons (cKOR+E+M). Neurons were infected with Cre-expressing lentiviruses (to …
Figure 2—figure supplement 1
Assessment of Munc13-1 levels in RIM + ELKS + Munc13 knockout neurons.
(A) Quantification of Synaptophysin distribution aligned to the PSD-95 peak in side-view synapses of cultured controlR+E+M and cKOR+E+M neurons from the experiment shown in Figure 2B–D, controlR+E+M …
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Figure 2—figure supplement 1—source data 1
Western blots for Figure 2—figure supplement 1.
(A) Original scans (5 s and 1 min) of Western blots shown in Figure 2—figure supplement 1F. (B) Raw grayscale scans (left) and brightness- and contrast-adjusted scans (right) of Western blots shown in Figure 2—figure supplement 1F.
- https://cdn.elifesciences.org/articles/79077/elife-79077-fig2-figsupp1-data1-v2.zip
Figure 2—figure supplement 2
Assessment of Synaptophysin fluorescence in RIM + ELKS + Munc13 knockout neurons after lentiviral infection at DIV2.
(A–E) Overview images of anti-Synaptophysin (confocal) and anti-PSD-95 (STED) staining (A) and quantification of Synaptophysin puncta density (B), intensity (C), size (D), and of the density of …
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Figure 2—figure supplement 2—source data 1
Western blots for Figure 2—figure supplement 2.
(A) Original scans (1 s and 30 s) of Western blots shown in Figure 2—figure supplement 2F. (B) Raw grayscale scans (left) and brightness- and contrast-adjusted scans (right) of Western blots shown in Figure 2—figure supplement 2F.
- https://cdn.elifesciences.org/articles/79077/elife-79077-fig2-figsupp2-data1-v2.zip
Figure 3
Synaptic ultrastructure after RIM + ELKS + Munc13 knockout.
(A–E) Sample images (A) and analyses (B–E) of synaptic morphology of high-pressure frozen neurons analyzed by electron microscopy; docked vesicles (B), total vesicles (C), bouton size (D), and …
Figure 4 with 2 supplements
Neurotransmitter release is strongly impaired after RIM + ELKS + Munc13 ablation.
(A–C) Sample traces (A) and quantification of miniature excitatory postsynaptic current (mEPSC) frequencies (B) and amplitudes (C) in controlR+E+M and cKOR+E+M neurons, 27 cells/3 cultures each. (D–F…
Figure 4—figure supplement 1
Assessment for the kinetics of spontaneous vesicle release in RIM + ELKS + Munc13 knockout neurons.
(A–C) Sample traces of the average miniature excitatory postsynaptic current (mEPSC) of a single controlR+E+M or cKOR+E+M neuron (A) and quantification of mEPSC rise times (B, 20%–80%) and decay …
Figure 4—figure supplement 2
Workflow of the hierarchical bootstrap analyses.
(A) Analysis pipeline for the hierarchical bootstrap on the example of NMDAR-EPSCs in controlR+E, cKOR+E, controlR+E+M, and cKO R+E+M (data from Figure 4). For all cells, we resampled with …
Figure 5
The remaining functional RRP in RIM + ELKS-deficient synapses depends at least in part on Munc13.
(A, B) Sample traces (A) and quantification (B) of excitatory postsynaptic currents (EPSCs) triggered by hypertonic sucrose in controlR+E and cKOR+E neurons, the first 10 s of the EPSC were …
Figure 6 with 1 supplement
Vesicular release probability is not further impaired by combined RIM + ELKS + Munc13 knockout.
(A, B) Sample traces (A) and quantification (B) of excitatory postsynaptic current (EPSC) paired pulse ratios in controlR+E and cKOR+E neurons, controlR+E 15 cells/3 cultures, cKOR+E, 16/3. (C, D) …
Figure 6—figure supplement 1
Additional analyses of spontaneous vesicle release and inhibitory postsynaptic currents (IPSCs) evoked by stimulus trains in RIM + ELKS and RIM + ELKS + Munc13 knockout neurons.
(A–C) Sample traces (A) and quantification of miniature excitatory postsynaptic current (mEPSC) frequencies (B) and amplitudes (C) in controlR+E and cKOR+E neurons, controlR+E 21 cells/3 cultures, …
Author response image 1
(a, b) Cumulative IPSC amplitude plots in stimulus trains (original data from Figure 6 —figure supplements 1I-1L, n’s as in those figures). (c, d) Back-extrapolation to time zero yields the IPSC-amplitudes at Y-intercept to estimate the recovery-corrected pool size for each cell, back-extrapolation was based on the last ten (4150) responses. (e) Comparison of IPSC at Y-intercept normalized to their own controls in cKOR+E (absolute data from c) and cKOR+E+M (from d) neurons.
Author response image 2
(a-c) Sample STED images (a) and quantification (b, c) of side-view synapses stained for Munc13-1 (imaged in STED), PSD-95 (imaged in STED), and Synaptophysin (imaged in confocal). Munc13-1 primary antibody was not added during staining process as negative control (“no primary antibody”). Peak position and levels were analyzed in line profiles (600 nm x 200 nm) positioned perpendicular to the center of elongated PSD-95 structure and aligned to the PSD-95 peak, 20 synapses/1 cultures each.
Author response image 3
Comparison of hierarchical vs. non-hierarchical bootstrap for the analyses of NMDAR-EPSCs.
Author response image 4
(a, b) Comparison of RIM1 levels between confocal and STED images. In a, RIM1 levels in indicated conditions are from confocal images (from Figure S2A of 3). In b, RIM1 levels in indicated conditions are from STED images (data from Figure S2H of 3).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | Rims1tm3Sud/J (RIM1αβfl/fl) | Kaeser et al., 2008 | RRID:IMSR_JAX:015832 | |
| Genetic reagent (M. musculus) | Rims2tm1.1Sud/J (RIM2αβγfl/fl) | Kaeser et al., 2011 | RRID:IMSR_JAX:015833 | |
| Genetic reagent (M. musculus) | Erc1tm2.1Sud/J (ELKS1αfl/fl) | Liu et al., 2014 | RRID:IMSR_JAX:015830 | |
| Genetic reagent (M. musculus) | Erc2tm1.2Sud/J (ELKS2αfl/fl) | Kaeser et al., 2009 | RRID:IMSR_JAX:015831 | |
| Genetic reagent (M. musculus) | Unc13atm1.1Bros (Munc13-1fl/fl) | Banerjee et al., 2022 | MGI:7276178 | |
| Genetic reagent (M. musculus) | Unc13btm1Rmnd (Munc13-2-/-) | Varoqueaux et al., 2002 | RRID:MGI:2449706 | |
| Genetic reagent (M. musculus) | Unc13ctm1Bros (Munc13-3-/-) | Augustin et al., 2001 | RRID:MGI:2449467 | |
| Cell line (Homo sapiens) | HEK293T cells | ATCC | Cat# CRL-3216; RRID:CVCL_0063 | |
| Recombinant DNA reagent | pFSW EGFP Cre | Liu et al., 2014 | pHN131014; lab plasmid code (LPC): p009 | |
| Recombinant DNA reagent | pFSW EGFP ΔCre | Liu et al., 2014 | pHN131015; LPC: p010 | |
| Antibody | Anti-RIM (rabbit polyclonal) | SySy | Cat# 140003; RRID:AB_887774; lab antibody code (LAC): A58 | Immunofluorescence (IF) (1:500) |
| Antibody | Anti-PSD-95 (mouse monoclonal) | NeuroMab | Cat# 73-028; RRID:AB_10698024; LAC: A149 | IF (1:500) |
| Antibody | Anti- Synaptophysin (guinea pig polyclonal) | SySy | Cat# 101004; RRID:AB_1210382; LAC: A106 | IF (1:500) |
| Antibody | Anti-Munc13-1 (rabbit polyclonal) | SySy | Cat# 126103; RRID:AB_887733; LAC: A72 | IF (1:500); Western blot (WB) (1:1000) |
| Antibody | Anti-Synapsin-1 (mouse monoclonal) | SySy | Cat# 106001; RRID:AB_2617071; LAC: A57 | WB (1:4000) |
| Antibody | Anti-rabbit Alexa Fluor 488 (goat polyclonal) | Thermo Fisher | Cat# A-11034; RRID:AB_2576217; LAC: S5 | IF (1:200) |
| Antibody | Anti-mouse IgG2a Alexa Fluor 555 (goat polyclonal) | Thermo Fisher | Cat# A-21137; RRID:AB_2535776; LAC: S20 | IF (1:200) |
| Antibody | Anti-guinea pig Alexa Fluor 633 (goat polyclonal) | Thermo Fisher | Cat# A-21105; RRID:AB_2535757; LAC: S34 | IF (1:500) |
| Antibody | Anti-mouse peroxidase-conjugated (goat polyclonal) | MP Biologicals | Cat# 0855550; RRID:AB_2334540; LAC: S52 | WB (1:10000) |
| Antibody | Anti-rabbit peroxidase-conjugated (goat polyclonal) | MP Biologicals | Cat# 0855676; RRID:AB_2334589; LAC: S53 | WB (1:10000) |
| Software, algorithm | MATLAB | MathWorks | RRID:SCR_001622; https://www.mathworks.com/products/matlab.html | |
| Software, algorithm | Prism | GraphPad | RRID:SCR_002798; https://www.graphpad.com/scientific-software/prism | |
| Software, algorithm | Fiji/ImageJ | NIH | RRID:SCR_002285; https://imagej.net/software/fiji/downloads | |
| Software, algorithm | pClamp | Molecular Devices | RRID:SCR_011323; https://www.moleculardevices.com/products/software/pclamp.html | |
| Software, algorithm | MATLAB code for object recognition and analysis of 2D images | This paper | https://doi.org/10.5281/zenodo.6388196 | This code was adapted for 2D images from a previously generated code (Liu et al., 2018; Liu et al., 2022) and is freely accessible at zenodo.org |
| Software, algorithm | MATLAB code for hierarchical bootstrap | This paper | https://github.com/kaeserlab/Hierarchical_Bootstrap_Analysis_RB; Born, 2022 | This code was generated for this paper and is freely accessible at github.com |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/79077/elife-79077-mdarchecklist1-v2.pdf
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Source data 1
Numerical data for all analyses shown in the figures.
- https://cdn.elifesciences.org/articles/79077/elife-79077-data1-v2.xlsx
