A proline-rich motif on VGLUT1 reduces synaptic vesicle super-pool and spontaneous release frequency
- Interdisciplinary Institute for Neuroscience, Université de Bordeaux, France
- Interdisciplinary Institute for Neuroscience CNRS UMR 5297, France
- Max Planck Institute of Experimental Medicine, Germany
- Neuroscience Paris Seine NPS, Université Pierre et Marie Curie INSERM U1130 CNRS UMR8246, France
- Bordeaux Imaging Center, Université de Bordeaux, CNRS UMS 3420, INSERM US4, France
- Institut Pasteur, CNRS UMR 3571, Unité NISC, France
- Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), Direction de laRecherche Fondamentale (DRF), Institut de Biologie François Jacob (IBFJ), MolecularImaging Research Center (MIRCen), France
- Douglas Mental Health University Institute, McGill University, Canada
Figures
Figure 1 with 1 supplement
Dose dependent regulation of SV super-pool size by VGLUT1.
(A) Expression of Synaptobrevin2 fused to Enhanced Green Fluorescent Protein (Syb2EGFP) in hippocampal neurons at 18 days in culture. (B) examples of FRAP sequences from both Slc17a7 (VGLUT1) +/+ …
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Figure 1—source data 1
Raw data for FRAP experiments with VGLUT1 WT and KO culture.
- https://doi.org/10.7554/eLife.50401.004
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Figure 1—source data 2
Raw data for FRAP experiments with VGLUT1 rescued and VGLUT1 overexpressed culture.
- https://doi.org/10.7554/eLife.50401.005
Figure 1—figure supplement 1
VGLUT2 does not rescue SV super-pool size in VGLUT1 knock out neurons.
(A) Average FRAP kinetics obtained upon rescue of VGLUT1 KO cells by either VGLUT1venus or VGLUT2venus lentivirus. 24 synapses from VGLUT1venus and 20 synapses from VGLUT2venus were measured by FRAP …
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Figure 1—figure supplement 1—source data 1
Raw data for FRAP experiments with VGLUT1 and VGLUT2 rescued culture.
- https://doi.org/10.7554/eLife.50401.006
Figure 2
Structure of VGLUT1 and expression of mutants in neurons.
(A) schematic of VGLUT1 structure with 560 amino acids, 12 transmembrane domains, N- and C-termini facing the cytoplasmic side, and N-glycosylation at the first luminal loop. In blue the three …
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Figure 2—source data 1
Raw data for Bouton size and intensity in VGLUT1 WT and mutants rescued culture.
- https://doi.org/10.7554/eLife.50401.008
Figure 3 with 2 supplements
Glutamate transport and SV tonicity are not involved in the reduction of SV super-pool size.
(A) Expression of VGLUT1mCherry, sVGLUT1mCherry and Syb2EGFP in hippocampal neurons. FRAP was performed on Syb2EGFP at axons, both on those with, and those without mCherry signal. Scale bar 5 µm. (B)…
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Figure 3—source data 1
Raw data for FRAP experiments with VGLUT1 WT, sVGLUT1 rescued and non-rescued culture.
- https://doi.org/10.7554/eLife.50401.013
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Figure 3—source data 2
Raw data for Electrophysiological recording with VGLUT1 WT and sVGLUT1 rescued culture.
- https://doi.org/10.7554/eLife.50401.014
Figure 3—figure supplement 1
VGLUT2 expression diminishes in hippocampal neurons until DIV17.
(A) Immunofluorescence of VGLUT2 on hippocampal neurons at DIV 6, 12, 17, and 22. Scale bar 40 µm. (B) Quantification of VGLUT2 expression during hippocampal neuron culture development. Note the …
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Figure 3—figure supplement 1—source data 1
Raw data for Electrophysiological recording with VGLUT1 WT and sVGLUT1 rescued culture.
- https://doi.org/10.7554/eLife.50401.015
Figure 3—figure supplement 2
Vesicular glutamate uptake function does not influence mIPSCs features.
(A) Spontaneous inhibitory activity in VGLUT1 KO rescued neurons. Example traces of mIPSC activity in wild type and sVGLUT1 rescue conditions. B: Comparison of the frequency of mIPSC events in wild …
Figure 4 with 2 supplements
The VGLUT1 PRD2 domain mediates SV super-pool size and mEPSC frequency reductions.
(A) Comparison of VGLUT1venus and VGLUT1P554A-venus rescues of VGLUT1 KO on SV exchange rates at synapses. 21 (WT) and 22 (P554A) synapses from each rescue were measured by FRAP and the average …
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Figure 4—source data 1
Raw data for FRAP experiments with VGLUT1 WT and P554A rescued culture.
- https://doi.org/10.7554/eLife.50401.019
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Figure 4—source data 2
Raw data for FRAP experiments with VGLUT1 WT, ΔPRD 1+2 and ΔPRD 2 rescued culture.
- https://doi.org/10.7554/eLife.50401.020
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Figure 4—source data 3
Raw data for FRAP experiments with VGLUT1 WT and DQL514AQA rescued culture.
- https://doi.org/10.7554/eLife.50401.021
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Figure 4—source data 4
Raw data for FRAP experiments with VGLUT1 WT and PP534AA rescued culture.
- https://doi.org/10.7554/eLife.50401.022
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Figure 4—source data 5
Raw data for FRAP experiments with VGLUT1 WT and S540A rescued culture.
- https://doi.org/10.7554/eLife.50401.023
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Figure 4—source data 6
Raw data for FRAP experiments with VGLUT1 WT and T544A rescued culture.
- https://doi.org/10.7554/eLife.50401.024
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Figure 4—source data 7
Raw data for Cumulative SV axonal transport in VGLUT1 WT, P554A and S540A rescued culture.
- https://doi.org/10.7554/eLife.50401.025
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Figure 4—source data 8
Raw data for SV axonal transport speed in VGLUT1 WT, P554A and S540A rescued culture.
- https://doi.org/10.7554/eLife.50401.026
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Figure 4—source data 9
Raw data for Electrophysiological recording with VGLUT1 WT and P554A rescued and non-rescued culture.
- https://doi.org/10.7554/eLife.50401.027
Figure 4—figure supplement 1
VGLUT1 is phosphorylated but not at the conserved 540-SYGAT motif.
(A) Effect of alkaline phosphatase on the electrophoretic mobility of VGLUT1 from forebrain homogenates (and positive control VIAAT ; Vesicular Inhibitory Amino-Acid Transporter). (B) regional …
Figure 4—figure supplement 2
VGLUT1 PRD2 domain removal does not affect mIPSCs features.
(A) Spontaneous inhibitory activity in VGLUT1 KO neurons rescued by wild type and VGLUT1 P554A constructs. Example traces of mIPSC activity in sham controls, wild type and VGLUT1P554A rescue …
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Figure 4—figure supplement 2—source data 1
Raw data for Electrophysiological recording with VGLUT1 WT and P554A rescued and non-rescued culture.
- https://doi.org/10.7554/eLife.50401.028
Figure 5 with 1 supplement
A tripartite complex between VGLUT1, endophilinA1 and intersectin1 mediates SV super-pool reduction.
(A) Schematic model of the competition experiment designed to test for the effective recruitment of intersectin1 at the VGLUT1/endophilinA1 complex (Pechstein et al., 2015). The three SH3 domains …
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Figure 5—source data 1
Raw data for FRAP experiments with SH3 domain mutant overexpressed VGLUT1venusculture.
- https://doi.org/10.7554/eLife.50401.031
Figure 5—figure supplement 1
EndophilinA1 accumulates at synaptic vesicle clusters of VGLUT1 synapses.
Subcellular distribution of EndophilinA1 in VGLUT1 immunopositive terminals in mossy fiber terminals of the cerebellum (A,B) and cortico-striatal projections (C,D). We used pre-embedding immunogold …
Tables
Table 1
List of mutant constructs tested.
https://doi.org/10.7554/eLife.50401.009| Mutation | Domain/Motif | Name | Putative function | |
|---|---|---|---|---|
| VGLUT1 | R80Q/R176K/R314Q | TM1;TM4;TM7 | sVGLUT1 | Glutamate transport |
| ∆504–560 | Whole C-terminus | ∆C-term | SV/VGLUT Trafficking | |
| DQL514AQA | PDZ type three binding | DQL514AQA | Unknown | |
| ∆530–560 | Proline Rich Domains 1+2 | ∆PRD1+2 | SH3 domain binding | |
| PP534AA | Proline Rich Domain1 | PP534AA | SH3 domain binding | |
| S540A | SYGAT | S540A | Unknown | |
| T544A | SYGAT | T544A | Unknown | |
| ∆550–560 | Proline Rich Domain 2 | ∆PRD2 | Endophilin binding | |
| P554A | Proline Rich Domain 2 | P554A | Endophilin binding | |
| EndoA1 290–352 | SH3 | SH3 | Binds PRD and ITSN1 SH3B | |
| EndoA1 290-352E329K,S336K | SH3 | SH3E329K,S336K | Binds PRD and ITSN1 SH3B | |
| ITSN1 903–971 | SH3B | SH3B | Endo SH3 binding through E329 and S336 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Slc17a7-/-(VGLUT1 KO) mice | PMID: 15103023 | available upon request to Dr Sonja M. Wojcik wojcik@em.mpg.de | |
| Strain, strain background (Mus musculus) | Slc17a7v/v(VGLUT1venus KI) mice | PMID: 22031900 | available upon request to corresponding author | |
| Transfected construct (Homo sapiens) | F(syn)W-RBN::Synaptobervin2-EGFP | PMID: 23581566 | available upon request to Dr. Etienne Herzog | Figure 1, Figure 3 and related results part. Lentiviral vector expressing Syb2EGPF. |
| Transfected construct (Rattus norvegicus) | F(syn)W-RBN::VGLUT1-venus | PMID: 23581566 | available upon request to Dr. Etienne Herzog | Figure 1, Figure 4 and related results part. Lentiviral vector expressing VGLUT1venus. |
| Transfected construct (Rattus norvegicus) | F(syn)W-RBN::VGLUT2-venus | This paper | available upon request to Dr. Etienne Herzog | Figure 1—figure supplement 1–S1 and related results part. Lentiviral vector expressing VGLUT2venus. |
| Transfected construct (Rattus norvegicus) | F(syn)W-RBN::VGLUT1 ∆C-term-venus | This paper | available upon request to Dr. Etienne Herzog | Figure 4 and related results part. Lentiviral vector expressing VGLUT1∆C-term-venus. |
| Transfected construct (Rattus norvegicus) | F(syn)W-RBN::VGLUT1 DQL514AQA-venus | This paper | available upon request to Dr. Etienne Herzog | Figure 4 and related results part. Lentiviral vector expressing VGLUT1DQL514AQA-venus. |
| Transfected construct (Rattus norvegicus) | F(syn)W-RBN::VGLUT1 ∆PRD1+2-venus | This paper | available upon request to Dr. Etienne Herzog | Figure 4 and related results part. Lentiviral vector expressing VGLUT1∆PRD1+2-venus. |
| Transfected construct (Rattus norvegicus) | F(syn)W-RBN::VGLUT1 PP534AA-venus | This paper | available upon request to Dr. Etienne Herzog | Figure 4 and related results part. Lentiviral vector expressing VGLUT1PP534AA-venus. |
| Transfected construct (Rattus norvegicus) | F(syn)W-RBN::VGLUT1 S540A-venus | This paper | available upon request to Dr. Etienne Herzog | Figure 4 and related results part. Lentiviral vector expressing VGLUT1S540A-venus. |
| Transfected construct (Rattus norvegicus) | F(syn)W-RBN::VGLUT1 T544A-venus | This paper | available upon request to Dr. Etienne Herzog | Figure 4 and related results part. Lentiviral vector expressing VGLUT1T544A-venus. |
| Transfected construct (Rattus norvegicus) | F(syn)W-RBN::VGLUT1 ∆PRD2-venus | This paper | available upon request to Dr. Etienne Herzog | Figure 4 and related results part. Lentiviral vector expressing VGLUT1∆PRD2-venus. |
| Transfected construct (Rattus norvegicus) | F(syn)W-RBN::VGLUT1 P554A-venus | This paper | available upon request to Dr. Etienne Herzog | Figure 4 and related results part. Lentiviral vector expressing VGLUT1P554A-venus. |
| Transfected construct (Rattus norvegicus) | AAV9::VGLUT1mCherry-miniSOG | This paper | available upon request to Dr. Etienne Herzog | Figure 3 and related results part. Lentiviral vector expressing VGLUT1mCherry-miniSOG. |
| Transfected construct (Rattus norvegicus) | AAV9::sVGLUT1mCherry-miniSOG | This paper | available upon request to Dr. Etienne Herzog | Figure 3 and related results part. Lentiviral vector expressing sVGLUT1mCherry-miniSOG. |
| Transfected construct (Homo sapiens) | AAV9::mCerulean3-EndophilinA1SH3 | This paper | available upon request to Dr. Etienne Herzog | Figure 5 and related results part. Lentiviral vector expressing mCerulean3-EndophilinA1SH3. |
| Transfected construct (Homo sapiens) | AAV9::mCerulean3-EndophilinA1SH3E329K, S336K | This paper | available upon request to Dr. Etienne Herzog | Figure 5 and related results part. Lentiviral vector expressing mCerulean3-EndophilinA1SH3E329K, S336K. |
| Transfected construct (Homo sapiens) | AAV9::mCerulean3-IntersectinSH3B | This paper | available upon request to Dr. Etienne Herzog | Figure 5 and related results part. Lentiviral vector expressing mCerulean3-IntersectinSH3B. |
| Antibody | GFP, Mouse, monoclonal | Roche | Cat. 11814460001 RRID:AB_390913 | 1:1000 |
| Antibody | VIAAT, Guinea pig, polyclonal | SYSY | Cat. 131004 RRID:AB_887873 | 1:1000 |
| Antibody | VGLUT2, Guinea pig, polyclonal | Millipore | Cat. AB2251 RRID:AB_1587626 | 1:2000 |
| Antibody | VGLUT1, guinea pig, polyclonal | Merck | Cat. AB5905 RRID:AB_2301751 | 1:5000 |
| Antibody | EndophilinA1, rabbit, polyclonal | PMID: 16606361 | available upon request to Dr. Etienne Herzog | 1:500 |
| Peptide, recombinant protein | FastAP Thermosensitive Alkaline Phosphatase | Thermo Scientific | Cat. EF0651 | |
| Peptide, recombinant protein | Haltphosphatase inhibitor cocktail | Thermo Scientific | Cat. 78420 | |
| Chemical compound, drug | Phos-tag Acrylamid | Wako | Cat. AAL-107 | |
| Software, algorithm | FRAP Analysis Plugin | This paper | The plugin is available at: https://github.com/fabricecordelieres/IJ-Macro_FRAP-MM | |
| Software, algorithm | KymoToolbox Plugin | PMID: 23374344 | The plugin is available at: https://github.com/fabricecordelieres/IJ-Plugin_KymoToolBox |
Additional files
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Supplementary file 1
Supplementary tables collating statistical analysis.
- https://doi.org/10.7554/eLife.50401.032
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Supplementary file 2
Seal test recording of every cell in the electrophysiology analysis.
- https://doi.org/10.7554/eLife.50401.033
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Transparent reporting form
- https://doi.org/10.7554/eLife.50401.034
