Protein composition of axonal dopamine release sites in the striatum
- Department of Neurobiology, Harvard Medical School, United States
Figures
Figure 1 with 2 supplements
In vivo biotin identification (iBioID) for release site proteins in dopamine axons of the mouse striatum.
(A) Schematic of the experiment with AAVs for Cre-dependent BirA fusion protein expression (AAV2/5-DIO-BirA-fusion), followed by in vivo biotinylation, affinity purification, and analyses by mass …
Figure 1—figure supplement 1
Sample images for ELKS2β-BirA expression.
Sample confocal images of a striatal section after viral expression of EKLS2β-BirA (images identical to the ones shown in Figure 1C) or without viral expression of a BirA fusion protein; images were …
Figure 1—figure supplement 2
BirA fusion proteins are self-biotinylated and purified with iBioID.
(A) Western blots of striatal lysates (input) and the pellet of the affinity-purified fractions (pulldown) are shown. The striata were harvested from mice that were injected with AAVs expressing …
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Figure 1—figure supplement 2—source data 1
Western blots for Figure 1 – figure supplement 2A.
(A) Original scan (5 min) of anti-RFP Western blots shown in Figure 1—figure supplement 2A. (B) Grayscale scans (left) and brightness and contrast-adjusted scans (right) of Western blots shown in Figure 1—figure supplement 2A. Stars denote bands that are likely degradation products of the BirA-tdTomato fusion protein. Arrows denote bands that are likely cross-reactive; they are present independent of BirA-tdTomato expression and are strong in intensity because a long exposure is shown and a large amount of input was loaded. (C) Original scan (3 s) of anti-β-actin Western blots shown in Figure 1—figure supplement 2A. (D) Grayscale scans (left) and brightness and contrast-adjusted scans (right) of Western blots shown in Figure 1—figure supplement 2A. Arrowheads denote bands that likely represent synapsin isoforms.
- https://cdn.elifesciences.org/articles/83018/elife-83018-fig1-figsupp2-data1-v2.zip
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Figure 1—figure supplement 2—source data 2
Western blots for Figure 1 – figure supplement 2B.
(A) Original scan (5 min) of anti-HA Western blots shown in Figure 1—figure supplement 2B. (B) Grayscale scans (left) and brightness and contrast-adjusted scans (right) of Western blots shown in Figure 1—figure supplement 2B.
- https://cdn.elifesciences.org/articles/83018/elife-83018-fig1-figsupp2-data2-v2.zip
Figure 2 with 4 supplements
Protein composition of release sites in dopamine axons.
(A) Venn diagram listing genes that encode protein hits enriched at least 2.0-fold over BirA-tdTomato with RIMPPCP-BirA, ELKS2β-BirA, or CaVβ4-BirA iBioID baits. Bait-encoding genes are shown …
Figure 2—figure supplement 1
Venn diagram of the dopamine release site proteome with a 1.5-fold enrichment threshold.
Venn diagram listing genes that encode protein hits enriched ≥1.5-fold over BirA-tdTomato (instead of 2.0-fold shown in Figure 2) with RIMPPCP-BirA, ELKS2β-BirA, or CaVβ4-BirA iBioID baits. …
Figure 2—figure supplement 2
Venn diagram of the dopamine release site proteome with a 2.5-fold enrichment threshold.
Venn diagram listing genes that encode protein hits enriched ≥2.5-fold over BirA-tdTomato (instead of ≥2.0-fold shown in Figure 2) with RIMPPCP-BirA, ELKS2β-BirA, or CaVβ4-BirA iBioID baits. …
Figure 2—figure supplement 3
Assessment of release site hits listed in SynGO across enrichment thresholds.
(A–C) Venn diagrams showing hits (without gene names) that are ≥1.5-fold (A), ≥2.0-fold (B, Venn diagram identical to the “synaptic” diagram shown in Figure 2B), or ≥2.5-fold (C) enriched. Proteins …
Figure 2—figure supplement 4
Neuroplastin antibody labeling is enhanced in Bassoon-containing dopaminergic synaptosomes.
(A) Representative confocal images of striatal synaptosomes stained with anti-Bassoon antibodies to mark release sites, anti-tyrosine hydroxylase (TH) antibodies to label dopamine synaptosomes, and …
Figure 3 with 2 supplements
Enrichment of release site proteins after conditional ablation of RIM or of Synaptotagmin-1 from dopamine neurons.
(A) Strategy for ablation of RIM1 and RIM2 (RIM cKODA) or Synaptotagmin-1 (Syt-1 cKODA) from dopamine neurons using conditional mouse genetics (Banerjee et al., 2020; Liu et al., 2018). (B) The …
Figure 3—figure supplement 1
Venn diagrams for the RIM cKODA dataset.
(A) Venn diagram listing genes that encode protein hits enriched ≥2.0-fold over BirA-tdTomato with RIMPPCP-BirA, ELKS2β-BirA, or CaVβ4-BirAiBioID baits in RIM cKODA mice. Bait-encoding genes are …
Figure 3—figure supplement 2
Venn diagrams for the Syt-1 cKODA dataset.
(A) Venn diagram listing genes that encode protein hits enriched ≥2.0-fold over BirA-tdTomato with RIMPPCP-BirA, ELKS2β-BirA, or CaVβ4-BirAiBioID baits in Syt-1 cKODA mice. Bait-encoding genes are …
Figure 4 with 1 supplement
RIM cKODA disrupts the protein composition of release sites in dopamine axons.
(A) The average log2 fold change of identified proteins over the same proteins in the BirA-tdTomato conditions, all proteins with a synaptic localization annotation in SynGO for each bait and …
Figure 4—figure supplement 1
Analyses of enrichment of synaptic vesicle and postsynaptic proteins across genotypes.
(A) The average log2 fold change of identified proteins over the same proteins in the BirA-tdTomato conditions, all proteins with a synaptic vesicle localization annotation in SynGO for each bait …
Figure 5
STRING diagrams illustrate functional categories of release site proteins and their disruption in RIM cKODA mice.
(A) STRING diagram of the enriched proteins that have a synaptic SynGO annotation in the control dataset. Physical or functional interactions determined by either empirical data or predictive …
Figure 6
Genes associated with Parkinson’s disease in the dopamine release site proteomes.
(A) Enrichment of proteins associated with monogenic forms of Parkinson’s disease (Blauwendraat et al., 2020; Day and Mullin, 2021; Marras et al., 2016) in the control dataset. Hits above the …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | B6.SJL-Slc6a3tm1.1(Cre)Bkmm/J (DATIRES-Cre) | Bäckman et al., 2006 | RRID:IMSR_JAX:006660 | |
| Genetic reagent (M. 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) | C57BL/6Ntac-Syt1tm1a(EUCOMM)Wtsi/WtsiCnrm (Syt-1fl/fl) | Zhou et al., 2015 | RRID:IMSR_EM:06829 | The identifier refers to the line before flp recombination |
| Cell line (Homo sapiens) | HEK293T cells | ATCC | Cat#: CRL-3216; RRID:CVCL_0063 | |
| Recombinant DNA reagent | pAAV2/5-syn-DIO-RIM-PRM-PDZ-PxxP-C2A-BirA | This study | LK18005; lab plasmid code (LPC) p864 | This reagent was used to generate AAV viruses and can be obtained from the corresponding author |
| Recombinant DNA reagent | pAAV2/5-syn-DIO- ELKS2β-BirA | This study | LK17008; LPC p857 | This reagent was used to generate AAV viruses and can be obtained from the corresponding author |
| Recombinant DNA reagent | AAV2/5-syn-DIO-CaVβ4-BirA | This study | LK19004; LPC p868 | This reagent was used to generate AAV viruses and can be obtained from the corresponding author |
| Recombinant DNA reagent | AAV2/5-syn-DIO-BirA-tdTomato | This study | LK17011; LPC p860 | This reagent was used to generate AAV viruses and can be obtained from the corresponding author |
| Antibody | Anti-HA (rabbit polyclonal) | Cell Signaling Technology | CAT# 5017; RRID:AB_10693385, lab antibody code (LAC) A40 | Immunofluorescence (IF) (1:500) |
| Antibody | Anti-HA (mouse monoclonal) | BioLegend | CAT# 901501; RRID:AB_2565006, LAC A12 | IF (1:500) Western blot (WB) 1:500 |
| Antibody | Anti-tyrosine hydroxylase (guinea pig polyclonal) | SySy | CAT# 213 104; RRID:AB_2619897, LAC A111 | IF (1:1000) |
| Antibody | Anti-Bassoon (mouse monoclonal) | Millipore | CAT# ADI-VAM-PS003-F; RRID:AB_11181058, LAC A85 | IF (1:500) |
| Antibody | Anti-Neuroplastin (goat polyclonal) | R&D Systems | CAT# AF5360; RRID:AB_2155920, LAC A253 | IF (1:500) |
| Antibody | Anti-red fluorescent protein (rabbit polyclonal) | Rockland | CAT#600-401-379; RRID:AB_2209751, LAC A81 | WB (1:1000) |
| Antibody | Anti-β-actin (mouse monoclonal) | Sigma-Aldrich | CAT# A1978; RRID:AB_476692, LAC A127 | WB (1:10,000) |
| Antibody | Anti-pyruvate carboxylase (rabbit polyclonal) | Novus | CAT# NBP1-49536G; RRID:AB_11016707, LAC A252 | This antibody was used for depletion as described in the 'Materials and methods' |
| Software, algorithm | Prism9 | GraphPad | RRID:SCR002798; https://www.graphpad.com/scientific-software/prism | |
| Software, algorithm | Cytoscape v 3.8.2 | Cytoscape | RRID:SCR003032; http://cytoscape.org/ | |
| Software, algorithm | SEQUEST PRO | Thermo Fisher | https://scicrunch.org/resolver/SCR_014594 | |
| Software, algorithm | MATLAB code for object analysis | Liu, 2021 | https://github.com/kaeserlab/3DSIM_Analysis_CL |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/83018/elife-83018-mdarchecklist1-v2.pdf
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Source data 1
Source data for Figures 1—3 and figure supplements.
(A) Table of proteins detected in mass spectrometry in control mice. An alphabetical list of genes encoding the proteins identified by mass spectrometry and associated peptide counts in the control datasets is provided.
(B) DNA sequences. Sequences of the inserts of newly generated plasmids used for producing AAVs are provided. Only the inserts in the multiple cloning site 3′ of the human synapsin promotor of a standard AAV vector are included.
(C) Table of proteins for the Venn diagram in Figure 2. Genes encoding the bait proteins are shown in the first three rows. The table contains an alphabetical list ordered by enrichment group (enriched in all bait conditions, enriched in two bait conditions, enriched in one bait condition). To be included in the Venn diagram, enriched is defined as an average ≥2.0-fold number of peptides for a specific protein over the peptide count for the same protein in the BirA-tdTomato condition, calculated across biological repeats and mass spectrometry experiments as described in detail in the 'Materials and methods'.
(D) Table of proteins for the Venn diagram in Figure 2—figure supplement 1. Genes encoding the bait proteins are shown in the first three rows. The table contains an alphabetical list ordered by enrichment group (enriched in all bait conditions, enriched in two bait conditions, enriched in one bait condition). To be included in the Venn diagram, enriched is defined as an average ≥1.5-fold number of peptides for a specific protein over the peptide count for the same protein in the BirA-tdTomato condition.
(E) Table of proteins for the Venn diagram in Figure 2—figure supplement 2. Genes encoding the bait proteins are shown in the first three rows. The table contains an alphabetical list ordered by enrichment group (enriched in all bait conditions, enriched in two bait conditions, enriched in one bait condition). To be included in the Venn diagram, enriched is defined as an average ≥2.5-fold number of peptides for a specific protein over the peptide count for the same protein in the BirA-tdTomato condition.
(F) Table of proteins detected in mass spectrometry in RIM cKODA mice. An alphabetical list of genes encoding the proteins identified by mass spectrometry and associated peptide counts in the RIM cKODA datasets is provided.
(G) Table of proteins detected in mass spectrometry in Syt-1 cKODA mice. An alphabetical list of genes encoding the proteins identified by mass spectrometry and associated peptide counts in the Syt-1 cKODA datasets is provided.
(H) Table of proteins in RIM cKODA mice for the Venn diagram in Figure 3—figure supplement 1. Genes encoding the bait proteins are shown in the first three rows. The table contains an alphabetical list ordered by enrichment group (enriched in all bait conditions, enriched in two bait conditions, enriched in one bait condition). To be included in the Venn diagram, enriched is defined as an average ≥2.0-fold number of peptides for a specific protein over the peptide count for the same protein in the BirA-tdTomato condition.
(I) Table of proteins in Syt-1 cKODA mice for the Venn diagram in Figure 3—figure supplement 2. Genes encoding the bait proteins are shown in the first three rows. The table contains an alphabetical list ordered by enrichment group (enriched in all bait conditions, enriched in two bait conditions, enriched in one bait condition). To be included in the Venn diagram, enriched is defined as an average ≥2.0-fold number of peptides for a specific protein over the peptide count for the same protein in the BirA-tdTomato condition.
- https://cdn.elifesciences.org/articles/83018/elife-83018-data1-v2.xlsx
