Uncovering the electrical synapse proteome in retinal neurons via in vivo proximity labeling
- College of Optometry, University of Houston, United States
- Moran Eye Center/Ophthalmology, University of Utah, United States
- Animal Navigation/ Neurosensorics, Institute for Biology and Environmental Sciences, University of Oldenburg, Germany
- Research Center Neurosensory Science, University of Oldenburg, Germany
- Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, United States
- Institute-Associated Research Group: Cell Adhesion and Cell Polarity, Institute of Medical Biochemistry, ZMBE, University of Münster, Germany
- Cell Signal Unit, Okinawa Institute of Science and Technology, Japan
Figures
Figure 1
Generation of a Cx35.1-V5-TurboID zebrafish line.
(A) Cartoon illustrating the generation of Cx35.1-V5-TurboID fish via Tol2-mediated transgenesis and outline of in vivo biotinylation experiments. To induce efficient proximity labeling, zebrafish were intraperitoneally injected with 30 µl of 5 mM biotin (PBS) for three consecutive days. Afterward, the animals were sacrificed, and the retinas were isolated for streptavidin pull-downs. (B) Confocal scans of entire zebrafish retinas, the outer retina including ZPR1-labeled photoreceptors to confirm successful targeting of Cx35.1-V5-TurboID to photoreceptor gap junctions. Neutravidin Oregon Green labeling was used to validate efficient proximity biotinylation of Cx35.1 and surrounding molecules in biotin-injected Cx35.1-V5-TurboID fish. Reagent or antibody used for labeling is shown in the gray box above each image. Scale 20 µm, magnified inset: 5 µm. (C) Western blot of streptavidin pull-downs probed with V5 antibodies and streptavidin-HRP. The arrows indicate the position of the Cx35.1-V5-TurboID construct, which is detected with streptavidin-HRP and a V5 antibody. (D) String diagram illustrating the protein-protein network surrounding Cx35. Proteins that were two times or more abundant in comparison to the control condition were included in the string diagram. (E) Colocalization of Cx35.1 and Cx34.7-GFP in the outer plexiform layer of the zebrafish retina suggests that a subset of photoreceptor gap junctions contain both connexins. Scale: 5 µm; inset scale: 0.5 µm.
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Figure 1—source data 1
Raw images of western blots shown in Figure 1.
Relevant bands were labeled with arrows.
- https://cdn.elifesciences.org/articles/105935/elife-105935-fig1-data1-v1.zip
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Figure 1—source data 2
Raw images of western blots shown in Figure 1.
- https://cdn.elifesciences.org/articles/105935/elife-105935-fig1-data2-v1.zip
Figure 2
A GFP-directed TurboID approach uncovers the Cx36 interactome in AII amacrine cells.
(A) Cartoon illustrating different in vivo BioID approaches used to target Cx36. In strategy 1, we expressed an AAV Cx36-V5-TurboID construct with an internal insertion site under the control of the human SYN-promoter. This strategy greatly resulted in overexpression artifacts causing aberrant protein localization Scale: 20 µm, magnified inset: 5 µm. In strategy 2, we adapted the GFP-directed TurboID strategy developed by Xiong et al., 2021 to shuttle a V5-dGBP-TurboID construct to Cx36-EGFP containing gap junctions in retinas of the Cx36-EGFP transgenic strain. (B) Vertical sections of AAV infected retinas confirming cell type-specificity of our AAV vectors. The HKamac promoter developed by Khabou et al., 2023 is mainly active in AII amacrine cells and does not show any expression in bipolar cells (SCGN, magenta). V5-dGBP-TurboID colocalizes with Cx36-EGFP in AAV transduced retinas, confirming the successful delivery of TurboID to the gap junction. Scale: 10 µm, magnified inset: 5 µm. (C) Western blot confirming successful biotinylation and capture of Cx36-EGFP, biotinylated proteins, and V5-TurboID-dGBP. (D) String diagram illustrating the protein-protein interaction network associated with Cx36 in AII amacrine cells. Proteins that were three times or more abundant compared to the control condition were included in the string diagram.
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Figure 2—source data 1
Raw images of western blots shown in Figure 2.
Relevant bands were labeled with arrows.
- https://cdn.elifesciences.org/articles/105935/elife-105935-fig2-data1-v1.zip
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Figure 2—source data 2
Raw images of western blots shown in Figure 2.
- https://cdn.elifesciences.org/articles/105935/elife-105935-fig2-data2-v1.zip
Figure 3
Localization of scaffold and endocytosis proteins identified by BioID at Cx36 gap junctions in AAV transduced AII amacrine cells expressing GFP.
(A) Colocalization of Cx36 and scaffold proteins including known interactors such as ZO-1, ZO-2, and cingulin. Besides these known interactors, we identified Sipa1l3, a PDZ domain containing protein implicated in cell adhesion and cytoskeletal organization. Sipa1l3 shows abundant colocalization with Cx36. (B) Colocalization of Cx36 and components of the endocytosis machinery. Among all proteins we have tested, we found frequent colocalization for EpsS15I1, an endocytic adapter protein, and Cx36. Scale: 10 µm. Magnified inset: 1 µm. (C) Bar graphs showing the degree of colocalization as the percentage of Cx36 puncta that overlap with Cgn and Sipa1l3. Colocalization was quantified for the original image and a horizontally flipped image to exclude random overlap. Bar graphs with SEM, ***p<0.05 for Cx36 and Cgn and ****p<0.0001 for Cx36 and Sipa1l3. Significance for Cx36 and Cgn was determined using a Wilcoxon matched-pairs signed rank test. Significance for Cx36 and Sipa1l3 was determined using a two-tailed paired t-test. N=10–12 regions of interest. (D) Bar graphs showing the degree of colocalization as the percentage of Cx36 puncta that overlap with endocytosis proteins the Ep15l1 and Hip1r. Bar graphs with SEM, ****p<0.0001 for Cx36 and Eps15l1 and ***p<0.05 for Cx36 and Hip1r. Significance was determined using a two-tailed paired t-test. N=10–12 regions of interest.
Figure 4 with 5 supplements
Localization of trafficking, cytoskeleton-associated, adhesion, and synaptic proteins identified by BioID at Cx36 gap junctions in AAV transduced AII amacrine cells expressing GFP.
(A) Proteins implicated in membrane trafficking Sj2bp and Syt4 colocalize with Cx36 in AII amacrine cells. (B) Several cytoskeleton-associated proteins and regulators such as Map6, Dock7, or Gprin1 colocalize with Cx36 in AII cell dendrites. (C) The adhesion molecule Bai1 was often colocalized with Cx36 in AII cell dendrites. (D) Often components of chemical synapses such as Shank2 and Glur2-3 were found in the periphery of gap junction plaques in AII amacrine cells. Scale: 10 µm. Magnified inset: 1 µm.
Figure 4—figure supplement 1
Colocalization of Cx36 and Sipa1l1 in the IPL.
(A) Colocalization of Cx36 and Sipa1l1 puncta in arboreal dendrites of AII amacrine cells. (B) Additional example of Cx36/Sipa1l1 colocalization in the IPL. Scale: 10 µm, magnified inset: 2.5 µm.
Figure 4—figure supplement 2
Localization of additional proteins captured with BioID.
(A) Ajm1 labels the base of AII cell somas but does not directly colocalize with Cx36. (B) Cap1 is associated with Cx36 in AII cell dendrites. (C) Nbea does not directly associate with Cx36 but is often found close to Cx36 clusters. Scale: 10 µm, magnified inset: 2.5 µm.
Figure 4—figure supplement 3
Localization of Ncam1, AF6, Rtn4, and Trim9 cluster in proximity to Cx36 in the inner retina.
(A, B) Ncam1 and Af6 labeling is found adjacent to Cx36 containing gap junctions in the inner retina. (C, D) Rtn4 and Trim9 colocalize with Cx36 in the IPL. Scale: 10 µm, magnified inset: 2.5 µm.
Figure 4—figure supplement 4
Localization of Golga4 in the somas of AII amacrine cells.
Golga4 labeling is absent from the IPL and detected in the INL including somas of AII amacrine cells. Scale: 10 µm, magnified inset: 2.5 µm.
Figure 4—figure supplement 5
Partial colocalization of Cx36 and Stxbp1 in the IPL.
Stxbp1 labeling broadly covers the entire IPL and colocalizes occasionally with Cx36. Scale: 10 µm, magnified inset: 2.5 µm.
Figure 5
Sipa1l3, Sj2bp, and Eps15I1 interact with Cx36.
(A) Coexpression of Sipa1l3 with ZO-1, ZO-2, and Cx36. In transfected HEK293T cells, Sipa1l3 colocalizes with all three proteins. Colocalization of Sj2bp with Cx36 but not with a Cx36 mutant that lacks the PDZ binding motif. (B) Domain organization of constructs used for IP experiments. Co-precipitation of Cx36 and Sjbp2 and Sipa1l3 from lysates of co-transfected HEK293T cells. The PDZ binding deficient Cx36/S318Ter fails to bind Sjbp2 and Sipa1l3. IP experiments with ZO-1 and ZO-2 serve as a positive control. The proteins that were captured in each IP experiment are indicated with arrows in magenta. Cx36 dimers in IP samples are indicated with red arrows and monomers are indicated with blue arrows. (C) In transfected HEK293T cells, Cx36 is associated with endogenous proteins that were also detected in AII amacrine cells. Line scans next to each panel illustrate the close association of these proteins (Greenlees et al., 2015) with Cx36 (magenta). These proteins were also captured via GFP-directed proximity biotinylation. Scale: 10 µm. Magnified inset: 1 µm.
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Figure 5—source data 1
Raw images of western blots shown in Figure 5.
Relevant bands were labeled with arrows.
- https://cdn.elifesciences.org/articles/105935/elife-105935-fig5-data1-v1.zip
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Figure 5—source data 2
Raw images of western blots shown in Figure 5.
- https://cdn.elifesciences.org/articles/105935/elife-105935-fig5-data2-v1.zip
Figure 6
Localization of BioID ‘hits’ at AII amacrine cell/ON cone bipolar cell junctions.
(A) Cartoon illustrating the neurons involved in the primary rod pathway and the subcellular localization of AII/Cone bipolar cell junctions (dashed rectangle). (B) 3D reconstruction of AII amacrine cells from the RC2 connectomics dataset illustrating the localization of AII-AII gap junctions (cyan plaques in second image) and AII/ON Cone bipolar cell gap junctions (yellow plaques in second image). (C) EM micrographs of AII/ON Cone bipolar cell gap junctions. Pseudo-colored: Gap junction (cyan), AII cell (magenta), and ON cone bipolar cell (yellow). White box indicates area shown to the right at 40,000× magnification (0.27 nm/px resolution) with 20° tilt. The density profile for the ROI indicated by the yellow box confirms the pentalaminar (dark-light-dark-light-dark) structure of a gap junction. Also note the asymmetric cytoplasmic densities on the AII versus ON Cone bipolar cell sides. Scale: 0.5 µm. (D) Several of the proteins we identified in AII amacrine cells colocalize with Cx36 at AII/cone bipolar cell junctions. The right plot for each panel depicts an intensity scan of a horizontal region of interest in the middle of each gap junction (arrow). Scale: 10 µm. Magnified inset: 1 µm. (E) High-resolution scan highlighting the subsynaptic distribution of SIPA1L3. Scale: 5 µm. Magnified inset: 1 µm.
Figure 7
AII amacrine cell/ON cone bipolar cell contacts remain in Cx36 KO retinas and contain electrical synapse scaffolds.
(A–C) AII amacrine cell/ON cone bipolar cell contacts were visualized with GFP and SCGN. In Cx36 KO retinas, ZO-1, ZO-2, and Sipa1l3 still localize to AII/CBC contacts. (D) Bar graphs with SEM. Both Sipa1l3 and ZO-1 puncta density in the inner plexiform layer were reduced in Cx36KO, but puncta size was unchanged. Colocalization of Sipa1l3 and ZO-1 was reduced in Cx36KO. For each analysis, 22–32 regions of interest were quantified. (E, F) Cartoon illustrating. Scale: 10 µm. Magnified inset: 1 µm.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | AJM1, rabbit polyclonal | Thermo Fisher | Cat#PA5-145137 RRID:AB_3091817 | 1:100–250 |
| Antibody | BAI1, rabbit polyclonal | Novus Biologicals | Cat# NB110-81586 RRID:AB_1144873 | 1:100 |
| Antibody | Connexin Cx35/36, mouse monoclonal | Millipore | Cat#MAB3045 RRID:AB_94632 | 1:250 |
| Antibody | Connexin 36, mouse monoclonal | Thermo Fisher | Cat#37-4600 RRID:AB_2533320 | 1:250 |
| Antibody | CGN, rabbit polyclonal | Thermo Fisher | Cat# PA5-55661 RRID:AB_2639733 | 1:250 |
| Antibody | Dock7, rabbit polyclonal | Proteintech | Cat#13000-1-AP RRID:AB_10646476 | 1:100 |
| Antibody | EPS15l1, rabbit polyclonal | Thermo Fisher | Cat# PA5-65940 RRID:AB_2665047 | 1:250 |
| Antibody | GFP, chicken polyclonal | Thermo Fisher | Cat# A10262 RRID:AB_2534023 | 1:250 |
| Antibody | GFP, rabbit monoclonal | Cell Signalling | Cat#2956 RRID:AB_10692764 | 1:1000 |
| Antibody | Glur2-3, rabbit polyclonal | Sigma | Cat#07-598 RRID:AB_11213931 | 1:200 |
| Antibody | Gprin1, rabbit polyclonal | Proteintech | Cat#13771-1-AP RRID:AB_2114013 | 1:100 |
| Antibody | HIP1R, rabbit polyclonal | Sigma | Cat# AB9882 RRID:AB_10260185 | 1:250 |
| Antibody | ITSN1, rabbit polyclonal | Thermo Fisher | Cat# PA5-115432 RRID:AB_2900068 | 1:100–250 |
| Antibody | MAP6, rabbit polyclonal | Novus Biologicals | Cat# NBP2-14220 RRID:AB_3261703 | 1:100 |
| Antibody | Myc, rabbit polyclonal | Proteintech | Cat#16286-1-AP RRID:AB_11182162 | 1:1000–2000 |
| Antibody | NCAM1, rabbit monoclonal | Cell Signalling | Cat#50831 RRID:AB_2868490 | 1:100 |
| Antibody | Neurobeachin, rabbit polyclonal | Thermo Fisher | Cat#PA5-58903 RRID:AB_2644492 | 1:100 |
| Antibody | SCGN, sheep polyclonal | Biovendor | Cat# RD184120100 RRID:AB_10719237 | 1:500 |
| Antibody | SCGN, rabbit polyclonal | Cusabio | Cat#CSB-PA020821LA01HU | 1:250–500 |
| Antibody | SHANK2, guinea pig polyclonal | Synaptic Systems | Cat#162204 RRID:AB_2619861 | 1:500 |
| Antibody | Stxbp1, rabbit polyclonal | Proteintech | Cat#11459-1-AP RRID:AB_2196690 | 1:100 |
| Antibody | Rtn4, rabbit polyclonal | Abcam | Cat#ab47085 RRID:AB_881718 | 1:100 |
| Antibody | SIPA1L1, rabbit polyclonal | Proteintech | Cat#25086-1-AP RRID:AB_2714023 | 1:100 |
| Antibody | SIPA1L3, rabbit polyclonal | Proteintech | Cat#0544-1-AP RRID:AB_3086357 | 1:100 |
| Antibody | SJ2BP, rabbit polyclonal | Proteintech | Cat#15666-1-AP RRID:AB_22021149 | 1:100 |
| Antibody | Synaptotagmin4, rabbit polyclonal | Synaptic Systems | Cat#105 143 RRID:AB_2619771 | 1:100 |
| Antibody | vGlut1, guinea pig polyclonal | Sigma | Cat#AB5905 RRID:AB_2301751 | 1:500 |
| Antibody | V5, mouse monoclonal | Thermo Fisher | Cat#R969-25 RRID:AB_2556564 | 1:1000 |
| Antibody | ZO-1, mouse monoclonal | Thermo Fisher | Cat#33-9100 RRID:AB_2533147 | 1:250 |
| Antibody | ZO-2, rabbit polyclonal | Thermo Fisher | Cat#71-1400 RRID:AB_2533976 | 1:250 |
| Antibody | Anti-rabbit-HRP, goat polyclonal | Thermo Fisher | Cat#34160 RRID:AB_228341 | 1:1000 |
| Antibody | Anti-mouse-HRP, goat polyclonal | Thermo Fisher | Cat#34130 RRID:AB_228307 | 1:1000 |
| Antibody | Anti-chicken 488, donkey polyclonal | Jackson Immunoresearch | Cat#703-545-155 RRID:AB_2340375 | 1:250 |
| Antibody | Anti-rabbit Cy3, donkey polyclonal | Jackson Immunoresearch | Cat# 711-165-152 RRID:AB_2340606 | 1:250 |
| Antibody | Anti-mouse Cy5, donkey polyclonal | Jackson Immunoresearch | Cat# 715-175-150 RRID:AB_ 2340819 | 1:250 |
| Antibody | Anti-mouse DyLight 405, donkey polyclonal | Jackson Immunoresearch | Cat#715-475-140 RRID:AB_2340838 | 1:100–250 |
| Antibody | Anti-guinea pig 647, donkey polyclonal | Jackson Immunoresearch | Cat#706-605-148 RRID:AB_2340476 | 1:250 |
| Antibody | Anti-sheep 647, donkey polyclonal | Jackson Immunoresearch | Cat#713-605-003 RRID:AB_2340750 | 1:250 |
| Antibody | Anti-mouse 647, donkey polyclonal | Jackson Immunoresearch | Cat#715-605-151 RRID:AB_2340863 | 1:250 |
| Commercial assay or kit | Dynabeads MyOneStreptavidin C1 | Thermo Fisher | Cat#65001 RRID:SCR_021025 | |
| Commercial assay or kit | ChromoTek GFP-Trap Agarose | Proteintech | Cat#gta RRID:AB_2631357 | |
| Commercial assay or kit | ChromoTek DYKDDDDK Fab-Trap Agarose | Proteintech | Cat#ffa RRID:AB_2631357 | |
| Commercial assay or kit | Pierce Protein A/G Agarose | Thermo Fisher | Cat#20421 | |
| Recombinant DNA reagent | Venus-ZO-1-GFP | Addgene | Cat#56394 | |
| Recombinant DNA reagent | pEGFP-C3-ZO-2-GFP | Addgene | Cat#27422 | |
| Recombinant DNA reagent | FLAG-Eps15l1 | GenScript | Clone: OMu22488D | |
| Recombinant DNA reagent | FLAG-Gprin1 | GenScript | Clone: OMu09339D | |
| Recombinant DNA reagent | AAV_HKamac_V5-dGBP-TurboID | Generated in this study | ||
| Recombinant DNA reagent | AAV_HKamac_GFP | Generated in this study | ||
| Recombinant DNA reagent | AAV_HSyn_Cx36-V5-TurboID | Generated in this study |
Additional files
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Supplementary file 1
Excel workbook containing ranked list of all proteins that were detected via mass spectroscopy in Cx35.1-V5-TurboID fish in two experiments, plus SAINT analysis of these experiments.
- https://cdn.elifesciences.org/articles/105935/elife-105935-supp1-v1.xlsx
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Supplementary file 2
Excel workbook containing ranked list of all proteins that were detected via mass spectroscopy in Cx36-EGFP-directed proximity labeling in two experiments, plus SAINT analysis of these experiments.
- https://cdn.elifesciences.org/articles/105935/elife-105935-supp2-v1.xlsx
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Supplementary file 3
Comparison of electrical synapse components that were identified in zebrafish and mouse retinas.
- https://cdn.elifesciences.org/articles/105935/elife-105935-supp3-v1.docx
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Uncovering the electrical synapse proteome in retinal neurons via in vivo proximity labeling
eLife 14:RP105935.
https://doi.org/10.7554/eLife.105935.4
