Generation of a Cx35b-V5-TurboID zebrafish line.

(A) Cartoon illustrating the generation of Cx35-V5-TurboID fish via Tol2-mediated transgenesis and outline of in vivo biotinylation experiments. To induce efficient proximity labeling, zebrafish were intraperitoneally injected with 30µl of 5mM Biotin (PBS) for three consecutive days. Afterwards 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 Cx35b-V5-TurboID to photoreceptor gap junctions. Neutravidin Oregon Green labeling was used to validate efficient proximity biotinylation of Cx35b and surrounding molecules in biotin injected Cx35b-V5-TurboID fish. Scale 10µm. (C) Western blot of streptavidin pull-downs probed with V5 antibodies and Streptavidin-HRP. (D) String diagram illustrating the protein-protein network surrounding Cx35b. (E) Colocalization of Cx35b 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.

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 synapsin promoter. This strategy greatly resulted in overexpression artefacts causing aberrant protein localization. In strategy 2 we adapted the GFP-directed Turbo strategy developed by Xiong et al., (36) to shuttle a V5-dGBP-TurboID construct to Cx36-EGFP containing gap junctions in retinas of the Cx36-EGFP strain. (B) Vertical sections of AAV infected retinas confirming cell type-specificity of our AAV vectors. The HKamac promoter developed by Khabou et al. (40) 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: 5µm. (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.

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 EPS15I1, an endocytic adapter protein, and Cx36. Scale: 10 µm. Magnified inset: 1 µm.

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.

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. (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 (56) with Cx36 (magenta). These proteins were also captured via GFP directed proximity biotinylation. Scale: 10 µm. Magnified inset: 1µm.

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 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). EM micrographs of AII/On Cone bipolar cell gap junctions. Pseudo colored: Gap junction (Cyan), AII cell (Magenta) and ON cone bipolar cell (Yellow). (C) 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.

AII amacrine cell/ON cone bipolar cell contacts remain in Cx36 KO retinas and contain electrical synapse scaffolds.

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) 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. (E-F) Cartoon illustrating Scale: 10 µm. Magnified inset: 1µm.

Localization of additional proteins captured with BioID.

(A) AJM1 labels the base of AII cell somas but does 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 Cx36 clusters. Scale: 10 µm.

Localization of NCAM1 cluster in proximity to Cx36 in AII amacrine cells.

NCAM1 labeling does overlap with Cx36 but is found adjacent to gap junctions. Scale: 10 µm.