Ribeye A-domains inconsistently localize to synaptic ribbons at 3 dpf and 5 dpf.

Representative confocal maximum-intensity projection images of immunolabel or GFP in lateral line NM hair cells. Scale bars: 3 μm (main panels), 1 μm (D, inset). (A–A′) Ribeye a (A-domain)-myc alone (A) and merged with Ribeye b labeling synaptic ribbons (A′) in posterior lateral line NM1 hair cells of a 3 dpf larva. (B) Ribeye a (A-domain)-myc (cyan) and Vglut3 (magenta) immunolabel in a cross-section of an anterior lateral line NM at 5 dpf. (C–C′) Ribeye b (A-domain)-GFP alone (C) and merged with Ribeye a immunolabel (C′) in NM1 hair cells of a 3 dpf larva. White boxes indicate regions used in (D–D′′). (D–D′′) Insets of region in C′ showing basally-localized Ribeye b (A-domain)-GFP in relation to synaptic ribbons labeled with an antibody to Ribeye a. (D) Two adjacent hair cells: one with low Ribeye b (A-domain) expression next to another hair cell with moderate expression. A domain-GFP colocalizes with endogenous Ribeye a. (D′) A hair cell with moderate Ribeye b (A-domain) expression. (A domain)-GFP colocalizes with endogenous Ribeye a. (D′′) A hair cell with high Ribeye b (A-domain) expression. Read arrows indicate basally localized A-domain aggregates that do not colocalize with endogenous Ribeye. (E–E′) Ribeye a (A-domain)-myc (E), Ribeye b (A-domain)-GFP (E′), and merged with Ribeye b immunolabel (E′′) in NM1 hair cells of a 3 dpf larva. Note that the Ribeye b antibody labels both endogenous Ribeye b and exogenous Ribeye b (A-domain).

Ribeye B-domain/CtBP2s localizes to synaptic ribbons at 3 dpf.

Representative images of immunolabel or fluorescent tag in posterior lateral line NM1 hair cells of 3 dpf larvae. Scale bars: 3 μm (main panels), 1 μm (insets). (A) Ribeye (B-domain)-GFP (cyan) and Ribeye a antibody labeling of synaptic ribbons (magenta). Note that B-domain-GFP does not completely colocalize with Ribeye a immunolabel. (B) Ribeye (B-domain)-myc (cyan), Ribeye a (blue), and Ribeye b (red) immunolabel. Dashed circles indicate nuclear localization of B-domain. Note that Ribeye a and b immunolabel colocalizes with each other (magenta), but only partially colocalizes with B-domain (white). Red arrows indicate resolvable indentations in the synaptic ribbons that do not contain exogenous B-Domain, but do contain Ribeye. (C) Ribeye a (full length)-mcherry (cyan) and Ribeye b immunolabel (magenta). Full length exogenous Ribeye a-mcherry appears throughout the synaptic ribbon and colocalizes with Ribeye b immunolabel (white).

Ribeye B-domain/CtBP2s disrupts endogenous Ribeye retention at synaptic ribbons in 5 dpf hair cells.

(A-B) Representative images of immunolabel or fluorescent tag in posterior lateral line NM3 hair cells of 5 dpf larvae. Scale bars: 3 μm (main panels), 1 μm (insets). (A, A′) Ribeye a (blue) and Ribeye b (red) antibody labeling of synaptic ribbons, and anti-myc antibody labeling of B domain-myc (cyan) in a WT (A) and a transgenic (A′) larva. Dashed circles indicate weak nuclear localization of B-domain. (B, B’) Ribeye (B-domain)-myc (cyan), Ribeye b (magenta), and MAGUK (yellow) immunolabel in a WT (B) and a transgenic (B′) larva. Red asterisks indicate strong nuclear localization of B-domain. Red arrows indicate cells with moderate levels of B-domain in the cytosol. (C) Box plots of cumulative immunolabel intensities of presynaptic Ribeye immunolabeled spheres and postsynaptic MAGUK immunolabeled patches in 5 dpf transgenic B domain-myc larvae and WT siblings. These plots show the median value (horizontal bar), the upper and lower quartiles (box), and the range (whiskers). Whiskers indicate the 10^th and 90^th percentiles. ****P<0.0001, defined by a Mann-Whitney U Test. Each plot represents a population of intensity measurements collected from NM3 hair cells of 7–8 individual larvae. (D) Relative expression level of ribeye b transcripts in the posterior lateral line of 5 dpf transgenic B domain-myc larvae and WT siblings. Expression data was normalized to b-actin expression. The level of gene expression in WT siblings was normalized to one. Error bars are s.e.m.

CtBP1 localizes to synaptic ribbons, but does not disrupt endogenous Ribeye.

Representative images of immunolabel in posterior lateral line NM3 hair cells of 5 dpf larvae. Scale bars: 3 μm (main panels), 1 μm (insets). (A) Ribeye b antibody labeling of synaptic ribbons (magenta) and MAGUK antibody labeling of postsynaptic densities (yellow) in a WT sibling larva. Anti-myc (cyan) immunolabel was performed as a negative control. (B–C) CtBP1-myc (cyan), Ribeye b (magenta), and MAGUK (yellow) immunolabel in two representative transgenic larvae. (B) CtBP1-myc (cyan) is strongly localized to the nucleus with weak synaptic localization. The red arrow indicates a synaptic ribbon containing CtBP1-myc. Note that Ribeye immunolabel intensity in all four synaptic ribbons appears comparable. (C) CtBP1-myc (cyan) is weakly localized to the nucleus with strong synaptic localization. Presynaptic Ribeye immunolabel intensity is not reduced compared to WT (A).

Ribeye B-domain/CtBP2s and CtBP1 localizes to the basal end of synaptic ribbons facing the postsynaptic density.

Isosurface renderings of ribbon synapses extrapolated from z-stack confocal images of Ribeye b (magenta), GFP or myc (cyan), and MAGUK (yellow). Dashed arrows indicate the ribbon synapses used from the images to generate the 3D renderings. (A–A′) Ribeye (B-domain)-GFP (cyan) with Ribeye b (magenta), and MAGUK (yellow) in 3 dpf larvae. Note that B-domain-GFP within synaptic ribbons appears adjacent to patches of MAGUK. (B–B′) Ribeye (B-domain)/CtBP2s-myc (cyan) with Ribeye b (magenta), and MAGUK (yellow) in 5 dpf larvae. Note that B-domain-myc within synaptic ribbons also appears adjacent to patches of MAGUK. (C–C′) CtBP1-myc (cyan) with Ribeye b (magenta), and MAGUK (yellow) in 5 dpf larvae. Synaptic ribbon localization of CtBP1 appears comparable to Ribeye (B-domain)/CtBP2s.