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Sheets et al., 2014 - Characterization of Ribeye Subunits in Zebrafish Hair Cells Reveals That Exogenous Ribeye B-Domain and CtBP1 Localize to the Basal Ends of Synaptic Ribbons. PLoS One   9:e107256 Full text @ PLoS One

Fig. 2 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).

Fig. 3 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).

EXPRESSION / LABELING:
Antibodies:
Fish:
Anatomical Term:
Stage: Protruding-mouth

Fig. 4 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 10th and 90th 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.

EXPRESSION / LABELING:
Antibodies:
Fish:
Anatomical Term:
Stage: Day 5

Fig. 5 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).

Fig. 6 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.

EXPRESSION / LABELING:
Antibody:
Fish:
Anatomical Term:
Stage Range: Protruding-mouth to Day 5
Acknowledgments:
ZFIN wishes to thank the journal PLoS One for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ PLoS One