Kolmogorov-Smirnov assessments were used to determine the statistical significance. a fluorescent protein module destabilizes the resulting hybrid protein and causes a complete loss of synaptic ribbons. Our results thus demonstrate an essential role of the RIBEYE B-domain in enabling RIBEYE assembly into synaptic ribbons, reinforcing the notion that RIBEYE is the central organizer of synaptic ribbons. or whether this process also depends on RIBEYE B-domain. In order to understand whether RIBEYE B-domain plays a role in the assembly of synaptic ribbons, we made use of RIBEYEKI knockin (KI) mice (Maxeiner et al., 2016). In these KI mice, RIBEYE B-domain has been replaced by GCaMP3 in the KI GNE 2861 allele, thus making these animals an ideal tool to study the role of the RIBEYE B-domain. We found that in the absence of the RIBEYE B-domain, synaptic ribbons are not assembled and the RIBEYE A-domain/GCaMP3 fusion protein is destabilized. Thus, the RIBEYE B-domain is essential for the assembly of synaptic ribbons. Materials and Methods Mice All animal care and use procedures were reviewed and approved by the local animal authorities (Landesamt fr Verbraucherschutz; Gesch?ftsbereich 3; 66115 Saarbrcken, Germany; GB 3-2.4.1.1-K110/180-07). Mice were anesthetized with isoflurane and killed by cervical dislocation in ambient light before organ collection. The RIBEYE knockin (KI) mice that were analyzed in the present study were generated by Maxeiner et al. (2016). In the GNE 2861 RIBEYE KI, the alternative exon 1b MGC129647 of the mouse CtBP2/RIBEYE gene encoding RIBEYE A-domain was fused in frame with cDNA encoding for the Ca2+-indicator GCaMP3 (Tian et al., 2009) concluded by a STOP codon. As a consequence, the RIBEYE B-domain was replaced by GCaMP3 in the recombinant RIBEYE KI allele (Maxeiner et GNE 2861 al., 2016). All possible genotypes at the recombinant RIBEYE locus (WT: RBEWT/WT; heterozygous KI: RBEWT/KI and homozygous KI: RBEKI/KI) were analyzed in the RIBEYE KI mice as GNE 2861 indicated in the respective experiments/figures. The genotypes were obtained by breeding heterozygous mice with each other (RBEWT/KI X RBEWT/KI). Mice were kept under standard light/dark cycle and supported with standard food and water 0.001). In the box and whisker plots (D2) of the data from (D1), mean values are labeled by horizontal dotted yellow lines and median values by horizontal solid green lines. The boxes represent 25th-75th percentiles, and whiskers 1.5 times IQR. The statistical analysis was performed by Students 0.05; *** 0.001). In the box and whisker plots (D2) of the data from (D1), means values are shown as horizontal yellow dotted line and median values by horizontal green solid line. The boxes represent the 25th-75th percentiles and whiskers represent 1.5 times IQR. The statistical analyses were performed by Mann-Whitney = number of mice; n = number of analyzed confocal images. Scale bar: 5 m. Open in a separate window Figure 4 Absence of RIBEYE A-domain immunosignals in the retina of RBEKI/KI mice. (A1CA3,B1CB3,C1CC3) 0.5 m-thin GNE 2861 retina sections from the indicated littermate mice (RBEWT/WT, RBEWT/KI, and RBEKI/KI) with mouse anti-SV2 and rabbit anti-RIBEYE A-domain (tau, Maxeiner et al., 2016). (C1) RIBEYE A-domain immunosignals were completely absent in the OPL and IPL of RBEKI/KI mice. (D1,D2) Quantification of RIBEYE A-domain immunofluorescence signals (as integrated density). Quantification of the immunofluorescence signals in the OPL confirmed the absence of RIBEYE A-domain immunosignals in the OPL of RBEKI/KI mice (D1). RIBEYE A-domain immunosignals in the OPL of RBEWT/KI mice were significantly reduced in comparison.