Panel A shows a schematic illustrating tethered and docked synaptic vesicle organization around ribbon from multiple orientations. Panel B shows a protein domain diagram of Piccolino with amino and carboxyl terminal antibody binding sites. Panel C shows confocal microscopy images of Piclo carboxyl terminal and RIBEYE localization within the outer plexiform layer. Panel C prime shows stimulated emission depletion microscopy images comparing Piclo carboxyl terminal and RIBEYE localization at ribbon synapses. Panel C double prime shows a line graph comparing pixel intensity profiles of Piclo carboxyl terminal and RIBEYE signals. Panel D shows confocal microscopy images of Piclo amino terminal and RIBEYE localization within the outer plexiform layer. Panel D prime shows stimulated emission depletion microscopy images comparing Piclo amino terminal and RIBEYE localization at ribbon synapses. Panel D double prime shows a line graph comparing pixel intensity profiles of Piclo amino terminal and RIBEYE signals. Panel E shows electron microscopy images and schematic illustrating Piclo carboxyl terminal localization along the synaptic ribbon. Panel E prime shows electron microscopy images and schematic illustrating Piclo amino terminal localization along the synaptic ribbon. Panel E double prime shows a scatter plot comparing distances of Piclo carboxyl and amino terminals from the ribbon.
Piccolino is oriented at rod photoreceptor synapses. (A) Schematic 3D representation of a rod photoreceptor SR viewed from the side, front and top. (B) Schematic representation of Piccolino indicating binding sites for Pclo4 (Pclo N-term) and Pclo49 (Pclo C-term) antibodies. (C and D) Representative fluorescence micrographs of WT retinae double-labeled with Pclo C-term (C) and Pclo N-term (D) antibodies co-stained with the SR marker RIBEYE. (C′ and D′) High-power views of STED microscopy images of a single SR viewed from the top labeled with Pclo C-term (C′) and Pclo N-term (D′) antibodies co-stained with the SR marker RIBEYE. (C″ and D″) Line intensity profiles corresponding to lines in C′ and D′. (E and E′) Representative pre-embedding immunoelectron micrographs of rod photoreceptor SRs labeled with Pclo C-term (E) and Pclo N-term (E′) antibodies. Arrowheads (➤) point to individual immunogold puncta. (E″) Quantification of distance for individual immunogold puncta to the center of SRs. Data are n = 157 puncta from three individual experiments (Pclo C-term) and n = 262 puncta from four individual experiments (Pclo N-term). Mean values were compared using an unpaired t test, ***P < 0.001. Scale bar = 2 µm in D for C and D, 0.5 µm in D′ for C′ and D′ and 0.2 μm in E′ for E and E′. AU, arbitrary units; ab, antibody; FWHM, full-width at half maximum.