n-Syb localizes to synaptic endosomal compartments in addition to synaptic vesicles. (A) Intracellular compartments in a wild-type synaptic terminal. Cysteine string protein (CSP), Synaptotagmin (Syt), and the t-SNARE Syntaxin 1A (Syx) function in synaptic vesicle (SV) exocytosis. Early endosomes (EE) and endosomal vesicles (EV) are marked by Rab5 and Avalanche/Syntaxin 7 (Syx7). Early endosomes undergo degradation via multivesicular body (MVB) formation, marked by Rab7 and Hrs, and lysosomes (Ly) marked by carnation (Car), deep orange (Dor), and sunglasses (Sun). Recycling endosomes (RE) are marked by Rab11. (B) Colocalization of 16 markers with n-Syb in developing photoreceptor terminals at ∼P+30%. Shown are the percentiles of individually distinguishable compartments for immunolabeling with 13 antibodies (black bars) that are positive for n-Syb coimmunolabeling. Green bars show similar quantification of n-Syb colocalization for compartments marked by YFP-tagged Rab5, Rab7, and Rab11. (C–H′′′′) Immunolabeling of photoreceptor terminals at ∼P+30%. (C) Immunolabeling for n-Syb, Syx7, and Hrs (green). Single channels are shown in C′–C′′′. (D–F) Colabeling of n-syb-Gal4>YFP-Rab5 (D), YFP-Rab7 (E), and YFP-Rab11 (F) expression with n-Syb antibody. Single YFP channels are shown in (D′–F′). All arrows show compartments with colocalization, and arrowheads indicate lack of colocalization. (G–H′′′′) High-resolution single sections from deconvolved 3D confocal datasets (Hiesinger et al., 2001). Note that individual compartments are discernible both in the cell body (cb) region and at synapses (syn) that are positive for various combinations of endosomal markers: n-Syb, V100, Syx7, and Rab5 (green in H). Original sections are shown in G and H, deconvolved sections are shown in G′ and H′, and single channels are shown in G′′–G′′′′ and H′′–H′′′′. Bars: (C–E and G) 5 µm; (H) 2 µm. All error bars are SEM. WT, wild type.