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Fig. 10. RBO is required for activity-dependent endosome formation in synapses. FM1-43 photoconversion generates an electron-dense marker that is clearly visible by electron microscopy. Dye was incorporated into NMJ synapses after a 2-minute high-[K⁺] stimulation at 37°C, photoconverted and examined in wild type (OR) and rbo^ts. (A) Inset shows a light-microscopy image of muscle 6/7 NMJ as photoconverted, ready to be sectioned at the electron-microscopic level. Top panels show representative images of control and mutant bouton profiles. Many FM-labeled vesicles and endosomes can be seen throughout the boutons. Asterisks (^*) mark labeled endosomes in wild type; note the absence of label in rbo^ts. Lower panels show higher-magnification images of endosomes (^*) and SVs (thin arrows). In rbo^ts, no labeling of endosomes was observed (^*), but abundant SVs were labeled. Scale bars: 250 nm. (B) Quantification of FM-labeled SVs per bouton section. (C) Quantification of FM-labeled endosomes per bouton profile. Significance indicated: ^**P<0.01, ^***P<0.001.

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