Figure 8.

Calcineurin activity is essential for SV budding from bulk endosomes. A, Cultures were loaded with HRP (10 mg/ml) for 2 min in the presence of KCl (50 mm) and washed immediately to remove excess HRP. Cells were then stimulated twice with KCl (50 mm, 30 s each) to release all available SVs (Immediate Unload) and left to rest for 30 min. CsA (10 μm) was added during the KCl unload and also the rest period. Cultures were fixed after HRP loading (Load), the immediate unload (Unload), or the rest period (Rest), as indicated by arrows. B, Representative electron micrographs are shown. Scale bar, 500 nm. Black and white arrows indicate HRP-labeled SVs and endosomes, respectively. C, Bar graph displays the mean number of HRP-labeled SVs per nerve terminal ±SEM (number of independent experiments: Load, 5; Unload-Control, 3; Unload-CsA, 3; Rest-Control, 3; Rest-CsA, 3; ***p < 0.001, **p < 0.01, one-way ANOVA). D, Frequency distribution of endosome diameter after Rest in the presence or absence of CsA. At least 130 HRP-labeled endosomes were used for diameter measurements per independent experiment. ***p < 0.001, two-way ANOVA.