Figure 5.

Calcium within bulk endosomes is required for SV budding. A, Cultures were loaded with HRP (10 mg/ml) and either TMR-Dextran (50 μm) or Rhod-Dextran (50 μm) for 2 min in the presence of KCl (50 mm). Cells were washed immediately and then stimulated twice with KCl (50 mm, 30 s each) to release all available SVs (Immediate Unload) and left to rest for 30 min. Cultures were fixed after HRP loading (Load), the immediate unload (Unload), or the rest period (Rest), as indicated by arrows. B, Bar graph displays the mean number of HRP-labeled SVs per nerve terminal ±SEM (number independent experiments = 3 for both; **p < 0.01, *p < 0.05, one-way ANOVA). C, D, Frequency distribution of endosome diameter for Load, Unload, and Rest in cells loaded with either TMR-Dextran (C) or Rhod-Dextran (D). At least 70 HRP-labeled endosomes were used for diameter measurements per independent experiment. ***p < 0.001, two-way ANOVA.