Figure 7. CCP number following stimulation is drastically elevated at dynamin mutant synapses, but in both WT and KO synapses they are selectively localized in the plasma membrane.
(A and B) CCP number in cultures incubated with CTX-HRP selectively after the high K+ stimulation. CCPs positive for HRP reaction product are indicated by the dark portion of each bar. Note that in these samples, where only very few vacuoles are labeled (see Figure 1G,H), nearly all CCPs are labeled, indicating their localization on the plasma membrane. (C and D) CCP number in cultures stimulated in the presence of soluble HRP and washed at the end of the stimulus. Note that CCPs observed after stimulation are negative for HRP reaction product (dark portion of each bar), once again indicating their selective localization on the plasma membrane. The HRP-positive CCPs in the resting (R) and stimulated (K+) samples reflect accessibility to the extracellular medium of arrested plasma membrane CCPs in dynamin KO synapses. (E) Electron micrograph of a Dyn1 KO synapse incubated for 5 min with CTX-HRP selectively during recovery (as for field A). Most CCPs are HRP reaction product positive (red arrows). (F) Dyn1 KO synapse incubated with soluble HRP during stimulation and then washed and examined at 3 min (as for field C). All CCPs are HRP reaction product negative. Scale bar = 100 nm. ****, ***, **, * indicate p-values of <0.0001, <0.001, <0.01, and <0.05, respectively. n.s., ‘not significant’, N.D., ‘not determinated’. The standard error of the mean is shown in each graph. Black asterisks refer to comparisons between total vesicles, colored asterisks to comparisons between HRP-labeled vesicles. Bars: standard error of the mean.