Figure 2
CAPS-1 C2 and PH ___domain and C-terminal truncation mutants do not support neuronal DCV exocytosis. (a) Representative images of neuronal DCV labeling with NPY-mCherry in CAPS-2 KO (control) and CAPS DKO neurons. Boxed area is enlarged on the right. (b) Number of DCVs in CAPS DKO hippocampal neurons infected with wild type or mutant CAPS-1 constructs and control neurons. One-way ANOVA: p = 0.42 (not significant, ns). (c) Sholl analysis of the number of DCVs in distal neurites of CAPS DKO neurons infected with wild type or mutant CAPS-1 constructs and control neurons. (d) Schematic representation of the method to measure neuronal DCV exocytosis. Neurons, co-infected with NPY-mCherry and CAPS-1, are stimulated with 16 trains of 50 AP at 50 Hz (blue bars), which induces DCV exocytosis (sudden disappearance of a fluorescent punctum, middle panels). Representative trace of DCV exocytosis is depicted on the right. (e–g) Average DCV exocytosis events per cell before, during and after stimulation in (e) control, CAPS DKO and DKO + WT, (f) DKO + WT, DKO + K428E and DKO + G476E and (g) DKO + WT, DKO + RKK and DKO + ΔC neurons. (h) Cumulative plot of DCV exocytosis events in CAPS DKO neurons infected with wild type or mutant CAPS-1 constructs and control neurons. Shaded area represents SEM. (i) Average DCV exocytosis events per cell in CAPS DKO neurons infected with wild type or mutant CAPS-1 constructs and control neurons. One-way ANOVA: p = 5.9 *10−9 (***); post-hoc Dunnett’s test: control vs DKO + WT: p = 0.27 (ns), control vs DKO (+CAPS-1 mutants): p ≤ 8.5 *10−5 (***), DKO vs DKO + CAPS-1 mutants: p ≥ 0.96 (ns). (j) DCV release probability in CAPS DKO neurons infected with wild type or mutant CAPS-1 constructs and control neurons. One-way ANOVA: p = 2.2 *10−8 (***); post-hoc Dunnett’s test: control vs DKO + WT: p = 0.045 (*), control vs DKO (+CAPS-1 mutants): p ≤ 1.9 *10−4 (***), DKO vs DKO + CAPS-1 mutants: p ≥ 0.98 (ns). Detailed information (average, SEM, n and statistics) is shown in Table S1.
