Fig. 2. Expression of endophilin 1 and endophilin 2 rescued exocytosis in endophilin TKO cells.

a, b Expression of endophilin 1 and endophilin 2 full-length rescued the exocytic defects seen in endophilin TKO cells. Panel arranged as in Fig. 1i, with three groups: endophilin TKO (red traces; N = 4, 4 mice (24 cells)), TKO + endophilin 1 (green traces; N = 4, 4 mice (23 cells)) and TKO + endophilin 2 (blue traces; N = 4, 4 mice (25 cells)). Control KOWTKO data from Fig. 1I (gray trace; N = 4, 6 mice (32 cells)) are superimposed. Note that both endophilin 1 and endophilin 2 can rescue exocytosis; rescue with endophilin 2 is indistinguishable from control KOWTKO cells. c–e Burst and sustained component, as well as RRP size, were rescued upon expression of endophilin 1 and 2, respectively. f, g The altered kinetics of the RRP in TKO was rescued (f), while the time constant of the SRP was not significantly changed (g) upon expression of endophilin 1 and 2, respectively (b–g) Kruskal–Wallis test with Dunn’s multiple comparison test. h Exemplary traces from amperometric recordings of endophilin TKO and endophilin TKO expressing endophilin 2. Insets show magnified view. i Schematic of analyzed amperometric spike parameters. j–q Amperometry analysis (210 s recording per cell) reveals problems in vesicle fusion: number of fusion events per cell (j), single spike amplitude (k) and charge (l) were significantly decreased in endophilin TKO cells, while the kinetics of single fusion events, such as duration at half-maximal amplitude (m), rise time (n), and decay time (o), was unchanged. The stability of the fusion pore was also altered, as shown by shorter foot amplitude (p) while the foot duration (q) was not changed. TKO: 2 mice (16 cells), TKO + endophilin: 2 mice (12 cells)—each cell is a biological replicate. Error bars denote SEM. N = number of independent replicates. Unpaired two-sided t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ns not significant.