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. 2020 Mar 9;11:1266. doi: 10.1038/s41467-020-14993-8

Fig. 7. Endophilin’s role in exocytosis is mediated, at least in part, through its interaction with intersectin-1.

Fig. 7

a–d Distribution of ITSN-1 and ITSN-2 was altered in the endophilin TKO cells. Representative confocal images of chromaffin cells stained for ITSN-1 (a) and ITSN-2 (c) under resting and stimulated (depolarization by high K+) conditions (note that different cells are shown for each condition in the panel). Intensity line profiles below indicate ITSN-1 and ITSN-2 intensities along the line (the approx. line position is marked in yellow in a and c). Quantification of ITSN-1 (b) and ITSN-2 (d) intensities in the cytosol vs. near the membrane (see Methods and Suppl. Fig. 7) revealed an altered distribution of ITSN-1 and ITSN-2 in endophilin TKO cells that did not further change upon stimulation. ITSN-1 (N = 3, 3 mice each, WT resting (26 cells), TKO resting (33 cells), WT high K (32 cells) and TKO high K (36 cells), and ITSN2 (N = 3, 3 mice/condition, WT resting (29 cells), TKO resting (30 cells), WT high K (30 cells) and TKO high K (28 cells). b, d note that WT resting vs. WT high K+, WT resting vs. TKO resting and TKO resting vs. TKO high K+ were compared). e, f The altered distribution of ITSN-1 in endophilin TKO cells was rescued by expression of endophilin 1 (green traces), but not by expression of endophilin 1-ΔITSN (endophilin E329K + S336K mutant that does not bind ITSN-1) (pink traces). N = 3, 3 mice, TKO (25 cells), TKO + A1FL (30 cells) and TKO + A1 ΔITSN (35 cells). g–m Expression of endophilin 1  in endophilin TKO cells rescued exocytosis as inspected by combined capacitance and amperometry measurements (see Fig. 2), but the same effect could not be achieved by expressing an endophilin mutant that does not bind ITSN1 (E329K + S336K). Data shown in g–m are from four independent experiments, four mice each condition, cells TKO (39), TKO + A1-FL (36), and TKO + A1 ΔITSN (21). Scale bars 3 µm. Error bars denote SEM. N = number of independent replicates. One-way ANOVA with Tukey’s post-hoc test, *p < 0.05, **p < 0.01; ***p < 0.001, ns not significant.