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. 2019 May 20;8:e41711. doi: 10.7554/eLife.41711

Figure 6. Fusion pore regulation by dynamin1 and amisyn is cAMP-dependent.

(a–b) Example image sequence of transient recruitment of dynamin1-GFP (a, lower) or mCherry-amisyn (b, lower) to granules (upper, labeled with NPY-tdmOrange2 or NPY-EGFP) during K+-stimulated exocytosis in presence of forskolin. (c) Average time course (± SEM) of dynamin1-GFP (dyn) fluorescence during 34 flash-type exocytosis events (red) and eight full-fusion type events (black) in presence of forskolin; and nine flash events in presence of fsk + ESI09 (blue); data points represent average of five frames and time is relative to the flash onset in the granule signal. (d) Cumulative frequency histograms and medians (inset, with p for Kolmogorov-Smirnov test) of NPY release times in presence of fsk in cells expressing dynamin1-EGFP (red), dynamin with added ESI09 (blue) or control (black). 119 (CTR), 42 (dyn), 24 (dyn + ESI09) events. n of preps: 5 (C + fsk); 1 (dyn); 2 (dyn + ESI-09). (e) Fraction of flash events in (d). n, number of cells, p for Kruskal-Wallis/Dunns test. (f) Average time course (± SEM) of mCherry-amisyn (amis) fluorescence (red n = 274 flash events; black n = 46 full fusion events) or in presence of fsk + ESI09 (blue; n = 56 flash events). (g) Cumulative frequency histograms and medians (inset, with Kolmogorov-Smirnov test) of NPY release times in cells expressing mCherry-amisyn, amysin with ESI09, or control; fsk was present. 213 (CTR), 320 (amisyn), and 90 (amis +ESI09) events. n of preps: two for each. (h) Fraction of flash events in (g); p for Kruskal-Wallis/Dunn test. n, number of cells. (i) As in c, but without forskolin for control (black), dynamin (red), and dynamin with S223 (green); n = 37 flash events, n = 39 full fusion events for dyn and n = 40 flash events for dyn +S223. (j–k) As in (d–e), but for 38 (ctrl, black), 76 (dynamin1, red) and 55 (Dyn + S223, green) events in the absence of forskolin. n of preps: 4 (C-fsk); 2 (dyn); 2 (dyn + S223). (l) As in f, but without forskolin present; 65 flash events (red) and 73 full fusion events (black) for amisyn, and 154 flash events for amisyn + S223 (green). (m–n) As in (g–h), but for 123 (ctrl, black), 138 (amisyn, red) and 174 (amis + S223, green) events in the absence of forskolin. n of preps: 1 (C-fsk); 2 (amis); 2 (amis + S223).

Figure 6.

Figure 6—figure supplement 1. NPY and amisyn/dynamin1 recruitment profiles at the point of release.

Figure 6—figure supplement 1.

(a) Exocytosis events separated for flashes (left, green) and full fusions (right, gray) for dynamin1-GFP and NPY-tdmOrange2 expressing INS-1 cells in presence of forskolin from Figure 6. n, number of events (b) As in a, but for mCherry-amisyn and NPY EGFP expressing INS-1 cells from Figure 6. n, number of events (c) As in a, but in absence of forskolin. n, number of events (d) As in b, but in absence of forskolin. n, number of events.
Figure 6—figure supplement 2. Quantification of overexpression.

Figure 6—figure supplement 2.

Ins1-cells expressing mCherry-amisyn or dynamin1-GFP were fixated and immunostained using anti-amisyn or anti-dynamin1 and fluorescence was quantified for both labels by TIRFM of single cells. (a) Example images of immunostaining (upper) and mCherry-amisyn (lower). (b) Average fluorescence (cell-background) for immunostaining (white) and mCherry-amisyn (gray). (c) Plot of mCherry-amisyn vs immunostaining fluorescence; each symbol represents one cell. The offset at the y-axis corresponds to cells that only express endogenous amisyn. (d–e) as b-c but for dynamin1.