Fig. 3: Exo70 optogenetics with the iLID system recapitulates tethering with the CRY2-CIB system.

a Schematic of Exo70 optogenetics using the iLID system. b Exo70-KK–mCherry-SspB optogenetics with or without LOV2-SsrA (“iLID”). Cells were activated at 2 Hz with 100-ms pulses of 488-nm light (1.5 W/cm²). Maximum-intensity projection of movies (top). Scale bars, 6 μm. Kymographs of Exo70 channel (bottom). Red arrowheads, stuck vesicles. Vertical scale bar, 6 μm. c Ensemble average image sequence of fusing vesicles from four cells (left). Scale bar, 2 μm. Exo70-KK–mCherry-SspB traces (red), time aligned to fusion (right). Averages (bold line) of cell averages (light lines) are shown. n = 4 cells for both + iLID and – iLID optogenetics. d Three fusion modes (FF, KR and KS) observed with coexpression of iLID (top) and two fusion modes (FF and KR) observed without coexpression iLID (bottom) using 100 mM HEPES. Ensemble average image sequences from four cells for both iLID conditions (left). Scale bars, 2 μm. TfRc-pH (green) and Exo70-KK–mCherry-SspB (red) traces (average of cell averages) for different fusion modes (right). Mean ± SEM. Black dashed lines, zero baseline. e TfRc-pH traces (green) from the + iLID condition in d overlaid on an expanded timescale. Note that the TfRc-pH signal for KS remains elevated after fusion. f Tethering half-times for different fusion modes (left). Mean ± SEM (*P = 0.015, cyan *P = 0.012, NS = not significant, two-tailed Student’s t-test). Rescue of FF by Exo70 iLID optogenetics (right). Mean ± SEM (***P = 7.5 × 10⁻⁴, two-tailed Student’s t-test).