Fig. 5: Rab11 optogenetics in Exo70 KD cells does not rescue full fusion.

a Schematic of Rab11 optogenetics using the CRY2-CIB system. b Rab11 optogenetics with or without Exo70 KD. Maximum-intensity projection of movies (top). Scale bars, 6 μm. Kymographs of Rab11 channel (bottom). Red arrowheads, stuck vesicles. Vertical scale bar, 6 μm. c Images of a single vesicle undergoing very long tethering (left, black) and fusion (right, green). Colored circles in the traces (bottom) correspond to dashed circles in the image frames (top). Scale bar, 2 μm. d Average image sequence of fusing vesicles from one cell (left). Scale bar, 2 μm. CRY2-Rab11 traces, time aligned to fusion (right). Averages (bold line) of cell averages (light lines) are shown. n = 5 cells for both scram and Exo70 KD. For comparison, the thick red line redisplays the average Exo70 trace from the Exo70-KK–CRY2 ( +CIB) optogenetics experiment. Dashed cyan lines, zero baseline. e Three fusion modes observed with CRY2-Rab11 activation (+Exo70 KD) using 100 mM HEPES. Average image sequence of FF, KR and KS events from one cell (left). Scale bar, 2 μm. TfRc-pH (green) and CRY2-Rab11 (red) traces (average of cell averages) for all fusion modes (right). Mean ± SEM. Red dashed lines, zero baseline. f Tethering half-times for different fusion modes. Mean ± SEM (NS = not significant, two-tailed Student’s t-test. g Frequency of fusion modes with Rab11 optogenetics at different light doses. Note that 1.5 W/cm² was used in b to f. Mean ± SEM (NS = not significant, two-tailed Student’s t-test). h Schematic summary of Exo70-KK (Fig. 2) and Rab11 optogenetics results. During active tethering, optimal engagement of exocyst leads to FF (i). This model entails coupling between vesicle tethering and fusion (dots). Passive tethering occurs when the exocyst is suboptimally engaged (ii) or when vesicles are artificially tethered without the exocyst (iii). Note, for clarity, only two copies of exocyst are depicted.