Fig. 1: Membrane binding by Exo70 influences the mode of vesicle fusion.

a Localization of endogenous Exo70 in HeLa cells. Solid and open arrowheads, selected examples of triple-colocalizing and Sec8-only spots, respectively (left). Scale bars, 6 and 2 μm for whole and zoomed images, respectively. Pearson’s correlation of Exo70 and Sec8 with Rab11 (right). Data represent mean ± SEM (*P = 0.049, **P = 0.0017, two-tailed Student’s t-test). b Two-color TIRFM of Exo70-WT–mCherry and transferrin receptor-pHluorin (TfRc-pH). Maximum-intensity projection of movie (top). Scale bar, 6 μm. Inset, zoom of dashed box. Kymograph for Exo70-WT (middle; region outlined by rectangle) and -KK (bottom). Arrowheads, tethering duration. Vertical scale bar, 6 μm. c Average image sequence of fusing vesicles from one cell (top). Arrow, fusion onset. Dashed white circles, initial tethering. Scale bar, 2 μm. Exo70-WT (black) and Exo70-KK (red) traces, time aligned to fusion (bottom). Averages (bold line) of cell averages (light lines) are shown. n = 5 cells for both Exo70-WT and -KK. Asterisk denotes elevated tethering signal for Exo70-KK. d Rescue of vesicle fusion by Exo70 constructs. n = 5 cells for scram, Exo70 KD, Exo70-KK rescue, and n = 6 cells for Exo70-WT rescue. Mean ± SEM (*P = 0.0011, cyan *P = 0.029, **P = 0.0091, two-tailed Student’s t-test). e Schematic of TfRc-pH (green) and transferrin (Tf)-A568 (purple) signals during full fusion (FF) and kiss-and-run (KR). f Effect of 100 mM HEPES on vesicle reacidification (compare dashed boxes). n = 26 vesicles for short tethering with both 25 and 100 mM HEPES, and n = 24 and 40 vesicles for long tethering with 25 and 100 mM HEPES, respectively. Mean ± SEM. g Effect of 100 mM HEPES on the average TfRc decay with Exo70-WT and -KK (left). n = 5 cells for both Exo70-WT and -KK in 25 mM HEPES, and n = 6 cells for both Exo70-WT and -KK in 100 mM HEPES. Mean ± SEM (*P = 0.011, cyan *P = 0.011, orange *P = 0.42, two-tailed Student’s t-test). Arrow, fusion onset. Dashed box highlights difference in decay for the first time point (0.5 s) after fusion onset. Frequency of KR with Exo70-WT and -KK (right). Mean ± SEM (***P = 2.2 × 10⁻⁶, two-tailed Student’s t-test). h Summary of tethering times. Mean ± SEM (*P = 0.038, cyan *P = 0.048, **P = 0.0011, cyan **P = 0.0015, two-tailed Student’s t-test). Number and sample size of experiments, here and elsewhere, are shown in Supplementary Table 1.