Video 6

Movie S6. M10 cells transfected for TfR-pHluorin and stably expressing Rab11-mCherry (top part) or stably expressing eYFP-Langerin/Rab11-mCherry (bottom part) were imaged for 3D TIRF reconstruction (SI Imaging Model and Reconstruction). A multiangle TIRF sequences of five image stacks have been recorded with a 25-ms exposure time per plane. Movies last for only 10 s and are extracted from 120-s series with a time interval of 152 ms between two stacks. Fusion events automatically detected from the 2D TIRF image sequences (events 75 and 102 from Movie S5) are realigned at frame 30 and are displayed on the top left side of the sequences in 21 × 21 pixels regions defined around the detected events. Under each 2D regions, kymographs represent the time evolution of the intensity in the region around the docking–fusion events (30 frames before and 30 frames after). In the upper part, two subsequent events of docking–fusion with different intensities are detectable in the same region. Note that docking already started 5 s before fusion in the TfR-pHluorin/Rab11-mCherry cell, whereas docking starts (red channel) only 1.52 s before fusion for the eYFP-Langerin/Rab11-mCherry. On the Right, 3D representations of the corresponding events after multiangle reconstruction are shown. Note that two docking–fusion processes for the TfnR/Rab11a vesicles are visible in the upper reconstruction.