Extended Data Fig. 3: SEC14L2 compartments are dynamically recruited into the ER-associated RAB5-labeled early endosome fission site.

a-b, STED imaging and line scan analysis of endogenous SEC14L2 in COS-7 cells overexpressing EGFP-RAB5 (a) or EGFP-RAB4 (b) after 16 h of transfection. Boxed regions are magnified in the insets. Arrows indicate contact sites between vesicles. Right, example intensity profiles of the lines. SEC14L2, magenta; EGFP-RAB5/RAB4, green. c, Predicted frequency of SEC14L2 compartment-endosome contacts due to chance. A still image was randomly extracted from a 2-min movie and the amount of the SEC14L2 compartment surface covered by endosomes determined. Statistical data are shown as the Mean ± SEM at the right from n = 7 cells over 2 independent experiments. d, Overlay of FIBSEM images with confocal pictures of the cell with zSec14l3-mCherry (purple) and iRFP-SEC61β (yellow) overexpression. e, Magnification of the region in the white box in d. Segmentation of the ER (yellow), SEC14L2 compartments (zSec14l3⁺ in purple), and endosomes (cyan). f, Contours and line scan analysis of the relative fluorescence intensity of zSec14l3, RAB5, and SEC61β at three time points as shown in Fig. 2b, t = 10 s (pre-fission), t = 12 s (fission), t = 18 s (post-fission). Arrows in the contours at the top right corner indicate plotted lines. Images in Fig. 2b and the line scan analysis reveal that a dynamic SEC14L2 compartment is recruited to the dividing surface between RAB5-positive endosomes before fission (t = 12 s).

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