Internalized exosomes undergo a stop-and-go movement along the ER. (a) Cumulative intracellular exosome trajectories color coded for exosome speed (0–0.5 µm/s) in an overlay of DIC with confocal fluorescence images (left) and side by side (right). (b) Cumulative intracellular trajectories (green) show exosome residency mainly near regions of peripheral ER (B and C) but largely absent from the perinuclear region (A). (c) Fraction of intracellular exosomes for which at least one contact with the ER was detected. (d) Representative examples for predominant modes of exosome–ER interactions, with a corresponding speed trace illustrating exosome stop-and-go movement (top left). (e) Characterization of 148 individual exosome–ER contacts for interaction mode and spatial distribution (left), contact duration (middle), and number of interactions per vesicle (right). (c and e) Pooled data from three independent experiments. (d) Representative data from at least five different experiments. (f) Overlay of DIC with confocal fluorescence images (150 min after exosome addition) show exosomes localizing closely to ER filaments, tips, cavities, and branches (arrows). All images, human primary fibroblasts: green, CD63-emGFP HEK293 exosomes; red, ER Tracker. (g) TEM imaging shows CD63-Apex2–labeled exosomes upon uptake in human primary fibroblasts typically within vesicles close to rough ER and cytoskeleton. Bottom right, CD63-Apex2–transfected HEK293 parent cells.