Tf-endosome–mitochondrial interaction does not depend on intraendosomal iron release. (A) Double-labeled fluorescence image from a representative time-lapse video of MitoTracker orange–labeled MDCK-PTR cells pulsed with AF488 lock-hTf (a); corresponding 3D rendering of AF488 lock-hTf–endosomes and mitochondria (b); the 3D-rendered image selecting kiss and run undergoing lock-hTf–endosomes (c; see Video 8); magnified ROI (d; white box in c); magnified ROI (white box in d) where the selected lock-hTf–endosome (yellow spot) is in close proximity to a mitochondrion (e). Bars: (a–d) 10.0 µm; (e) 1.0 µm. (B) Videomicrograph of the kiss and run interaction between the selected lock-hTf–endosome and mitochondrion (A e). Graph shows a definite decrease in lock-hTf–endosomal instantaneous speed (blue line) during the kiss phase (dashed boxes), defined by DT = 0 (red line). See Video 9. Video 10 shows two additional lock-hTf–endosomal kiss and run events with mitochondria displaying a similar trend of decreased endosomal instantaneous speed upon interaction. Bar, 0.5 µm. (C and D) Mean hTf-endosomal (C: 311 kiss and run events, 131 endosomes, and 6 cells) and lock-hTf–endosomal (D: 603 kiss and run events, 356 endosomes, and 10 cells) instantaneous speeds were significantly higher during run noninteractions than during their kiss phases. Error bars: 95% confidence interval; **, P < 0.001, Student’s t test.