Tf-endosome–mitochondria interactions facilitate functional iron transfer. MDCK-PTR cells with iron sensor RDA-labeled mitochondria were pulsed with AF647-Tf for 2 min at 37°C and chased, and time-lapse images of 15-s duration (107-ms interval) were acquired at 5 min after the Tf pulse to capture the early time points of Tf-endosomal trafficking. (A and B) Videomicrographs of distinct kiss interactions between 3D-rendered Tf-endosomes (white spots) and surface-rendered mitochondria (asterisks), proceeding from left to right at 107-ms intervals. The rendered mitochondrial color, indicated by the color bars, is based on the mitochondria’s mean fluorescence intensity. See Videos 3 and 4. Graphs represent Tf-endosomal interaction in terms of its distance from the mitochondrion (blue lines) and corresponding change in mean intensity of interacting (red lines) and noninteracting mitochondria (green lines). The dotted lines mark the beginning of the kiss interaction, after which the interacting mitochondrial mean intensity gradually quenches, whereas that of the noninteracting mitochondria remains unchanged. Bars, 0.5 µm. (C) Normalized mean intensity of interacting mitochondria (n = 12) is significantly reduced after the kiss interaction. (D) Mean intensity of noninteracting mitochondria (n = 31) remains unchanged over time. Error bars: 95% confidence interval; **, P < 0.001, Student’s t test. a.u., arbitrary units.