Figure 2

Translocation of Pep-1-labeled mitochondria (Pep-1-Mito) into MELAS cybrid cells. (A) Illustration showing mitochondrial translocation from medium into cells. Red (Mitotracker Red) and green (Mitotracker Green) fluorescence represent donor Pep-1-Mito and innate mitochondria, respectively. Asterisks indicate aggregates of Pep-1-Mito complexes suspended in medium. Reconstructed three-dimensional (3D) confocal images showing co-localization of donor and innate mitochondria inside MELAS cybrid cells, indicated by arrows (yellow fluorescence). (B) Mitochondria labeled with fluorescent proteins. Donor mitochondria tagged with GFP (Mito^GFP, green fluorescence) and innate mitochondria tagged with HcRed1 (Mito^RFP, red fluorescence). Nuclei were stained with DAPI (blue fluorescence). Donor Pep-1-labeled Mito^GFP (Pep-1-Mito^GFP) inside recipient MELAS cybrid cells after 2 days; possible sites of fusion between donor and innate mitochondria (yellow fluorescence) are indicated by arrows. (C) 3D visualization of Pep-1-Mito^GFP internalization and innate Mito^RFP in MELAS cybrid cells tracked over time using confocal microscopy combined with differential interference contrast (DIC). Z-axis yields a longitudinal view showing Pep-1-Mito^GFP stuck to the cell membrane after 0.5 h and penetration into the cell after 1 h, as indicated by an arrow. Blank arrow indicates co-localization of Pep-1-Mito^GFP and Mito^RFP after 2 h and 4 h. Star symbol indicates decayed fluorescence of Pep-1-Mito^GFP suspended in medium after 8 h culture. Insert frame in z-stack image shows quantified Pep-1-Mito^GFP and Mito^RFP expression at each time point, represented by mean area of red fluorescence (pixel) per cell in same section thickness (μm).