Figure 2
Abnormal mitochondrial ultrastructure and mitochondrial fusion of Drp1 cells. (a) Transmission electron micrographs of Vector and Drp1 cells showing a high degree of mitochondrial interconnectivity and abundant, well-organized cristae patterns in the former cells and small, sparse mitochondria with a lack of interconnectivity and poorly formed cristae in the latter cells. (b) Immunoblotting for select mitochondrial membrane and matrix proteins in Vector and Drp1 cells. Proteins included sirtuins (sirt) 3 and 5, mitochondrial fission factor (MFF) and medium-chain acyl-CoA-dehydrogenase (MCAD). Typical results are depicted. (c) Quantification of the western blots shown in panel (b). Three separate immunoblots typical of those depicted in panel (b) were densitometrically scanned and normalized to β-actin. The values obtained for Vector cells were then arbitrarily set to 1. The results of relative mean band intensities±1 S.E. are shown. (d) Higher rates of mitochondria fusion in Drp1 cells. Vector and Drp1 cells stably expressing DsRed2-Mito or EGFP-Mito were mixed in equal numbers and fused with PEG for 1 min. After washing, cells were incubated in medium containing CHX for 2 h, fixed, stained with DAPI and examined by confocal microscopy. Mitochondrial fusion in the resultant heterokaryons was identified by the merging of green and red signals. The histogram indicates the degree of mitochondrial fusion in each cell pair and was generated by calculating the percentage of co-localized fluorescent signals from at least 20 randomly selected heterokaryons with the use of the CoLocalizer Pro software package. Fluorescence micrographs below the histogram show typical images of mitochondrial fusion in each cell type. The rapidly alternating fission and fusion activity in the Drp1 cells when compared with the Vector cells can best be appreciated in the time-lapse movies in Supplementary Video S2
