Figure 7

Rescue of mitochondrial fragmentation phenotype due to mortalin knockdown by Parkin overexpression requires an intact autophagic machinery leading to reduced cell death. (a) Fluorescence microscopy in fixed cells (n=94 cells) was used to analyze mitochondrial morphology in mortalin and control siRNA-treated MEFs with or without overexpressed wt mortalin, PINK1 or Parkin. Mortalin siRNA-treated cells have a smaller mitochondrial form factor, aspect ratio and mitochondrial size (area per mitochondrion). The reintroduction of either wt mortalin or Parkin rescued this mitochondrial fragmentation phenotype. Reintroduction of PINK1 resulted only partially in the rescue of mitochondrial fragmentation. This suggests that successful rescue through Parkin overexpression is dependent on ATG5 (compare also Figure 7b), whereas PINK1 does not appear to be affected by ATG5. (b) Fluorescence microscopy in fixed cells (n=102 cells) was used to analyze mitochondrial morphology in mortalin and control siRNA-treated ATG5 KO MEFs with or without overexpressed wt mortalin, PINK1 or Parkin. Mortalin siRNA-treated cells have a smaller mitochondrial form factor, aspect ratio and mitochondrial size (area per mitochondrion). Only the reintroduction of wt mortalin rescued this mitochondrial fragmentation phenotype, whereas neither overexpression of PINK1 nor of Parkin did. (c) SH-SY5Y cells were treated with control siRNA or mortalin siRNA and analyzed for apoptotic cell death via Annexin V staining measured by FACS analysis. Knockdown of mortalin resulted in an increased Annexin V signal. Overexpression of wt mortalin or Parkin in cells treated with mortalin siRNA led to a full rescue of this phenotype. PINK1 overexpression lowered the amount of Annexin V staining, but did not significantly rescue the observed phenotype in mortalin knockdown cells. (d) Knockdown of mortalin in HeLa cells was achieved by inducible miRNA system. Myc-Parkin was transiently transfected. The clustering of mitochondria was assessed by counting the number of mitochondrial particles per cell in those with a positive signal for miRNA only or for miRNA+myc-Parkin in knockdown mortalin cells as well as control cells. Knockdown of mortalin resulted in fragmentation of the mitochondrial network, whereas the exogenous introduction of myc-Parkin led to mitochondrial clustering around the nuclear region. Cells overexpressing myc-Parkin are encircled in red. Cells overexpressing without exogenous expression of myc-Parkin are encircled in yellow