Fig. 4.
Genetic interactions between Pink1 and fission genes in modulating mitochondrial dynamics in Drosophila and mammalian cells. (A–D) Live imaging of mitochondria in S2R cells subjected to the following genetic manipulations: gfp RNAi (A), dPink1 RNAi (B), Drp1 RNAi (C), dPink1 and Drp1 double RNAi (D). Note the similar phenotype of mitochondrial aggregation (arrows) caused by dPink1 RNAi and Drp1 RNAi despite the lower percentage in the case of dPink1 RNAi. The arrowheads point to thin continuous mitochondrial tubules extending beyond mitochondrial clusters, which appeared exclusively in D. S2R cells are not homogenous in size. Two types of cells, smaller-sized (Left) and bigger-sized (Right), are shown for each genetic manipulation. The effects are similar in both sized cells. (E and F) Live imaging of mitochondria in S2R cells subject to the following genetic manipulations: dPink1 RNAi plus EGFP transfection (E); dPink1 RNAi plus EGFP-dFis1 transfection (F). EGFP-dFis1 (E) but not EGFP (F) suppressed mitochondrial clustering induced by dPink1 RNAi. The arrow marks aggregated mitochondria. (G) Seven categories of mitochondrial morphology in mammalian COS-7 cells ranging from extreme fusion (type I) to extreme fission (type VII), as revealed by MitoTracker Red staining. The categories are arbitrarily defined based on the following mitochondrial characteristics: I, perinuclear aggregates together with long continuous tubules; II, extensive, long tubular network; III, intermediate-length tubular network; IV, a mixture of short tubular mitochondria and punctate units; V, small round tubules with holes in the center; VI, small punctate dots; VII, spherical aggregates similar to that see in DA neurons overexpressing Pink1 or Parkin. To highlight the difference between categories I and VII, the long tubular mitochondria threads present in I but not VII are marked by arrows. (H and I) Statistical analysis showing that hPink1 overexpression promotes mitochondrial fission, which is prevented by the overexpression of Drp1K38A (H), whereas hPink1 RNAi promotes mitochondrial fusion in COS-7 cells, which is rescued by hFis1 or hDrp1 overexpression (I). The ratio of cells exhibiting type II and III morphology typical of fusion versus type V and VI morphology typical of fission were quantified. *, P < 0.01, one-way ANOVA test.