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. 2014 Jun 5;10(6):e1004391. doi: 10.1371/journal.pgen.1004391

Figure 1. Phosphorylation of the Parkin Ubl domain regulates mitochondrial morphology.

Figure 1

(A) Parkin is phosphorylated by PINK1 in insect cells. S2 cells transfected with the indicated plasmids with or without Drosophila PINK1 were treated with or without 30 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 1 h. The cell lysate was subsequently separated on a Phos-tag gel, followed by western blotting with anti-Drosophila Parkin. (B) The phosphorylation status of Parkin affects the mitochondrial length in muscle tissue. Fluorescent and TEM images of the indirect flight muscle in the indicated genotypes of 14-day-old adult flies are shown. To visualize the mitochondria, the mitoGFP (green) transgene was co-expressed, and the muscle tissue was counterstained with phalloidin (magenta). Mitochondria in the TEM images are outlined with broken lines to highlight their morphology. Scale bars = 10 µm in the fluorescent images and 2 µm in the TEM images. (C) Mitochondrial morphology of 14-day-old PINK1 mutant flies expressing mock, WT Parkin and phospho-mutants. The inset shows a high-magnification TEM image of PINK1-/-; Parkin SE with intact mitochondrial matrices. Scale bars = 10 µm in the fluorescent images and 2 µm in the TEM images. (D) The length of the long axis of the muscle mitochondria was calculated. The data represent the mean ± SE from three flies (n = 25 in each). ** p<0.01 vs. all other genotypes, # p<0.01 vs. mock with PINK1+/+, § p<0.01 vs. PINK1RNAi; WT or SA Parkin, * p<0.05, N.S., not significant. (E) Mitochondrial morphology of 14-day-old Parkin mutant flies expressing mock, WT or SA Parkin. Scale bars = 10 µm in all fluorescent images and 2 µm for Parkin-/- and WT Parkin; Parkin-/- and 5 µm for SA Parkin; Parkin-/- in the TEM images. Arrowheads indicate large mitochondrial aggregates. (F) The length of the long axis of the muscle mitochondria was calculated. The data represent the mean ± SE from three flies (n = 25 in each). * p<0.05. The genotypes are as follows: (B) UAS-mitoGFP/+; MHC-GAL4/+ (control), UAS-mitoGFP/UAS-Parkin; MHC-GAL4 (WT, SA and SE Parkin). (C) PINK1B9/Y; UAS-LacZ; MHC-GAL4 (PINK1-/-), PINK1B9/Y; UAS-Parkin; MHC-GAL4 (PINK1-/-; WT, SA or SE Parkin). UAS-mitoGFP; MHC-GAL4, UAS-PINK1 RNAi crosses were used rather than PINK1B9 crosses for fluorescent images. (E) UAS-mitoGFP/UAS-LacZ; Da-GAL4, ParkinΔ21/Parkin1 (Parkin-/-), UAS-mitoGFP/UAS-Parkin; Da-GAL4, ParkinΔ21/Parkin1 (Parkin-/-; WT or SA Parkin).