Figure 5. Phosphorylation of Drp1^S616 is essential for PINK1‐mediated mitochondrial structures and function in Drosophila .

• A, B
  Drp1^WT and Drp1^S616D, but not Drp1^S616A, rescue PINK1‐null induced crushed thorax in Drosophila. Representative thorax images of PINK1^WT and PINK1 deficiency (PINK1^KO) Drosophila expressing either mhc‐gal4 (Mhc/+), mhc‐gal4‐driven human Drp1^wt (Mhc>hDrp1^WT), or mhc‐gal4‐driven human Drp1^S616A (Mhc>hDrp1^S616A) or mhc‐gal4‐driven human Drp1^S616D (Mhc>hDrp1^S616D) are shown. Images were taken either under light microscopy (A, upper panels, yellow arrowhead indicates the collapsed thorax) or SEM (B, lower panels). Quantitation of crushed thorax is shown (B). > 200 flies for each experiment are analyzed. Scale bar = 200 μm. One‐way ANOVA followed with Tukey's test. ***P < 0.001. Data were presented as mean ± SEM of three independent experiments. For each condition, > 200 flies were quantified.
• C
  Drp1^WT and Drp1^S616D, but not Drp1^S616A, restore abnormal ATP production in PINK1‐null flies. ATP production was measured using muscle lysates generated from PINK1^WT flies expressing mhc‐gal4 (Mhc/+) (as a control) and PINK1^KO flies expressing either mhc‐gal4 (Mhc/+), mhc‐gal4‐driven human Drp1^wt (Mhc>hDrp1^wt), or mhc‐gal4‐driven human Drp1^S616A (Mhc>hDrp1^S616A) or mhc‐gal4‐driven human Drp1^S616D (Mhc>hDrp1^S616D). One‐way ANOVA followed with Tukey's test. ***P < 0.001, ns: no significance. Data were presented as mean ± SEM of three independent experiments.
• D–G
  Drp1^WT and Drp1^S616D, but not Drp1^S616A, rescue mitochondrial structures in PINK1^KO flies. Representative images of mito‐GFP (green) labeled mitochondria (D) and TEM analysis (F) of IFMs from PINK1^WT flies expressing mhc‐gal4 (Mhc/+) (as a control) and PINK1^KO flies expressing either mhc‐gal4 (Mhc/+), Mhc‐gal4‐driven human Drp1^wt (Mhc>hDrp1^wt) or Mhc‐gal4‐driven human Drp1^S616A (Mhc>hDrp1^S616A) or mhc‐gal4‐driven human Drp1^S616D (Mhc>hDrp1^S616D) are shown. Scale bar = 10 μm (D) and 1 μm (F). Average mitochondrial size was quantified, and > 3 flies/group and 3–6 pictures of different microscopic fields from each fly were analyzed per repeat. One‐way ANOVA followed with Tukey's test. *P < 0.05, ***P < 0.001. Data were presented as mean ± SEM of three independent experiments (E). Normal and abnormal mitochondrial number per mm² were quantified. > 3 flies/group and 3–6 pictures of different microscopic fields from each fly were analyzed per repeat. Bars represent the mean ± SEM of three independent experiments (G).
• H, I
  Drp1^WT and Drp1^S616D, but not Drp1^S616A, suppress cell death in PINK1^KO flies. Representative TUNEL staining (Red) images of IFM sections from PINK1^WT and PINK1^KO flies expressing either Mhc‐gal4 (Mhc/+), Mhc‐gal4‐driven human Drp1^wt (Mhc>hDrp1^WT), or Mhc‐gal4‐driven human Drp1^S616A (Mhc>hDrp1^S616A) or mhc‐gal4‐driven human Drp1^S616D (Mhc>hDrp1^S616D) are shown. Nuclei were counter‐stained with DAPI (blue), scale bar = 10 μm. Apoptotic cells (%) in different genotype flies were quantified (I). > 5 flies/group and 3–6 pictures of different microscopic fields from each fly were analyzed per repeat. One‐way ANOVA followed with Tukey's test. ***P < 0.001. Data were presented as mean ± SEM of three independent experiments.
• J
  Expression and phosphorylation of hDrp1 in Drosophila. Fly muscle lysates generated from PINK1^WT or PINK1^KO flies expressing mhc‐gal4‐driven hDrp1^WT (WT) and hDrp1^S616A (S616A) were immunoblotted with antibodies against either phospho(Ser616)‐Drp1(pS616) (to detect phosphorylated hDrp1^S616), Drp1 (to detect both endogenous and exogenous Drp1), and myc‐tag (to detect exogenous Drp1). α‐tubulin was detected as a loading control. Flies expressing mhc‐gal4 (−) were included as an expression control.