PINK1 and Drp1 microscope images of control shRNA or PINK1 shRNA lines. At low magnification (upper panels), an association of mitochondria (M) with lysosomes (L) is seen, particularly in the PINK1 deficient cells. Occasional vacuoles are also seen, indicated by asterisks. N = nucleus. At higher power (lower panels), the mitochondrial cristae in PINK1 deficient cells are disrupted (arrows) while normal mitochondrial morphology is seen in control cells. Scale bars are shown for each figure and are 1 mm in the top panels and 0.5 mm in the lower panels. (E) Counting abnormal cristae across multiple experiments shows that approximately twice as many cells in the PINK1 deficient lines showed mitochondrial cristae abnormalities compared to the control shRNA (*, P = 0.032 by t-test, n = 8). doi:10.1371/journal.pone.0005701.g002 expressing the second PINK1 shRNA sequence (data not shown). Cell counts show a decrease in the proportion of cells with fragmented or truncated mitochondria after Drp siRNA (P = 0.001 by two-way ANOVA; Fig. 5E). Finally, mitochondrial length was measured and was lower in PINK1 deficient cells compared to control (P,0.001 by one-way ANOVA) and increased after Drp siRNA (P,0.001; Fig. 5F). Next, we addressed what would happen if levels of Drp1 were increased in PINK1 deficient cells. We transiently expressed YFP- Drp1 in the same cell lines and then imaged mitochondria to detect YFP, using non-transfected (YFP negative) cells in the same cultures as controls (Fig. 6A–D). In both control and PINK1 shRNA lines, YFP-Drp1 was recruited to foci on the surface of mitochondria as described previously [40–42]. This punctate staining pattern was detected with both N- and C- terminal YFP fusions of Drp1, but not with YFP alone (data not shown) and represent fission competent foci of active Drp1. Twenty-four hours after transfection, PINK1 deficient lines showed more disrupted mitochondrial networks compared to control lines or untrans- fected cells (compare Figs. 6B and 6D). The amount of YFP-Drp1 recruited into mitochondrial fractions was similar in control and PINK1 deficient lines by immunoblot (Fig. 6E). Furthermore, we also noted that increased expression of YFP-Drp1 resulted in an accumulation of both transfected and endogenous Drp1 protein in mitochondria, again presumably due to the formation of fission- competent Drp1 oligomers at the mitochondrial surface, and again this effect is similar in both control and PINK1 shRNA cell lines. Mitochondrial connectivity was quantitated by FRAP using an HA-tagged Drp1 construct to avoid interference of the YFP tag on FRAP. We confirmed that .90% of mito-YFP positive cells were HA-Drp1 positive (data not shown). As in the experiments above with mitotracker, YFP labeled mitochondria showed extensive fragmentation in PINK1 deficient cell lines when challenged by Drp1 overexpression (Fig. 6F). Drp1 overexpression decreased FRAP in both control shRNA and PINK1 shRNA cell lines, with the lowest mobile fraction values seen in the latter (Fig. 6G, 6H). Therefore loss of PINK1 and overexpression of Drp1 have additive effects. FRAP experiments also showed that overexpres- sion of PINK1 limits Drp1-mediated mitochondrial fragmentation (Fig. 6J). The sensitivity of PINK1 deficient cells (Fig. 6I) and resistance of PINK1 overexpressing cells (Fig. 6K) to Drp1- mediated fragmentation was confirmed using counts of mitochon- drial morphology. We also overexpressed the mitochondrial fusion proteins Optic Atrophy 1 (OPA1) and Mitofusin 2 (Mfn2) and found that either could recover mitochondrial connectivity in PINK1 knockdown cells (Supplementary Fig. S4). Overall, these results suggest that PINK1 deficient mitochondria are more sensitive to mitochondrial fission. Drp1 phosphorylation and activity are altered in the absence of PINK1 due to calcineurin activation Next, we wanted to know if Drp1 activity was modified in PINK1 deficient cells. As Drp1 activity can be affected by serine phosphorylation [40–43], we examined the phosphorylation status of Drp1 in PINK1 deficient cells by enriching for phosphoproteins and blotting for endogenous Drp1. We confirmed the phospho- enrichment by blotting for p-S637 Drp1 and used DJ-1, which is not basally phosphorylated [44], as a negative control (Fig. 7A). Drp1 showed a loss of phosphorylation in the absence of PINK1 (Fig. 7A). Quantification across multiple experiments showed an ,30% loss of endogenous Drp1 phosphorylation in PINK1 deficient cells (Fig. 7B). Phosphorylated amounts of OPA1, Mfn1 and Mfn2 and HtrA2 were also examined, but no differences between cell lines were found (Supplementary Fig. S5). Subcellular fractionation of control versus PINK1 shRNA cells were analyzed by western blotting and show mitochondrial recruitment of endogenous Drp1 is not influenced by the loss of PINK1 (Fig. 7C). BMH-crosslinking experiments did not show any alterations in Drp1 oligomerization in PINK1 deficient cells (Fig. 7D). Using in vitro GTPase assays, we found increased GTPase activity of Drp1 in cells expressing PINK1 shRNA (Fig. 7E; P,0.01 for cell lines), which may be related to the decreased phosphorylation seen in the same lines. Calcineurin (CaN) increases Drp1 activity via its phosphatase activity [41]. Neither calcineurin catalytic a subunit, regulatory calcineurin-b subunit or calmodulin protein levels were altered between PINK1 deficient cells and controls (data not shown). However, cellular CaN activity was significantly increased in PINK1 deficient cells (Fig. 8A; P,0.001 by ANOVA, n = 5). To address whether CaN activity influences Drp1, we treated cells with 1 mM of the CaN inhibitor FK506 for 1 hour and separated phosphorylated and total proteins and immunoblotted for Drp1 as in figure 7B. Quantification of multiple experiments confirmed the decreased ratio of phosphorylated to total Drp1 in PINK1 deficient cells compared to control cells and showed that this measure of Drp1 phosphorylation was increased to a level similar to controls after FK506 treatment (P,0.05 by ANOVA, n = 6; Fig. 8B). We also treated cells with 1 mM FK506 for 1–3 hours and imaged mitochondria using mito-YFP (Fig. 8C). Quantitatively, the lower FRAP signal in PINK1 deficient mitochondria was increased by FK506 treatment (Fig. 8D) such that by 3 h the difference between control and PINK1 shRNA lines was not significant. The effect of FK506 in PINK1 shRNA lines was significant at 1 h (P,0.05) and 3 h (P,0.001 by ANOVA, n = 60 cells measured over two experiments). Cell counts were also performed and confirmed that FK506 decreases the number of truncated or fragmented mitochondria (P = 0.0002; Fig. 8E). We investigated whether the rescue in connectivity would affect mitochondrial membrane potential and therefore we treated control and PINK1 shRNA lines with FK506 1 mM for 1 and 3 h and measured TMRM. There was no increase in TMRM intensity in PINK1 deficient cells (Fig. 8F), suggesting that that calcineurin-mediated dephosphorylation of Drp1 does not influ- ence these events. Discussion Mitochondrial dysfunction has been linked to neuronal cell loss in PD and it seems likely that the genes associated with recessive parkinsonism help maintain mitochondrial function under stressful conditions. Here, we first confirm previous reports suggesting that PINK1 is protective against mitochondrial toxins [14,30,31,33] PLoS ONE | www.plosone.org 6 May 2009 | Volume 4 | Issue 5 | e5701