in PINK1 deficient cells and their appropriate control shRNA line (Fig. 1F). The control shRNA line showed a similar amount of decrease in viability after rotenone exposure (55.64+/20.25%, n = 3 experiments with 10,000 cells analyzed/experiment) as the LacZ control for the overexpression (51.0+/25.6% n = 3), suggesting a lack of off-target effects of the shRNA. In contrast, PINK1 shRNA cells showed a greater loss of viability after rotenone exposure, to 47.4+/21.71% of cells being viable (P = 0.009 by t-test comparing the proportion of viable cells in control shRNA with PINK1 shRNA lines). These results suggest that PINK1, via its kinase activity, has beneficial effects on mitochondrial function under stressed conditions that improve cell viability. The above results show that our PINK1 lentiviral constructs have the expected effects in living cells. One small point of difference with the literature [14,30,31] is that in these cell lines, PINK1 does not protect against staurosporine-induced apoptosis (Supplementary Fig. S2D). With rotenone exposure, events typically associated with apoptotic cell death such as Bax multimerization and PARP cleavage are not observed, but are seen following staurosporine treatment (Supplementary Fig. S2E, F). FACS analyses showed both apoptosis and necrosis occurred in our model (Fig. 1C, F). These results suggest that the effect of PINK1 is not on apoptosis per se but on mitochondrial function. Next we examined mitochondrial morphology in these knockdown cell lines using the approach outlined by Exner et al [12], who divided the mitochondrial morphologies of PINK1 deficient cells into intact, truncated and fragmented categories (Fig. 2A). Rotenone was used as a stress-inducing agent, which has been shown to cause mitochondrial truncation and fragmentation in various cell types [25,34,35]. Increased expression of wild type PINK1 limited the number of cells with fragmented/truncated mitochondria after exposure to rotenone (Fig. 2B; P,0.0001 overall for cell lines/treatments by two-way ANOVA). PINK1 knockdown increased the number of cells containing truncated and fragmented mitochondria, and rotenone had an additive effect (Fig. 2C; P = 0.016 for differences between cell lines/treatments). It should be noted that the number of cells showing a full fission phenotype was low (10–20% of total) and so the effect of PINK1 deficiency on fission may be indirect, perhaps suggesting a general effect on mitochondria that can result in a number of aberrant morphologies. Using transmission electron microscopy (TEM; Fig. 2D), we saw swollen cristae and a possible association of mitochondria with lysosomes in PINK1 deficient cells, similar to previous reports [14,15]. We counted that 44+/27.8% of cells had mitochondria containing swollen cristae in the PINK1 deficient cells, which was significantly (Fig. 2E; P = 0.032 by t- test) higher than in control shRNA lines (19+/27.1%). Collec- tively, these results show that PINK1 has subtle but measureable effects on mitochondrial morphology. PINK1 limits mitochondrial fragmentation after rotenone exposure To provide a more quantitative estimate of functional mitochondrial connectivity, we used fluorescence recovery after photobleaching (FRAP) in living cells. Using mito-YFP to label mitochondria, cells expressing wild-type, but not kinase dead, PINK1 maintained connected mitochondrial morphology in the presence of rotenone (Fig. 3A). Under control conditions, we saw a loss of fluorescence upon photobleaching that partially recovered over time (Fig. 3B). Rotenone treatment in control M17 cell lines transfected with the mito-YFP vector alone (vector in Fig. 3B) significantly lowered the recovery of fluorescence, consistent with loss of connectivity in the mitochondrial network as reported PINK1 and Drp1 previously [25,34,35]. The same effect was seen in cell lines expressing recessive G309D or kinase dead PINK1 (data not shown). In contrast, FRAP in cells expressing wild-type PINK1 was similar with or without rotenone treatment, showing that PINK1 functionally protects against rotenone-induced mitochon- drial fragmentation (Fig. 3C). We calculated the mobile fraction of mito-YFP from the FRAP data, which confirmed that PINK1 protects against loss of connectivity in the mitochondrial network in a kinase-dependent fashion (Fig. 3D). The difference between rotenone treated and untreated cells was significant for control M17 (P,0.01), G309D and kinase dead PINK1 stable lines (P,0.05) but not for wild-type PINK1. A similar protection against rotenone-induced loss of mitochondrial connectivity was seen when wild-type PINK1 was transiently transfected into na¨ıve M17 cells (data not shown), arguing against clonal effects. To confirm that this effect is seen with endogenous PINK1, we examined cells stably expressing PINK1 shRNA, in this case using a scrambled shRNA sequence as a control (Fig. 3E–H). PINK1 knockdown cells had a partially fragmented and swollen morphology and a lower FRAP compared to control shRNA cells. After rotenone exposure, mitochondria were swollen and lost connectivity, particularly in the absence of PINK1 (Fig. 3E–G). We confirmed the effect in separate experiments with an independent shRNA sequence and a different shRNA control cell line (Supplementary Fig. S3), thus excluding a non-specific shRNA effect. We also compared the control shRNA line to parental M17 cells and did not see a statistically significant effect between the two but found that the PINK1 deficient cells had significantly lower mobile fraction values when compared to either cell line (Supplementary Fig. S3C). One possible interpretation of previous studies of loss of PINK1 in Drosophila is that there is an increased rate of mitochondrial fusion [22,23,36]. To address this directly, we estimated mitochondrial fusion in control and PINK1 deficient cells using photoactivatable GFP, where fusion results in a loss of GFP fluorescence over time. Both parental M17 and control shRNA lines showed a similar loss of pA-GFP fluorescence when quantified over time whereas PINK1 deficient cells had a slower loss of pA-GFP (Fig. 4A, B). Using two-way ANOVA, the differences in pA-GFP signal were significantly different over time, as expected (P,0.001) but also differed significantly between lines (P,0.001; see Fig. 4B for post-hoc comparisons between cell lines at each time point). Therefore, knockdown of PINK1 does not result in an increase but, instead, a slight decrease in the apparent rate of mitochondrial fusion. Effects of Drp1 on mitochondrial connectivity We asked whether loss of Dynamin related protein 1 (Drp1), a GTPase involved in various aspects of mitochondrial morphology and function, would antagonize the effects of PINK1 deficiency. Using siRNA to Drp1, we achieved knockdown to ,30% of controls and is similar in control or PINK1 shRNA lines (Fig. 5A). This partial knockdown resulted in an elongated mitochondrial network in both cell lines (Fig. 5B), similar to previous studies in other cell types [37–39]. This partial knockdown prevented mitochondrial fragmentation associated with loss of PINK1 expression in the absence of other stressors. Calculating the mobile fraction of mitoYFP, the difference between control shRNA and PINK1 shRNA lines remained significant (P,0.05 by one-way ANOVA with Student-Newmann Kuell’s post-hoc test) after expression of a control siRNA directed against GFP. The mobile fraction in PINK1 shRNA cells was significantly different after Drp1 RNAi compared to GFP RNAi (P,0.01; Fig. 5D). A similar rescue of the mobile fraction deficit was seen in cell lines PLoS ONE | www.plosone.org 4 May 2009 | Volume 4 | Issue 5 | e5701