PINK1 and Drp1 rotenone (closed circles) or under control conditions (open circles, DMSO was used as a vehicle for rotenone). Cells transfected with G309D or kinase dead PINK1 also responded to rotenone (data not shown). In B and C, each point is the average of .30 separate measurements and is representative of at least triplicate experiments for each line/treatment. Error bars indicate the SEM. (D) Mobile fraction of mito-YFP was measured in the indicated cell lines (colors as in B, filled boxes are rotenone treated). In this and all other plots of mobile fraction, summaries are of 24–30 cells. The box indicates the upper and lower quartiles, central line indicates the median and range bars indicate the 10th to 90th percentile range. Cells lying outside of this range are shown as single points. Differences between treatments were significant overall (P,0.0001 by ANOVA) and Student-Newman-Kuells’ post-hoc test was used to evaluate rotenone effects in each line. *P,0.05; ** P,0.01; ***P,0.001; ns = not significant (P.0.05). (E) Cells expressing an shRNA against PINK1 showed some basal evidence of fission, with some fragmented mitochondria visible, and exaggerated responses to rotenone and mitochondrial swelling. Scale bar in the lower right panel is 2 mm, applies to all fluorescence micrographs. (F–H) FRAP analyses as (in B,C) showing control shRNA lines (F) and shRNA against PINK1 (G). Open symbols are untreated cells, closed symbols were treated with 100 nM rotenone for 24 hours. (H) Mobile Fraction values were calculated from FRAP curves as in (D). Differences between treatments were significant overall (P,0.0001 by ANOVA; n = 30 cells, representative of at least three experiments per cell line) and Student-Newman Kuells’ post-hoc test was used to evaluate rotenone effects in each line. *P,0.05; ** P,0.01; ***P,0.001; ns = not significant (P.0.05). Additional shRNA sequences are shown in Supplementary Figure S2. doi:10.1371/journal.pone.0005701.g003 and that PINK1 deficiency results in lower Dym, [12,14]. Furthermore, we demonstrate that in a mammalian system, mitochondrial fragmentation is promoted by PINK1 silencing through a mechanism that involves calcineurin-mediated dephos- phorylation of the GTPase, Drp1. The dynamic regulation of mitochondrial morphology is important in maintaining the health of the organelle and of the cell [25,45,46]. Mitochondria with low Dym are preferentially degraded by autophagic turnover [46–49] and recent data suggests that parkin [7] and PINK1 [15] can promote this process for compromised organelles. Parkin can rescue loss of PINK1 phenotypes [8–10,12] and it is possible that both proteins form a linear pathway to remove damaged mitochondria. Our data would therefore be consistent with a model whereby lack of PINK1 or parkin results in the accumulation of mitochondria with lower Dym. Many aspects of mitochondrial function are dependent on Dym. This includes the homeostatic control of cytosolic Ca2+ by mitochondria [50,51], a process that involves interactions with the endoplasmic reticulum [52], and requires the mitochondrial dynamics protein Mfn2 [53]. Recent results have suggested that cells overexpressing mutant forms of PINK1, which may have a dominant negative effect, have a number of alterations in mitochondria that are dependent on uptake of Ca2+ into the organelle [54]. Moreover, cells lacking PINK1 have increased calcium efflux from mitochondria via the mitochondrial Na+/Ca2+ exchanger [16]. Our data shows that PINK1 deficient cells have higher activity of the Ca2+-dependent phosphatase calcineurin, which is known to dephosphorylate Drp1 and influences susceptibility to cell death [41,55]. Drp1-dependent mitochondrial fragmentation is influenced by changes in calcium levels [56] and by phosphorylation at Ser637 [40]. We propose that calcineurin, which we speculate might be activated by increased local Ca2+ near compromised mitochondria, will dephosphorylate Drp1 and thus lead to mitochondrial scission as a secondary event. This hypothesis would be consistent with our data that the calcineurin inhibitor FK-506 rescues FRAP defects but has only modest effects on Dym (Figure 9), although this will need to be confirmed in further studies. It should be noted that this proposal does not necessarily mean that PINK1 controls Dym directly as it may simply be the case that PINK1 deficient cells accumulate a population of mitochondria with low Dym due to decreased mitophagy. As well as derangements of Dym and calcium, several recent results indicate Figure 4. PINK1 deficient cells have fewer mitochondrial fusion events. (A) Control shRNA (upper panels) or PINK1 shRNA (lower panels) cells were transfected with a mitochondrially directed, photoactivatable GFP which was then photoactivated within a small region of interest (see Materials and Methods). Sequential images of the single cells (0, 15, 30, 45 and 60 minutes as indicated above the images) after photoactivation are shown. When cells are fusion-competent, decreases in fluorescence are seen over time in the region of photoactivation. Note that the fluorescence intensity is equal across the cell in the control line in the upper panel by 30 minutes, whereas the PINK1 deficient cell retains areas of higher intensity, indicating decreased fusion. Each scale bar is 5 mm and applies to all fluorescence micrograph in that series. (B) Quantification of experiments as in A (N = 3 experiments, with n = 9–10 cells measured per time point per experiment) shows the loss of fluorescence over time for parental M17 cells (black triangles), control shRNA (blue circles) or PINK1 shRNA (pink squares) cell lines. The difference between cell lines was significant by two-way ANOVA (P,0.001), as was the effect of time (P,0.001). Bonferonni post-hoc tests were used to compared PINK1shRNA lines to the control shRNA lines *P,0.05; ** P,0.01; ***P,0.001. Error bars indicate the SEM between experiments. doi:10.1371/journal.pone.0005701.g004 PLoS ONE | www.plosone.org 8 May 2009 | Volume 4 | Issue 5 | e5701