PINK1 and Drp1 the average of .30 separate measurements and error bars indicate the SEM. (D) Mobile fraction of mito-YFP was measured in control shRNA or PINK1 shRNA cell lines either after expression of a control siRNA (against GFP) or after expression of an siRNA against Drp1. Boxplots summarize data from n = 24–30 cells and are representative of duplicate experiments. Differences between treatments were significant overall (P,0.0001 by ANOVA) and Student-Newman Kuells’ post-hoc test was used to evaluate the differences between control shRNA and PINK1 shRNA after each treatment. *P,0.05; ** P,0.01. (E) Counts of mitochondrial morphology as in figure 2 were performed on n = 60 cells from duplicate experiments. Drp1 siRNA decreased the number of cells with truncated or fragmented mitochondria. Differences were analyzed by two-way ANOVA using morphology and cell line/ treatment as factors, P = 0.001 overall for the different cell groups. (F) Mitochondrial length was measured from images of unfixed, mito-YFP transfected cells. Boxes indicate the upper and lower quartiles, central line indicates the median and range bars indicate the 10th to 90th percentile range. Differences between treatments were significant overall (P,0.0001 by ANOVA, n = 14–29) and Student-Newman Kuells’ post-hoc test was used to evaluate the differences between control shRNA and PINK1 shRNA after each treatment. *** P,0.001. doi:10.1371/journal.pone.0005701.g005 that increased reactive oxygen species contribute to mitochondrial dysfunction in PINK1 [15] and parkin [13] deficiency models. Therefore, the overall model of PINK1 influence on mitochon- dria, as we have outlined in figure 9, probably includes several inter-related events. Our data differ somewhat from Drosophila models, where increased Drp1 dosage suppresses effects of PINK1 deficiency, leading to the proposal that PINK1 promotes fission [22,23,36,57]. In contrast, measuring mitochondrial connectivity in mammalian cells, we find that decreasing Drp1 had a suppressive effect and, measuring fusion, we observed slower loss of pA-GFP in PINK1 deficient cells. One possible explanation for the apparent difference between our results, which are consistent with some other results in human neuroblastoma cell lines [12,15], and those in Drosophila models is that the effects of PINK1 on mitochondrial morphology are indirect. It has been suggested that excess fusion can buffer accumulated damage in parkin-deficient fly muscle [58] and is a response to the damage rather than a cause. A similar interpretation of the various data from Drosophila, namely that PINK1 is not a core component of the fusion/fission machinery, has been proposed [36]. If the primary event is accumulation of compromised mitochondria, then the effects on morphology are secondary and may vary depending on tissue type and stage. In the case of the mammalian cells used here, the output is a loss of connectivity due to secondary effects on Drp1. Using electron microscopy, we see an approximate doubling in the number of cells with mitochondria that contained swollen cristae. Similar results are also seen in other cell lines [54], and in one recent study using EM an association of autophagic vacuoles with mitochondria with fewer cristae per unit length was noted [15]. Mitochondrial swelling is very prominent in the flight muscles of PINK1 deficient Drosophila [8]. Whether this is related to fission or fusion is unclear, as swelling might be independent of either process, but this does indicate that some aspects of mitochondrial dysfunction are conserved in different species when PINK1 is removed. It is also important to note that parkin can rescue PINK1 deficiency in both Drosophila [8,9] and in mammalian cell lines [12,15]. However, there are clear species and/or tissue differences with respect to the effects of PINK1, as experiments in mouse brain have not revealed any obvious changes in mitochondrial morphology in the absence of exogenous stressors [18]. It is possible that the effect of PINK1 is to influence mitochondrial fusion, in which case the effects of Drp1 would be antagonistic but indirect. In an attempt to address this directly, we used photoactivatable mitochondrially directed GFP and mea- sured the rate of loss of pA-GFP, which should correlate to decreased rates of fusion. However, this data is not straightforward to interpret if PINK1 plays a role, for example, in mitochondrial transport. We have recently described that PINK1, probably on the cytoplasmic face of mitochondria [59], interacts with Milton and Miro [60], two proteins involved in mitochondrial transport. Therefore, the decreased pA-GFP may relate to either a decreased mitochondrial transport rate, decreased mitochondrial fusion or both. Further experiments, perhaps using dual labeled mitochon- dria, are required to resolve this issue. However, what is relatively clear is that loss of PINK1 does not result in increased mitochondrial fusion [36], at least in this model system. An important point that arises from comparing different studies on mitochondrial function under circumstances where PINK1 activity has been depleted is that there are many different assays to address function and morphology of this complex organelle. In the current study, we principally used FRAP and pA-GFP as ways to interrogate mitochondria in living cells, which we chose so that we could avoid the influence of fixation or other processing steps. However, each of these methods has important advantages and disadvantages that should be considered in terms of the physical nature of mitochondria. For example, in some experiments we measured mitochondrial length and found that, as expected, lowering Drp1 levels increases mitochondrial length. However, at the light microscope level it is difficult to separate two nearby and juxtaposed mitochondria from one convoluted organelle, given their morphology. Therefore, we used FRAP to estimate total connected matrix volume, which is related to morphology and in our hands this is sensitive to relatively subtle changes. However, one of the limitations of this technique is the limited dynamic range and the wide variety of signal across different cells in a population, which necessitates measuring relatively large numbers of cells and validation by adding known fusion or fission molecules. As discussed above, the pA-GFP measures, which in principle should directly measure fusion, might also be influenced by mitochondrial transport. Therefore, our results need to be confirmed by different techniques performed in similar models. In this context, Dagda et al used a different estimate of mitochondrial connectivity, namely perimeter/area ratio mea- surements from fluorescently labeled mitochondria and found that PINK1 decreases connectivity [15]. Overall, our data support a role of PINK1 in limiting the damaging effects of loss of mitochondrial function. These results will allow us to investigate pathways relevant to dopaminergic cell death in further detail. Materials and Methods Cell lines and lentiviral vectors Human BE(2)-M17 (ATCC designation CRL-2267) human neuroblastoma cells [61–63] were used as a model for dopami- nergic neuron-like cells as they express dopamine synthesis enzymes [64] and have measurable levels of dopamine (DW Miller, unpublished observations). Cells were maintained in Optimem I media (Invitrogen) with 10% FBS. Cell lines were selected with 5 mg ml21 blasticidin (Invitrogen) for several weeks until non-transduced cells were dead. We used cells within fifteen passages of transduction. Both the overexpression vectors and the shRNA vectors encode blasticidin resistance cassettes, so the selection and maintenance are the same for both sets of lines. PLoS ONE | www.plosone.org 10 May 2009 | Volume 4 | Issue 5 | e5701