cells have defects including mitochondrial fragmentation, swelling and cristae abnormalities that increase over time [14,15]. Some of these defects appear to be related to a poor ability of PINK1 deficient mitochondria to buffer calcium [16], which in turn may be related to a decreased turnover of impaired mitochondria via mitophagy [15]. PINK1 knockout mice have no mitochondrial morphological defects [17] but their mitochondria are functionally impaired [18], as are mitochondria in cell lines where PINK1 is knocked down [19,20]. The above literature suggests that loss of PINK1 can be associated with functional and morphological effects on mito- chondria. The overall morphology of mitochondria is influenced by a number of mechanisms, including the relative rates of mitochondrial fission and fusion. Both mitochondrial fission and fusion are highly regulated processes that are critical for the function of mitochondria and may be especially important in neurons [21]. Several recent results suggest that PINK1 may alter the balance between fusion and fission, although there is some controversy about how exactly PINK1 may function. Increased expression of proteins that promote fission, namely dynamin- related protein 1 (Drp1), rescues PINK1 deficient phenotypes in Drosophila models [22,23]. In contrast, PINK1 prevents rather than promotes fission in C elegans [24]. Recent studies in mammalian cells suggest that dominant negative Drp1 constructs antagonize the effects of loss of PINK1 [15] and the morphological effects of PINK1 deficiency are more closely associated with mitochondrial fission rather than fusion [12,15], although Yang et al reported a fusion-like phenotype in COS7 cells [23] and another study reported no morphological effects related to lack of PINK1 [20]. The fact that PINK1 protects against rotenone-induced damage, which is reported to be associated with mitochondrial fission in mammalian cells [25], indirectly suggests that PINK1 might antagonize pro-fission processes. Table 1 lists some of the previous studies regarding the role of PINK1 in the balance of mitochondrial fission and fusion. The first aim of the current study was to therefore measure mitochondrial connectivity directly in living human dopaminergic neuroblastoma cells to attempt to PINK1 and Drp1 resolve whether the phenotype relates to mitochondrial fission or fusion. Mechanism(s) involved in PINK1-dependent maintenance of morphology and cell viability are not currently well defined [26]. As PINK1 is a kinase, it is likely that there are key substrate(s) that affect mitochondrial morphology. One possible PINK1 substrate, TRAP1 [27], is mitochondrial. PINK1 has also been proposed to modulate the phosphorylation status of another mitochondrial protein Omi/HtrA2, possibly through indirect mechanisms [28]. It is not clear if these substrates contribute to the morphological effects of loss of PINK1. Therefore, the second aim of the current study was to address the underlying mechanism by which PINK1 influences mitochondrial morphology. Using live cell imaging, we show that overexpression of PINK1 protects against loss of connectivity induced by rotenone in a kinase-dependent fashion and also that PINK1 deficiency induces mitochondrial fragmentation. We show that the activity of the fission GTPase Drp1, which is controlled by the phosphatase calcineurin, contributes to the effects of PINK1 on mitochondrial morphology. Results PINK1 influences mitochondrial function To address the function of PINK1 in living cells, we generated stable dopaminergic neuroblastoma lines that express wild-type PINK1, a recessive mutant, G309D and an artificial variant lacking kinase activity [29] (Fig. 1A–C). Because PINK1 mutations are recessive, we also examined cells stably transduced with either of two shRNA sequences directed against PINK1, with a scrambled shRNA used as control (Fig. 1D–F). PINK1 overexpression lines had equivalent expression of protein, assessed using western blotting (Fig. 1A). We also performed immunocytochemistry for the V5 tag, which demon- strated that all cells in each clone expressed V5-PINK1, and RT- PCR, which showed that the exogenous constructs were expressed at approximately 4-fold over endogenous PINK1 (data not shown). Table 1. Effects of mitochondrial fusion and fission proteins on PINK1 phenotypes in different models. Reference System Fission/Fusion manipulation Poole et al [22] Drosophila Genomic knockout Drp1 LOF (heterozygote) Yang et al [23] Drosophila Genomic knockout Drp1 LOF (heterozygote) COS7 (African green monkey) cells, transient siRNA Deng et al. [36] Drosophila Genomic knockout Drp1 LOF (heterozygote) Dagda et al. [15] SY5Y (Human) cells, stable siRNA Drp1-dominant negative expression This study SY5Y (Human) cells, stable siRNA Drp1 expression Drp1 expression or Opa1/Mfn2 LOF Drp1 expression or Opa1/Mfn2 LOF Fis1 expression or Drp1 expression Drp1 expression or Opa1/Mfn2 LOF Drp1-knockdown, Opa1/Mfn2 expression Effect Lethality Suppresses PINK1 LOF phenotypes Enhances PINK1 LOF phenotypes Suppresses PINK1 LOF phenotypes Suppresses PINK1 LOF phenotypes Lethality Suppresses PINK1 LOF phenotypes Suppresses PINK1 phenotypes Additive to PINK1 LOF phenotypes Suppresses PINK1 phenotypes Interpretation PINK1 promotes fission PINK1 promotes fission PINK1 promotes fission/ limits fusion PINK1 limits fission PINK1 limits fission or promotes fusion Prior studies are listed where the effects of manipulating the expression or activity of fission and/or fusion proteins was measured against PINK1 loss of function mutations. LOF, loss of function. doi:10.1371/journal.pone.0005701.t001 PLoS ONE | www.plosone.org 2 May 2009 | Volume 4 | Issue 5 | e5701