PINK1 and Drp1 by two-way ANOVA using morphology and cell line/treatment as factors and P values for cell groups are given above each graph. (J) M17 or stable PINK1 WT cells were transfected with Drp1. FRAP curves (data not shown) were generated and mobile fractions were derived. (J) Differences between treatments were significant overall (P,0.0001 by ANOVA, n = 30 cells) and Student-Newman Kuells’ post-hoc test was used to compare each line with and without Drp1 expression. *P,0.05; ** P,0.01; ***P,0.001. Data is representative of duplicate independent experiments. (K) Counts of mitochondrial morphology were performed on 60 cells from duplicate experiments in control or PINK1 overexpressing cell lines. Differences were analyzed by two-way ANOVA using morphology and cell line/treatment as factors and P values for cell groups are given above each graph. doi:10.1371/journal.pone.0005701.g006 Where parental cells (here referred to as M17) were used, we did not include blasticidin in the growth media and all experiments using live cell imaging were performed similarly in the absence of blasticidin. The cDNA for human PINK1 and mutants described previously [29] were subcloned into pLenti6 (Invitrogen) and packaged into viral particles according to the manufacturer’s instructions. Cells were transduced at a multiplicity of infection (MOI) of approximately 1 and stable clones were established by selection with Blasticidin (Invitrogen). To knockdown endogenous PINK1, we made two separate shRNA constructs (target sequences 59-GCTGGAGGAGTATCTGATAGG, starting at nucleotide 550 of human PINK1 and 59-GGGAGCCATCGCC- TATGAAAT, starting at nucleotide 1411) and a control shRNA (59- CCTAGACGCGATAGTATGGAC). These sequences were cloned into pLenti6, packaged and used to transduce M17 cells as above. Knockdown was confirmed using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) methods described previously [29]. Western blotting General methods for western blotting for PINK1 expression using total SDS cell lysates have been described previously [29]. For mitochondrial and cytosolic separations, we used the mitochondrial isolation kit for cultured cells (Pierce), according to the manufacturer’s instructions. Phosphoprotein enrichment was performed and validated as described previously [65]. The following antibodies were used at given dilutions; monoclonal anti- Figure 7. Dephosphorylation and increased GTPase activity of Drp1 in PINK1 deficient cells. (A) Lysates from control shRNA lines or either of two PINK1 shRNA lines were separated by phospho-enrichment compared to total lysates and blotted for the proteins indicated on the left of the blots. Drp1 was in both phospho-enriched and total fractions, but less phospho-Drp1 was seen in the PINK1 deficient cells. We used phospho- Drp1 to confirm the phosphopurification was efficient and DJ-1 served as a negative control. (B) Quantification of Drp1 as a ratio of the phospho- enriched and total fractions shows that there was an ,30% decrease in phospho-Drp1 (n = 3) that is consistent between both shRNA sequences (*, P,0.05 by ANOVA). (C) Recruitment of endogenous Drp1 to mitochondrial fractions (mito) is similar in control and PINK1 shRNA cell lines (total: whole cell lysates, cyt: cytosolic fractions). Data are representative of duplicate experiments and molecular weight markers on the right of the blots are in kilodaltons. (D) Oligomerization status of Drp1 is not affected by PINK1 shRNA compared to control lines. Lysates were crosslinked with BMH or not treated to show equivalent loading of Drp1. Arrowhead shows Drp1 oligomers, arrow shows monomeric Drp1. Molecular weight markers are in kilodaltons. (E) GTPase activity of Drp1 immunopurified from control or PINK1 shRNA cell lines was followed over time and expressed as percentage of GTP converted to GDP. The difference between the lines was significant (P,0.01 by two-way ANOVA). Each point is the average of 3 replicates and is representative of two independent experiments. doi:10.1371/journal.pone.0005701.g007 PLoS ONE | www.plosone.org 12 May 2009 | Volume 4 | Issue 5 | e5701