PINK1 and Drp1 Figure 8. Calcineurin-mediated dephosphorylation of Drp1 contributes to mitochondrial phenotypes related to loss of PINK1. (A) Calcineurin enzyme activity was measured in postnuclear supernatants of extracts from control and PINK1 shRNA lines. Recombinant calcineurin (CaN) was used as a positive control. Activity is expressed as the release of Pi from a calcineurin substrate peptide, corrected for activity seen in the presence of EDTA to chelate calcium (***, P,0.001.) (B) Cell extracts from PINK1 shRNA lines, treated either with DMSO as a vehicle or 1 mM FK506 for 1 h and enrichment of Drp1 in the phosphorylated fraction was measured as in figure 6B. Quantification (n = 6) confirmed a lower relative amount of phospho-Drp1 in PINK1 shRNA cells compared to controls and a significant increase comparing PINK1 shRNA cells treated with DMSO to those treated with FK506 (*, P,0.05 by ANOVA). All other comparisons were not significant (P.0.05). (C) Mitochondrial morphology in control shRNA and PINK1 shRNA cells was assessed by mito-YFP expression. Cells were treated with vehicle only or 1 mM FK506 for 1 h or 3 h. Increasing length of FK506 treatment improves mitochondrial connectedness, especially in the PINK1 shRNA line. Scale bar is 2 mm and applies to all panels. (D) Mobile fraction values were estimated from FRAP curves either without treatment or after 1 h or 3 h treatment with 1 mM FK506. Each box is the average of 60 measurements from cells across duplicate experiments. *, P,0.05; **, P,0.01, ***, P,0.001 by one-way ANOVA with Student-Newman Kuells’ post- hoc test. For clarity, non-significant differences (P.0.05) are not indicated. (E) Counts of mitochondrial morphology as in figure 2 were performed on n = 60 cells from duplicate experiments in control shRNA or PINK1 shRNA cell lines after treatment with FK506 for 1 or 3 hours. 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. (F) Mitochondrial membrane potential was estimated in PINK1 knockdown cells using TMRM staining and FACS analysis with and without 1 or 3 h treatment with 1 mM FK506. There is a significant (*, P,0.05; ***, P,0.001, n = 6 independent experiments) difference between the lines under basal conditions or after 1 h treatment by one-way ANOVA with Student-Newman Kuells’ post-hoc test, although this is not significant (ns) at 3 h. doi:10.1371/journal.pone.0005701.g008 V5 (Invitrogen), 1:500; monoclonal anti-bactin (Sigma), 1:5000; monoclonal anti-Drp1 (BD Translabs), 1:1000; monoclonal anti- Opa1 (BD Translabs), 1:500; polyclonal anti-Omi/HtrA2 (Cell Signaling), 1:1000; polyclonal anti-Fis1 (Biovision) 1:1000; mono- clonal anti-Hsp60 (Stressgen), 1:1000; monoclonal anti-DJ1 (Stressgen), 1:1000. Mfn1 and Mfn2 antibodies were a generous gift from Dr. Richard Youle. Phospho-S637 Drp1 antibody was kindly provided by Dr. Craig Blackstone. Cell viability Cell viability was measured using Annexin V and PI staining to label apoptotic and necrotic cells respectively using the Vybrant Apoptosis Assay Kit (Molecular Probes). Cultured cells were harvested from 24 well plates by 37uC Tryple (Invitrogen) treatment and then suspended with PBS and we pooled floating cells with harvested cells to give an estimate of the total cell viability across the total population of cells in each well. The cells were washed by centrifugation at 15006g at 4uC for 5 min and pellets were resuspended in 100 ml 16 annexin-binding buffer mixed with annexin V-FITC and PI for 15 min at room temperature in the dark. 200 ml 16 annexin-binding buffer was then added to the stained cells, followed by mixing and keeping the tubes on ice. The stained cells were analyzed immediately by flow cytometry. As a positive control, cells were exposed to 20 mM H2O2 for 10 minutes at 37uC, which evokes rapid cell death. Flow cytometry data were plotted as a function of fluorescence intensity FL-1 (green) versus FL-3 (red) fluorescence. We used annexin V-FITC (emission 518 nm) versus PI (emission 617 nm) to identify populations of viable cells (annexin V2PI2), early apoptotic cells (annexin V+PI2), necrotic cells (annexin V2PI+) and late necrotic cells (annexin V+PI+). Flow cytometry analysis was performed with a FACS Calibur (Becton Dickinson, San Jose, CA) equipped with a 488-nm argon laser and a 635-nm red diode laser. Experiment data were analyzed in CellQuest software (Becton Dickinson) using 5–10,000 events per sample. Triplicate experiments were performed. MTT assays were performed as described previously [3]. Analysis of Mitochondrial Membrane Potential (Dym) by Flow Cytometry Cells were harvested, washed once with PBS and stained with 100 nM tetramethyl rhodamine methyl ester (TMRM) for 15 minutes at 37uC, then washed once with PBS and kept at 4uC during measurements. Cytofluorimetric analysis was per- formed using a FACScan flow cytometer (Becton-Dickinson, San Jose, CA) equipped with a 488 nm argon laser. TMRM signal was analyzed in the FL2 channel and the data were acquired on a logarithmic scale. Non-cellular debris and dead cells were gated out based on light-scattering properties in the Side- and forward- scatter parameters, and 10–20,000 events from live cells were collected for each analysis. Data were analyzed using the Figure 9. Proposed mechanism of altered mitochondrial morphology induced by loss of PINK1. doi:10.1371/journal.pone.0005701.g009 PLoS ONE | www.plosone.org 14 May 2009 | Volume 4 | Issue 5 | e5701