CellQuest software (BD) and exposure to 10–100 mM CCCP (3- chlorophenylhydrazone) for 10 minutes was used to set a threshold of fluorescence intensity for those cells with intact Dym (Supplementary figure S1). Results are expressed as % of all cells with TMRM fluorescence greater than the threshold set by CCCP. Confocal Microscopy Cells were seeded on 22-mm coverslips coated with poly-L- lysine. Mitochondria were stained with 200 nM Mitotracker CMXRos (Molecular Probes, Inc.) for 30 minutes at 37uC, fixed with 4% paraformaldehyde, washed and mounted with ProLong Gold Antifade reagent (Molecular Probes, Inc.). Counts for mitochondrial morphologies were performed by counting 45– 130 Mitotracker stained cells in each of three independent experiments. The observer was blind to transfection/treatment status. In some experiments, we additionally measured mitochon- drial length from images of cells transfected with mitochondrially- directed YFP using the imaging software ImageJ. We thresholded images based on background fluorescence and measured total length of objects above background. Transmission electron microscopy Cells were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer containing 0.1 M sucrose and 3 mM CaCl2, pH 7.4 at room temperature for 30 min then overnight at 4uC. After fixation, cells were rinsed in 0.15 M sodium cacodylate buffer containing 3 mM CaCl2, pH 7.4 and centrifuged. Pellets were then postfixed in 2% osmium tetroxide in 0.07 M sodiumcacodylate buffer containing 1.5 mM CaCl2, pH 7.4 at 4uC for 2 hour, dehydrated in ethanol followed by acetone and embedded in LX-112 (Ladd, Burlington, Vermont, USA). Sections were contrasted with uranyl acetate followed by lead citrate and examined in a Tecnai 10 transmission electron microscope (Fei, The Netherlands) at 80 kV. Digital images were taken by using a MegaView III digital camera (Soft Imaging System, GmbH, Mu¨ nster, Germany) Measurements of mitochondrial connectivity FRAP was performed as previously described [66,67]. Circular ROIs, 2.5 mm in diameter, were imaged over a perinuclear region of the cytoplasm that contained highly interconnected mitochon- dria, using a 1006 Plan-Apochromat 1.4/Oil DIC objective lens (Carl Zeiss) before and after photobleach with 4 iterations of 514- nm laser set to 100% power. Scans were taken in 0.25 second intervals, for a total of 40 images and the fluorescence intensity in imaged ROIs was digitized with LSM 510 software (Zeiss MicroImaging). Curves were corrected for both non-specific photobleaching (NSPB) that occurred during imaging and background, and normalized to the first image in the series. Each FRAP curve represents the average of $30 measurements representative of results obtained in 2–3 separate experiments. Mobile Fractions [68] were calculated as follows: Mobile Fraction = [(FRAPt2Background)/FRAPi][(NSPBi2Background)/ NSPBt]. Photoactivation using live cell imaging with confocal microscopy Assays for mitochondrial fusion rates were performed using modifications of techniques described previously [66]. Briefly, cells were transfected as above with photoactivatable mitochondrially targeted GFP (mito-paGFP). Cells were imaged using a 636 plan- apochromat 1.4/oil DIC objective lens (Zeiss). After collecting PINK1 and Drp1 baseline fluorescence images, two circular, 15 mm, regions were photoactivated in each cell using a 413-nm laser set to 50% power output and 100% excitation. Fluorescence images were then taken of the same cells (n = 9–10 per line with N = 3 experiments performed) at 15 minute intervals over one hour using a 488 nm laser with 5% excitation and 70% laser power, with a GFP filter for emission. Fusion of mitochondria was estimated from the loss of (photoactivated) mito-paGFP fluorescence, averaged across both regions of interest and normalized to the initial measurement, which was set at 100% (MetaMorph software, Molecular Devices). BMH Crosslinking of Drp1 Prior to harvest, cells were incubated with BMH crosslinker (20 mM, 30 min) followed by two rinses with DTT (20 mM) to quench the crosslinking reaction. Cell lysates were separated via gel electrophoresis and protein multimers were detected on immunoblots using monoclonal antibodies for Drp1, Bax -clone 3 (BD Transduction Labs) and DJ-1 (Stressgen). M17 cells treated with staurosporine (400 nM, 6 hr) served as a positive control for apoptosis-related multimerization of Drp1 and Bax. GTPase Assay Cells were lysed and centrifuged to remove insoluble material. Supernatants were immunoprecipitated with 4 mg anti-Drp1 antibody (BD Translabs) overnight with rocking at 4uC. Lysates were then incubated with protein G sepharose beads (Amersham) for 2 hrs at 4uC. Beads were washed 5 times with PBS supplemented with 300 mM NaCl and 1% Triton X-100, once in assay buffer (20 mM HEPES pH 7.2, 2 mM MgCl2, 1 mM DTT, 0.005% BSA), re-suspended in 40 ml of the same buffer and a32P-GTP (5 m Ci; GE healthcare) was added to each reaction. Samples were incubated at room temperature with vigorous shaking and 1 ml aliquots removed at time points from 0– 120 minutes and spotted onto TLC plates (Sigma). Samples were then subjected to rising thin layer chromatography under 1 M formic acid, 1.2 M LiCl for two hours. Plates were dried for 5 min and radioactive bands were detected by autoradiography using a phosphoscreen. [32P]-GDP and [32P]-GTP spots were identified using a Storm860 PhosphorImager with ImageQuant software (GE Healthcare). GTPase activity was expressed as loss of GTP at each time point. All data points represent the average of at least three independent experiments. Calcineurin activity assay Post-nuclear cell extracts were desalted by gel filtration to remove excess phosphate and nucleotides. Samples were used in the cellular calcineurin (PP2B) phosphatase assay according to the manufacturer’s instructions (Biomol). The detection of free- phosphate released is based on malachite green assay using human recombinant calcineurin as a positive control. Following background subtraction, OD620nm data was converted into the amount of phosphate released using standard curve line-fit data, where the amount of phosphate released = (OD620nm2Yint)/ slope. To determine the contribution of calcineurin, activity from samples treated with EGTA were subtracted from the total phosphatase activity for each sample. Statistical analyses Where a single output variable was compared for multiple groups, including all mobile fraction values in FRAP assays, we used one-way ANOVA to estimate P values for all groups. Where the overall P value was ,0.05, Student-Newman-Kuell’s post hoc tests were then used to perform multiple comparisons for each PLoS ONE | www.plosone.org 15 May 2009 | Volume 4 | Issue 5 | e5701