Fluo-PHB but not fluorescein or fluo-DB induces mitochondrial membrane depolarization.

<p>HeLa cell loaded with 25/ml of fluorescent probes and imaged with a confocal microscope over time. Left column shows TMRM fluorescence in mitochondria before treatment. Second and third columns show TMRM and fluorescein after addition of the probes. Fluorescein did not distribute inside the cells, while fluo-DB did not show preferential mitochondrial localization. Note that neither fluorescein nor fluo-DB affected mitochondrial membrane potential, which was decreased only in the presence of fluo-PHB. This is represented in the graphs that are in the left column that show TMRM fluorescence in arbitrary units (AU) collected from the mitochondrial regions of the intact cells as a function of time. Scale bar 20 µm.</p

Effect of fluo-PHB on mitochondrial membrane potential in a recording solution with sodium and potassium ions substituted by NMDG.

<p>Isolated mitochondria were loaded with 0.2 µM TMRM and fluo-PHB (18 ng/ml) was added either in ICM (a) or NMDG without sodium and potassium ions (b) or followed by addition of KCl (50 mM) (c).</p

Fluorescein-conjugated PHB (Fluo-PHB) is preferentially distributed in the mitochondria of HeLa cells.

<p>A) 1.8 ng/ml fluo-PHB was added to HeLa cells. Times indicated in the panels correspond to acquisition points after fluo-PHB was added. Scale bar 20 µm. B) HeLa were loaded with 25 nM TMRM and with 1.8 ng/ml of fluo-PHB. Scale bar 10 µm. C) Kinetics of distribution of fluo-PHB in the mitochondrial, nuclear and in the extracellular regions. Scale bar 20 µm. D) Chemical structure of fluo-PHB (poly([R]-3-hydroxybutyrate is shown).</p

Effect of fluo-PHB on the membrane potential of isolated mouse liver mitochondria.

<p>A) Isolated mouse liver mitochondria were suspended to the concentration of 1 mg/ml in 2 ml of solution containing 150 mM KCl and 10 mM NaCl pH 7.4 in the presence of rotenone (1 µM) and succinate (5 mM) and loaded with 0.2 µM TMRM. Following 30 min of equilibration period the intensity of the fluorescence was measured with a spectrofluorimeter with excitation set at 546 nm and emission at 590 nm. During the experiment increased amounts of fluo-PHB were added to the cuvette. Each arrow corresponds to the addition of 1.8 ng/ml of fluo-PHB. Each trace corresponds to a different treatment either with fluo-PHB alone (trace a) or in the presence of EGTA (1 mM) (trace b); RuR (1 µM) (trace c) or CSA (1 µM) (trace d) and then addition of CCCP. * CCCP was added to RuR (trace c) experiment at this point. Fluorescence was normalized to be equivalent to 1 at the beginning of the experiment and 0 in the presence of CCCP under conditions of complete membrane depolarization. B) isolated mitochondria in ICM (150 mM KCl, 10 mM NaCl pH 7.4 in the presence of 1 µM rotenone and 5 mM succinate) show no swelling when treated with the same amounts of fluo-PHB that induces depolarization (18 ng/ml). Isolated mitochondria were treated with 50 µM calcium to induce swelling (control), this swelling was inhibited when the isolated mitochondria were pre-incubated with 1 µM of CSA.</p

Investigation of the mitochondrial morphology following the addition of fluo-PHB and CCCP.

<p>A) fluo-PHB induced mitochondrial membrane depolarization occurs prior to mitochondrial swelling. HeLa cells were transiently transfected with GFP and then loaded with TMRM and fluo-PHB. Arrows point at the region which lost membrane potential but the shape of mitochondria was not changed compared to polarized mitochondria. Images were collected immediately after loading and at 400 s after addition of the Fluo-PHB; B) experimental conditions are similar to those shown in panel A) except in the lower panel the cells were imaged immediately following the addition of 10 µM CCCP. C) HeLa transiently transfected with pMito-GFP were treated with fluo-PHB (18 ng/ml) and after 400 s these cells were treated with ferutinin (25 µM) showing typical swelling of the mitochondria. Scale bar is 20 µm.</p

CSA delays fluo-PHB induced mitochondrial membrane depolarization.

<p>A) HeLa cells were loaded with 25 nM TMRM and treated (red trace, n = 12) or not (black trace, n = 20) with CSA (1 µM); this was followed by the addition of fluo-PHB (18 ng/ml). Cells were imaged with a laser confocal microscope. Traces show TMRM intensity collected from the mitochondrial regions. B) fluorescence intensity ratios in the presence of fluo-PHB between nuclear and mitochondrial regions of HeLa cells treated with CSA and non-treated control cells. (n = 40 for each group of cells);</p

The effect of fluo-PHB on [Ca²⁺]c and [Ca²⁺]m.

<p>Application of fluo-PHB (1.8 ng/ml) to wild type (WT) A) PINK1 knockdown B) human dopaminergic neuroblastoma cell line (SH-SY5Y) induced increase in [Ca²⁺]c, but [Ca²⁺]m changed only in cells with PINK1 deficiency. The measurements of the fluorescence shown in panels A and B were obtained from analyzing fluorescence from cytoplasmic and mitochondrial regions of the cells (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075812#pone.0075812.s006" target="_blank">Fig. S6</a> for details). Fluo-PHB-induced calcium signal in SH-SY5Y WT cells is not prevented by cell incubation in Ca²⁺-free medium (+0.5 mM EGTA) (C); 0.5 µM thapsigargin (D)– each trace represents fluorescence measured from individual cytoplasmic region of the individual cell; E) Fluo-PHB induced Ca²⁺-rise in mitochondria of permeabilized cells. Each trace on the panel E represents measurements of the fluorescence from individual mitochondrial region of interest.</p

Time taken to induce contracture following administration of the uncoupler, CCCP (10 µM) in PINK1+/+ and PINK1−/− adult cardiomyocytes.

<p>N = 5 independent experiments.*P<0.01.</p

The effect of PINK1 deficiency on TMRM fluorescence in adult cardiomyocytes.

<p>Representative fluorescent images of adult murine cardiomyocytes isolated from i) PINK1+/+ mice and ii) PINK1−/− mice demonstrating a lower mitochondrial membrane potential (decreased TMRM fluorescence) in PINK1−/− cardiomyocytes. N = 5 independent experiments.*P<0.05.</p

Cardiomyocytes isolated from PINK1−/− mice exhibit similar amounts of oxidative stress at baseline, but exhibit significantly greater amounts of oxidative stress following simulated ischemia-reperfusion injury (SIRI) when compared to cardiomyocytes isolated from PINK1+/+ mice.

<p>N = 4 hearts per group.*P<0.05 compared to PINK1+/+ at baseline. **P<0.05 compared to PINK1−/− following SIRI.</p