in cells of human carcinoma and many other eukaryotic cells. ... Fragmentation of the Mitochondrial Reticulum in HeLa Cells. O. K. Nepryakhina a. , A. Yu.
ISSN 0012-4966, Doklady Biological Sciences, 2008, Vol. 420, pp. 221–223. © Pleiades Publishing, Ltd., 2008. Original Russian Text © O.K. Nepryakhina, A.Yu. Kuznetsova, K.G. Lyamzaev, D.S. Izyumov, O.Yu. Pletjushkina, B.V. Chernyak, V.P. Skulachev, 2008, published in Doklady Akademii Nauk, 2008, Vol. 420, No. 4, pp. 559–561.
CELL BIOLOGY
Reactive Oxygen Species Generated in Mitochondria Induce Fragmentation of the Mitochondrial Reticulum in HeLa Cells O. K. Nepryakhinaa, A. Yu. Kuznetsovaa, K. G. Lyamzaevb, D. S. Izyumovb, O. Yu. Pletjushkinab, B. V. Chernyakb, and Academician V. P. Skulacheva, b Received January 14, 2008
DOI: 10.1134/S0012496608030241
Mitochondria form a ramified network of organelles in cells of human carcinoma and many other eukaryotic cells. This structure was discovered by Bakeeva et al. [1] and is referred to as the mitochondrial reticulum. The processes of fission and fusion of mitochondria determine the state of dynamic equilibrium of the mitochondrial reticulum. The mitochondrial reticulum is fragmented during cell division, apoptosis, and different stresses. There are a number of proteins (e.g., Drp1 and Fis1) involved in this process, however, the details of the mechanism are unclear yet (for review, see [2, 3]). The pathway of signal transmission from various stimuli that induce oxidative stress or apoptosis to the machinery of mitochondrial fission is of interest. The central role in this pathway may be played by reactive oxygen species (ROSs), because their content increases during oxidative stress and apoptosis. The main source of ROSs under these conditions is mitochondria (for review, see [4]).
dria during oxidative stress play a key role in the fission of the mitochondrial reticulum. The interaction of mitochondria-targeted antioxidants with human carcinoma cells (HeLa) was studied with the use of SkQR1, whose cationic residue contains a fluorescent substance (rhodamine 19). Flow cytofluorometry showed that the maximum accumulation of SkQR1 in the cells is reached within 2 h. Confocal fluorescent microscopy showed that SkQR1 was accumulated in mitochondria with a high selectivity and completely colocalized with the mitochondrial probe MitoTracker Green (Molecular Probes, United States). The protonophore uncoupler FCCP (carbonylcyanidep-trifluoromethoxyphenyl hydrazone), which dissipates the membrane potential, prevented accumulation of SkQR1 in the cells (Fig. 1). Microscopic studies also did not show any accumulation of SkQR1 in the mitochondria pretreated with FCCP. These data confirm the
In this work, we studied the role of mitochondrial ROSs in mitochondrial fission. A novel, very effective instrument to study mitochondrial ROSs is mitochondria-targeted antioxidants. These substances were initially developed by Murphy et al. [5]. They consist of a ubiquinone residue and decyltriphenylphosphonium. These substances may be accumulated in mitochondria due to the membrane potential (with the negative charge inside) of the inner membrane, reduce ROSs, and, then, regenerate after reduction by the mitochondrial electron transport chain. Our laboratory has developed more effective antioxidants; their active group includes plastoquinone instead of ubiquinone [6]. In this work, we used these antioxidants, namely, SkQ1 and SkQR1 (for formulas, see [6]) and showed that ROSs generated in mitochon-
Intensity of SkQR1 fluorescence, arb. unit 10 without FCCP 9 8 7 6 5 4 3 2 1 0
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Moscow State University, Leninskie gory, Moscow, 117234 Russia b Belozerskii Institute of Physico-Chemical Biology, Moscow State University, Leninskie gory, Moscow, 119899 Russia
Fig. 1. Accumulation of the antioxidant SkQR1 in HeLa cells. Cultured HeLa cells were treated with 50 nM SkQR1, incubated at 37°C, and analyzed using a Beckman Coulter FC500 flow cytofluorometer. FCCP at a concentration of 10 µM was added simultaneously with SkQR1.
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Fig. 2. SkQR1 prevents ROS accumulation in HeLa cells treated with hydrogen peroxide. The HeLa cells were incubated for 2 h with 2 nM SkQR1; then, 300 µM ç2é2 was added and, within 1 h, the ROS content was measured by flow cytofluorometry with the use of the fluorescent probe CM–H2DCFDA (5-(and 6-)chloromethyl-2',7'-dichlorodihydrofluorosceine diacetate) as described in [7]. The black line, control cells; the gray histogram, cells treated with ç2é2.
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5 µm Fig. 3. SkQ1 prevents mitochondrial fission induced by hydrogen peroxide. HeLa cells were preincubated for 2 h with 2 nM SkQ1 and, then, treated with 350 µM ç2é2 for 18 h. Mitochondria were stained with 1 µM MitoTracker Green (30 min) and analyzed using a Zeiss Axiovert 210 M microscope.
electrophoretic mechanism of accumulation of mitochondria-targeted antioxidants. Hydrogen peroxide enhances the ROS production in mitochondria of HeLa cells, because it is a powerful inducer of oxidative stress [7]. Incubation of the cells for 2 h with SkQR1 at a very low concentration (2 nM) prevented H2O2-induced ROS accumulation in the majority of the cells (Fig. 2). These data demonstrate a very high effectiveness of these mitochondria-targeted antioxidants in the cells. Hydrogen peroxide induces changes in the morphology of mitochondria of HeLa cells [8]. Initially, the mitochondrial reticulum disintegrates into single stretched filaments; then, fragmentation continues and results in the formation of a large number of small sphere-like organelles. Figure 3 illustrates the final stage of this process. The mitochondria-targeted antioxidants SkQ1 (Figs. 3 and 4) and SkQR1 (not shown) prevented mitochondrial fission. To evaluate the effect, we measured the number of cells with completely fragmented mitochondria. The maximum protection against mitochondrial fission was observed when we used 2 nM SkQ1 (Fig. 4). An increase in the concentration of SkQ1 decreased the protective effect and, at concentrations of 20–100 nM, SkQ1 enhanced mitochondrial fission. In the absence of hydrogen peroxide,
SkQ1 at these concentrations did not induce fission (Fig. 4). The effect of SkQR1 almost did not differ from the effect of SkQ1. The antioxidants trolox and N-acetylcysteine also prevented mitochondrial fission caused by hydrogen peroxide, but at concentrations 500 and 50 000 times larger than the concentration of SkQ1 (100 µM and 10 mM, respectively). It is possible to hypothesize that the very high efficiency of SkQ1 and SkQR1 is determined by their capacity for accumulating in mitochondria. Note that trolox and N-acetylcysteine prevented mitochondrial fission induced by hydrogen peroxide in the presence of SkQ1 or SkQR1 at high concentrations (not shown). It seems that excess accumulation of quinone derivatives in mitochondria enhances their prooxidant activity and increases the ROS level, which results in mitochondrial fission. We previously showed that inhibitors of respiratory complexes I (piericidin, rotenote) and III (myxothiazole) stimulated the ROS production induced by hydrogen peroxide [7]. Piericidin or myxothiazol alone induced a negligible increase in the ROS content but caused fragmentation of the mitochondrial reticulum in HeLa cells. SkQ1 or SkQR1 at a concentration of 2 nM, as well as 10 mM N-acetylcysteine, prevented this effect. These data indicate that mitochondrial fission is very sensitive to mitochondrial ROSs and that the resDOKLADY BIOLOGICAL SCIENCES
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REACTIVE OXYGEN SPECIES GENERATED Cells with fragmented mitochondria, % 90 80 70
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Fig. 4. Relationship between the effect of SkQ1 on mitochondrial fission and the antioxidant concentration. The cells were incubated as described in the legend to Fig. 3. The proportion of cells with a completely fragmented mitochondrial reticulum was evaluated in four independent experiments; in each experiment, 100–200 cells were analyzed.
piratory chain is the main source of ROSs in mitochondria. The protein kinase inhibitor staurosporin induced rapid and complete mitochondrial fission and, then, mass apoptosis of HeLa cells. In this case, SkQ1 and SkQR1 neither prevented mitochondrial fission nor
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protected the cells against death. It seems that the antioxidants do not affect the mechanism of mitochondrial fission but only interrupted triggering of this mechanism by ROSs. The mitochondrial fission during staurosporin-induced apoptosis seems to be caused by factors other than ROSs. On the basis of the data obtained, we concluded that fragmentation of the mitochondrial reticulum of HeLa cells during oxidative stress is induced by ROSs generated by the mitochondrial electron transport chain. ACKNOWLEDGMENTS We are grateful to all participants of the project “Practical Use of Skulachev’s Ions” for their help and fruitful discussion of the work. This study was supported by the Vol’noe Delo Foundation (sponsor, O.V. Deripaska) and Russian Foundation for Basic Research (project nos. 07-04-00335 and 05-04-49062). REFERENCES 1. Bakeeva, L.E., Chentsov, Yu.S., and Skulachev, V.P., Biochim. Biophys. Acta, 1978, vol. 501, no. 3, pp. 349– 369. 2. McBride, H.M., Neuspiel, M., and Wasiak, S., Curr. Biol., 2006, vol. 25, no. 16, pp. 551–560. 3. Scorrano, L., J. Bioenerg. Biomembr., 2005, vol. 37, no. 3, pp. 165–170. 4. Skulachev, V.P., Bakeeva, L.E., Chernyak, B.V., et al., Mol. Cell Biochem., 2004, vol. 256-257, nos. 1–2, pp. 341–358. 5. Kelso, G.F., Porteous, C.M., Coulter, C.V., et al., J. Biol. Chem., 2001, vol. 276, no. 7, pp. 4588–4596. 6. Skulachev, V.P., Biokhimiya, 2007, vol. 72, no. 12, pp. 1700–1714. 7. Chernyak, B.V., Izyumov, D.S., Lyamzaev, K.G., et al., Biochim. Biophys. Acta, 2006, vol. 1757, nos. 5/6, pp. 525–534. 8. Pletjushkina, O.Y., Lyamzaev, K.G., Popova, E.N., et al., Biochim. Biophys. Acta, 2006, vol. 1757, nos. 5–6, pp. 518–524.
