Interrelationships between mitochondrial fusion, energy metabolism and oxidative stress during development in Caenorhabditis elegans

Biochemical and Biophysical Research Communications 404 (2011) 751–755

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

Interrelationships between mitochondrial fusion, energy metabolism and oxidative stress during development in Caenorhabditis elegans Kayo Yasuda a,b, Philip S. Hartman c, Takamasa Ishii a, Hitoshi Suda d, Akira Akatsuka b, Tetsuji Shoyama d, Masaki Miyazawa a, Naoaki Ishii a,⇑ a

Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan Education and Research Support Center, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan Biology Department, Texas Christian University, Fort Worth, TX 76129, USA d School of High-Technology for Human Welfare, Tokai University, Nishino 317, Numazu, Shizuoka 410-0395, Japan b c

a r t i c l e

i n f o

Article history: Received 30 November 2010 Available online 7 December 2010 Keywords: Mitochondrial fusion Oxidative stress Development Energy metabolism fzo-1

a b s t r a c t Mitochondria are known to be dynamic structures with the energetically and enzymatically mediated processes of fusion and fission responsible for maintaining a constant flux. Mitochondria also play a role of reactive oxygen species production as a byproduct of energy metabolism. In the current study, interrelationships between mitochondrial fusion, energy metabolism and oxidative stress on development were explored using a fzo-1 mutant defective in the fusion process and a mev-1 mutant overproducing superoxide from mitochondrial electron transport complex II of Caenorhabditis elegans. While growth and development of both single mutants was slightly delayed relative to the wild type, the fzo-1;mev-1 double mutant experienced considerable delay. Oxygen sensitivity during larval development, superoxide production and carbonyl protein accumulation of the fzo-1 mutant were similar to wild type. fzo-1 animals had significantly lower metabolism than did N2 and mev-1. These data indicate that mitochondrial fusion can profoundly affect energy metabolism and development. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Mitochondria are often portrayed in textbooks as solitary, static organelles with a uniform, oblong spherical appearance. While they frequently adopt this morphology, mitochondria are much more pleomorphic and, in many organisms, can even form an interconnected reticulum (reviewed by [1,2]. In addition, mitochondria are known to be dynamic structures with the energetically and enzymatically mediated processes of fusion and fission responsible for maintaining a constant flux. The relative activities of fusion and fission can determine the range of mitochondrial morphologies, from small spheres to tubular networks. The balance between mitochondrial fusion, fission and mitophagy has been implicated in a variety of pathologies [1,3,4]. Mitochondrial fusion is mediated by at least two so-called mitofusins. Mammalian mfn1 and mfn2 have been shown to mediate distinct but cooperative functions in catalyzing mitochondrial fusion [5,6]. mfn1 and mfn2 are developmentally regulated in mammals [7]. fzo (fuzzy onions) is the Drosophila homolog of mfn1 and is expressed only in spermatids [8]. Not surprisingly, lossof-function mutations in fzo were shown to be responsible for ⇑ Corresponding author. Fax: +81 463 94 8884. E-mail address: [email protected] (N. Ishii). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.12.017

defects in spermatogenesis. These mutants are characterized by a failure of spermatid mitochondria to merge into two supersized organelles, which form a structure that appears onion-like in cross section [8]. Rolland and associates [9] have recently demonstrated in Caenorhabditis elegans that the BCL-2-like protein CED-9 must be functional in order for FZO-1 to mediate mitochondrial fusion. Mitochondrial dysfunction is assumed to affect energy metabolism and reactive oxygen species (ROS) production, which is an unwanted but inevitable byproduct of oxidative phosphorylation. An oxygen-hypersensitive mutant, mev-1, has been isolated [10,11] which the gene encodes a subunit of complex II of the electron transport chain. A variety of evidence suggests that the kn1 mutation may confer oxidative stress via superoxide anion overproduction [11,12]. This results in a series of phenotypes that include excessive apoptosis, hypermutability and ultrastructural mitochondrial abnormalities [11,13,14]. These phenotypes have been recapitulated in mammalian tissue culture [15]. Given the dynamic nature of mitochondria, and that mitochondrial fusion and fission have been implicated in a variety of pathologies [1,3], we decided to examine the interrelationships of mitochondrial fusion, energy metabolism and oxidative stress on development in C. elegans. To do so, we employed the deletion mutant fzo-1 that is homologous to fzo of Drosophila and mfn1 of mammals. Our observations substantiate the role of mitochondrial

752

K. Yasuda et al. / Biochemical and Biophysical Research Communications 404 (2011) 751–755

fusion in energy metabolism and oxidative stress. They also illustrate the complex interplay between oxidative stress, mitochondrial dynamics, and development.

2. Materials and methods 2.1. Strains and general methods C. elegans animals were cultured as previously described [18]. The wild-type strain (N2) was obtained from the Caenorhabditis Genetics Center. mev-1(kn1) and fzo-1(tm1133) have been isolated in our laboratory [10] and by the National Bioresource Project for the Nematode C. elegans in Japan (http://shigen.lab.nig.ac.jp/c.elegans/index.jsp) [16], respectively. Embryos (eggs) were collected from nematode growth medium agar plates using alkaline sodium hypochlorite [17]. The released eggs were allowed to hatch by overnight incubation at 20 °C in S basal buffer [100 mM NaCl, 50 mM potassium phosphate (pH 6.0)] [18,19]. The newly hatched larvae (L1-stage larvae) were cultured on NGM agar plates seeded with the Escherichia coli strain OP50. 2.2. Measurement of body length and growth L1 larvae were grown on seeded NGM agar plates at 20 °C. To determine body lengths, about 50 animals were killed daily by heat treatment at 70 °C for 30 s. Lengths were then determined by observation under a dissecting microscope. 2.3. Sensitivity to oxygen First-stage (L1) larvae were prepared and counted, plated on NGM agar plates seeded with bacteria to enable development, and incubated at different oxygen concentrations. Under atmospheric condition, the numbers of fourth stage (L4)-young gravid adults were counted for N2 on the fourth day, mev-1 and fzo-1 on the fifth day and the double mutant on the sixth day. The animals under hyperoxia were counted one day later. The chamber was ventilated with one atmosphere of 100% oxygen at a rate of 2 l/min until the desired concentration was achieved. Oxygen concentrations were measured by an oxygen analyzer (Iijima Products Manufacturing Co., Model G-101-Y). Survival was determined by counting the number of late-stage larvae (L4 larvae) and adults. L4 larvae were scored as survivors because we have observed that such late-stage larvae usually attain adulthood. Roughly 100 animals were examined for each experimental point. 2.4. Isolation of mitochondria and measurement of superoxide anion (O2 ) A flotation method was used to remove debris and dead animals from living animals [20]. In brief, NGM plates were washed and the contents were suspended in ice-cold S basal buffer and mixed with an equal volume of ice-cold 60% sucrose. While the most wild-type (N2) and single-mutant (mev-1 and fzo-1) animals reached adulthood, the mev-1;fzo-1 double mutant preparation also included L2 through L4 larvae. After centrifugation for 15 s at 3000 rpm, the floating animals were transferred to a fresh tube. They were washed three times with S basal buffer and once with isolation buffer (210 mM mannitol, 70 mM sucrose, 0.1 mM EDTA and 5 mM Tris–HCl, pH 7.4). The animals were homogenized in isolation buffer using a glass–glass homogenizer with the inclusion of glass beads (0.10–0.11 mm). The debris was removed by a differential centrifugation at 600g. The supernatant was then centrifuged at 7200g and the mitochondria-containing pellet was suspended in TE buffer [50 mM Tris–HCl (pH 7.4), 0.1 mM EDTA]. The supernatant was also retained and served as the cytoplasmic fraction.

O2 production was measured using the chemiluminescent probe MPEC (2-methyl-6-p-methoxyphenylethynyl-imidazopyrazinone) (ATTO Co., Tokyo, Japan) [15,21]. MPEC has an advantage of low background relative to MCLA (3,7-dihydro-2-methyl-6-(4methoxyphenol) imidazol [1,2-a]pyrazin-3-one) that is generally used. Forty micrograms of intact mitochondria was added to 1 ml of assay buffer (50 mM HEPES-NaOH, pH 7.4, 2 mM EDTA) containing 0.7 lM MPEC. Solutions were placed into a photon counter with an AB-2200 type Luminescencer-PSN (ATTO Co., Tokyo, Japan) and measured at 37 °C. 2.5. Measurement of carbonylated proteins Carbonylated proteins were measured using anti-DNPH (2, 4Dinitrophenyl hydrazine) antibodies. While the most wild-type (N2) and single-mutant (mev-1 and fzo-1) animals reached adulthood, the mev-1;fzo-1 double mutant preparation also included L2 through L4 larvae. Each fraction was treated with DNPH as described by Levine [22] with some modifications [15,23]. Total protein concentrations were determined using a BCA protein assay kit (Pierce, Rockford, IL). For Western analysis, the samples were transferred to nitrocellulose membranes by a slot blot method using Milliblot-S (Millipore Co., Tokyo, Japan). The carbonyl proteins were detected with affinity purified anti-DNPH antibodies. Immunoreactive proteins were visualized using the enhanced chemiluminescence system (ECL, Amersham Biosciences, Uppsala, Sweden). The filters were analyzed and quantified by LAS3000 mini and Multi Gauge program (Fuji Film Co., Japan). 2.6. Measurement of energy metabolism Energy metabolism was determined indirectly by assaying the oxygen consumption rate. Oxygen concentrations were measured using a 300 lm aluminum jacketed fiber optic probe that acted as a spectrometer-coupled chemical sensor for full spectral analysis of dissolved or gaseous oxygen pressure (FOXY-2000, Ocean Optics, Inc., FL) [24]. A fluorescence method measured the partial pressure of dissolved or gaseous oxygen. An optical fiber carried the excitation light produced by the blue LED to the thin film (ruthenium complex) coating the probe tip. The probe collected fluorescence generated at the tip and carried it via the optical fiber to a high-sensitivity spectrometer. When oxygen in the gas or liquid sample diffused into the thin film coating, it quenched the fluorescence. The degree of quenching correlated to the level of oxygen pressure. The oxygen consumption rate was measured on groups of 10–20 animals in a small closed chamber of about 3 ll vol. (1 mm height and 2 mm in diameter). Gravid adult animals of N2, mev-1 and fzo-1 and mev-1;fzo-1 double mutants as well as L3 larvae of the mev-1;fzo-1 double mutant were immersed in a 0.5 ll liquid solution whose composition was the same as the animals’ culture medium without agar, including E. coli. All oxygen measurements were carried out within 30 min at 22 ± 1 °C. Energy metabolic rate per animal (W) was calculated from oxygen consumption rate per animal by using an energy equivalent of 20.1 J/ml oxygen. 2.7. Statistical analyses The error bars on figures represent the mean ± standard deviation. All the experiments were repeated at least three times. Differences in means were determined by using two-tailed student’s t test or Mann–Whitney U test. Life-span data were analyzed for using Mann–Whitney U test. Survival data of oxygen sensitivity were assessed by Kruskal Wallis H test, followed by a post hoc test.

K. Yasuda et al. / Biochemical and Biophysical Research Communications 404 (2011) 751–755

753

1.6 1.4

Length (mm)

1.2 1 0.8 0.6 0.4 0.2 0

2

3

4

5

6

Days after hatching Fig. 1. Body length as a measurement of growth rate. The average of body length of wild type (d), mev-1 (s), fzo-1 (N) and the double mutant fzo-1;mev-1 (h) was determined each day, and plotted as an individual point (50 animals per strain). The daily mean values are indicated by the horizontal bars.

3. Results 3.1. Body length and growth The growth rates of N2, fzo-1, mev-1 and fzo-1;mev-1 were quantified by measuring the lengths of 50 animals of each strain at daily intervals (Fig. 1 and Table S1). Development of the populations was synchronized at the beginning of the first larval stage by allowing eggs to hatch in the absence of food. The larvae of all four strains were similar in size on day two. The mev-1 mutant grew slightly slower than did N2, as has been noted previously [10]. While the fzo-1 single mutant also grew slightly slower than did mev-1, the fzo-1;mev-1 double mutant grew extremely slowly. For example, while most N2 animals attained adulthood after 3–4 days and the single mutants were delayed by roughly one day, it took the double mutant six days before significant numbers of adults were observed. Specifically, at four days after hatching, 99.3% of N2 animals were adults while only 0.7% were L2s and L3s. Conversely, only 83.6% and 68.9% of mev-1 and fzo-1 animals had attained adulthood after five days of development. Finally, at six days after hatching, only 48.0% of the double mutant had completed development to the adult stage. There was also considerably more variation in the size of individual animals in the double mutant, suggesting the developmental rates of individual animals were highly variable. In fact, some of the shorter animals did not achieve adulthood and arrested development, even when allowed additional time to develop (data not shown). Finally, the lower average lengths of the double mutant were due to the high number of larvae in the populations on days four through six. 3.2. Effects of oxidative stress on development The same four strains were tested to see if the oxidative stress might differentially affect development. The response of mev-1 to hyperoxia (Fig. 2A) recapitulates our previous observation that this mutant is extremely hypersensitive to this form of oxidative stress [10]. The fzo-1 mutant was very slightly oxygen sensitive (Fig. 2A). The fzo-1;mev-1 double mutant was more sensitive to oxygen compared to mev-1, indicating that the fzo-1 mutation exacerbated the oxygen hypersensitivity of mev-1 during larval development (Fig. 2A).

Fig. 2. (A) Survival rate under hyperoxic stress. Each point shows the percentage of animals that attained adulthood when L1 larvae were grown on plates exposed to oxygen at various concentrations. wild type (d), mev-1 (s), fzo-1 (N) and the double mutant fzo-1;mev-1 (h). The vertical bars indicate the standard deviation of four to eight separate experiments.  and   show statistical significance at p < 0.01 and p = 0.05, respectively, compared to 21% oxygen condition in each mutant. (B) Superoxide anion production from isolated mitochondria of each mutant. The error bars indicate the standard deviation of five separate experiments. : p = 0.05 and : p > 0.05 compared to N2,  : p < 0.05 compared to mev-1, respectively. (C) The relative accumulation of carbonylated protein in each strain. All values are relative to wild type. The error bars indicate the standard deviation of six separate experiments. : p < 0.005 and : p > 0.05 compared to N2,  : p = 0.05 compared to mev-1, respectively.

754

K. Yasuda et al. / Biochemical and Biophysical Research Communications 404 (2011) 751–755

3.3. Measurement of superoxide anion (O2 ) Hypersensitivity to hyperoxia is often the result of increased production of superoxide anion. This is certainly the case with mev-1 [12] (Fig. 2B). The superoxide anion production of fzo-1 was experimentally identical to N2. Finally, the production of superoxide anion in the fzo-1;mev-1 double mutant was similar to that in N2. Thus, fzo-1 suppressed the excess superoxide production caused by the mev-1 mutation. 3.4. Accumulation of carbonylated protein Carbonylated protein was employed as a measure of oxidative damage. The carbonylated protein concentrations were determined in total protein isolated from young adults (Fig. 2C). The mev-1 mutant was previously shown to have a significantly higher level of carbonylated protein than N2 [25]. The level in the fzo-1 mutant appeared to be elevated but there was no statistical difference compared to N2 because of considerably more variation in the experimental results. Conversely, the fzo-1;mev-1 double mutant contained significantly more oxidative stress than N2 (p < 0.005) and was even very slightly higher than mev-1 (p = 0.05) . 3.5. Energy metabolism Oxygen consumption was employed as a proxy of energy metabolism and was measured in the four strains (Fig. 3). As we have shown previously [26], metabolism progressively declines as N2 adults age. Similar patterns of decline were observed with mev-1. In contrast, fzo-1 animals had significantly lower metabolism than did N2 and mev-1. Given the size heterogeneity of the double mutant, we preformed separate measurement of metabolism on gravid adults as well as on laggard larvae (primarily L3 larvae) that had not developed to adulthood even after six days. The gravid double-mutant animals consumed oxygen at rates similar to the fzo-1 single mutant while the L3 larvae had even lower rates (Fig. 3). 4. Discussion Mitochondrial fusion and fission are essential for various processes of cellular metabolism [1,3,4]. In Drosophila, the fuzzy onion gene (fzo) encodes a GTPase that catalyzes mitochondrial fusion [8]. We employed a deletion mutation in the C. elegans fzo-1 gene

Metabolic energies (nW/ animal)

100

N2 mev-1 fzo-1 fzo1;mev-1G fzo1;mev-1L

80

60

40

20

0 3

4

5

6

7

8

9

10

Age (days) Fig. 3. Oxygen consumption as a measure of metabolic rate at 25 °C. The error bars indicate the standard deviation of three separate experiments. G (gravid) and L (larvae) refer to gravid adults and L3 larvae of the double mutant fzo-1;mev-1 mutant, respectively.

in order to explore the relationship between mitochondrial fusion and oxidative stress in this free-living nematode. Mitochondrial fusion is clearly an important process during development in C. elegans. This statement is supported by the observation that the fzo-1 mutant was retarded in growth and development (Fig. 1, Table S1). In fact, these developmental defects frequently proved permanent as roughly 20% fzo-1 and 40% fzo-1;mev-1 L1s failed to achieve adulthood even after extended incubation periods (data not shown). In addition, even the animals that attained adulthood were somewhat shorter than the wild type, suggesting this mutation conferred a general sickliness. These results are consistent with the RNAi phenotype of fzo-1, which is that of slow growth, developmental delays and some embryonic lethality [27–29]. Similar growth delays have recently been noted by two other groups [30,31] with the same tm1133 allele as we used. Given the report that a deficiency of FZO-1 enhances apoptosis [32], we measured and found supernumerary apoptosis in fzo-1 embryos (Fig. S2). Interestingly, the number of apoptotic cells in fzo-1, mev-1 and fzo-1;mev-1 embryos were higher compared to the wild-type N2, but there were no significant differences between these three stains. Since the double mutant grew significantly slower than either single mutant but did not have additional supernumerary apoptosis, it is unlikely that the developmental delays were caused by excessive apoptosis. Instead, it likely reflects the lower metabolic rate of the double mutant (Fig. 3). Interestingly, Breckenridge and associates [33] recently reported that the fzo-1 mutation had no effect on the apoptosis that occurs in the anterior pharynx, indicating that mitochondrial fusion is not requisite for the process as it occurs during normal development in C. elegans. In contrast, our observation of elevated apoptosis during embryogenesis indicates that there are conditions in which altered mitochondrial fusion can prompt supernumerary apoptosis in embryos. The oxygen sensitivity of fzo-1 during larval development is almost the same as wild type except that it is slightly sensitive under 90% oxygen. Kanazawa and colleagues [30] demonstrated that fzo-1 was resistant to paraquat. They also showed that the eat-3 mutant, deficient in mitochondrial inner membrane fusion, was paraquat sensitive but this was suppressed by a drp-1 mutation that affected the outer membrane. This suggests that outer membrane fusion processes may mediate at least a part of paraquat sensitivity. We confirmed the paraquat resistance of fzo-1 (data not shown), but this observation may be of limited significance because these experiments using paraquat may not reflect physiological phenomena. O2 production (Fig. 2B) and the level of carbonylated proteins as a marker of oxidative stress (Fig. 2C) in fzo-1 were almost the same as wild type. By constructing a double mutant, we were able to examine the interactions between fzo-1 and mev-1 relating to mitochondrial structure and function with oxidative stress and metabolism. While both mev-1 and fzo-1 single mutants showed retarded development and low growth rates, the double mutant was significantly more impaired than either single mutant in terms of its development (Fig. 1, Table S1). The fzo-1;mev-1 double mutant was also sensitive to oxygen compared to mev-1, indicating that the fzo-1 mutation enhanced the oxygen hypersensitivity of mev-1 during larval development (Fig. 2A). This sort of genetic interaction is typically interpreted as signifying that mev-1 and fzo-1 affect growth through different pathways. In this case, it is known that mev-1 encodes a component of complex II of the electron transport system [11]. Strong circumstantial evidence suggests that this results in increased superoxide anion generation at or near complex II [12]. The fzo-1 mutation clearly exerts its effects through some other mechanism, hence its ability to sensitize the already hypersensitive mev-1 mutation. Interestingly, the fzo-1;mev-1 double mutant

K. Yasuda et al. / Biochemical and Biophysical Research Communications 404 (2011) 751–755

had a complex set of phenotypes that did not completely recapitulate either the fzo-1 or mev-1 single mutant. First, fzo-1;mev-1 had no additional apoptotic embryonic cells than either single mutant (Fig. S2). Thus, unlike with development, the two mutations were epistatic to one another with respect to apoptosis. Second, both O2 production and level of carbonylated proteins of fzo1;mev-1 were similar to wild type (Fig. 2B and C). While carbonylation assays can be susceptible to artifacts, this may due to the fact that fzo-1 and fzo-1;mev-1 had lower metabolic rates than either wild type or mev-1 (Fig. 3). Finally, ultrastructural analyses indicated that mev-1 and fzo-1 each have distinct mitochondrial structural abnormalities, and mitochondria of the double mutant have intermediate morphological abnormalities (Fig. S1). In conclusion, fzo-1 had strong effects on development. The inhibition of mitochondrial fusion caused a reduction of mitochondrial function that led to reductions of both energy metabolism and oxidative stress. These data illustrate the complexities that result from mutational inactivation of not only integral components of electron transport (e.g., mev-1) but also mediators of mitochondrial fusion (e.g., fzo-1).

[12]

[13]

[14] [15]

[16]

[17]

[18] [19] [20] [21]

Acknowledgments This work is supported by the grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the grant-in-aid for Aging Research from the Ministry of Health, Labor and Welfare, Japan and a project of the Tokai Medical Health Science. Appendix A. Supplementary data

[22]

[23]

[24]

[25]

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.12.017.

[26]

References

[27]

[1] D.C. Chan, Mitochondria: dynamic organelles in disease, aging, and development, Cell 125 (2006) 1241–1252. [2] B. Westermann, Mitochondrial membrane fusion, Biochim. Biophys. Acta 1641 (2003) 195–202. [3] B. Westermann, Merging mitochondria matters: cellular role and molecular machinery of mitochondrial fusion, EMBO Rep. 3 (2002) 527–531. [4] I. Kim, S. Rodriguez-Enriquez, J.J. Lemasters, Selective degradation of mitochondria by mitophagy, Arch. Biochem. Biophys. 462 (2007) 245–253. [5] Y. Eura, N. Ishihara, S. Yokota, K. Mihara, Two mitofusin proteins mammalian homologues of FZO, with distinct functions are both required for mitochondrial fusion, J. Biochem. 134 (2003) 333–344. [6] N. Ishihara, Y. Eura, K. Mihara, Mitofusin 1 and 2 play distinct roles in mitochondrial fusion reactions via GTPase activity, J. Cell Sci. 117 (2004) 6535– 6546. [7] A. Santel, S. Frank, B. Gaume, M. Herrler, R.J. Youle, M.T. Fuller, Mitofusin-1 protein is a generally expressed mediator of mitochondrial fusion in mammalian cells, J. Cell Sci. 116 (2003) 2763–2774. [8] K.G. Hales, M.T. Fuller, Developmentally regulated mitochondrial fusion mediated by a conserved, novel, predicted GTPase, Cell 90 (1997) 121–129. [9] S.G. Rolland, Y. Lu, C.N. David, B. Conradt, The BCL-2-like protein CED-9 of C. elegans promotes FZO-1/Mfn1, 2- and EAT-3/Opa1-dependent mitochondrial fusion, J. Cell Biol. 186 (2009) 525–540. [10] N. Ishii, K. Takahashi, S. Tomita, T. Keino, S. Honda, K. Yoshino, K. Suzuki, A methyl viologen-sensitive mutant of the nematode Caenorhabditis elegans, Mutat. Res. 237 (1990) 165–171. [11] N. Ishii, M. Fujii, P.S. Hartman, M. Tsuda, K. Yasuda, N. Senoo-Matsuda, S. Yanase, D. Ayusawa, K. Suzuki, A mutation in succinate dehydrogenase

[28]

[29]

[30]

[31]

[32] [33]

755

cytochrome b causes oxidative stress and ageing in nematodes, Nature 394 (1998) 694–697. N. Senoo-Matsuda, K. Yasuda, M. Tsuda, T. Ohkubo, S. Yoshimura, H. Nakazawa, P.S. Hartman, N. Ishii, A defect in the cytochrome b large subunit in complex II causes both superoxide anion overproduction and abnormal energy metabolism in Caenorhabditis elegans, J. Biol. Chem. 276 (2001) 41553– 41558. N. Senoo-Matsuda, P.S. Hartman, A. Akatsuka, S. Yoshimura, N. Ishii, A complex II defect affects mitochondrial structure, leading to ced-3- and ced-4dependent apoptosis and aging, J. Biol. Chem. 278 (2003) 22031–22036. P. Hartman, R. Ponder, H.H. Lo, N. Ishii, Mitochondrial oxidative stress can lead to nuclear hypermutability, Mech. Ageing Dev. 125 (2004) 417–420. T. Ishii, K. Yasuda, A. Akatsuka, O. Hino, P.S. Hartman, N. Ishii, A mutation in the SDHC gene of complex II increases oxidative stress resulting in apoptosis and tumorigenesis, Cancer Res. 65 (2005) 203–209. K. Gengyo-Ando, S. Mitani, Characterization of mutations induced by ethyl methanesulfonate UV, and trimethylpsoralen in the nematode Caenorhabditis elegans, Biochem. Biophys. Res. Commun. 269 (2000) 64–69. S.W. Emmons, M.R. Klass, D. Hirsh, Analysis of the constancy of DNA sequences during development and evolution of the nematode Caenorhabditis elegans, Proc. Natl. Acad. Sci. USA 76 (1979) 1333–1337. S. Brenner, The genetics of Caenorhabditis elegans, Genetics 77 (1974) 71– 94. J.E. Sulston, S. Brenner, The DNA of Caenorhabditis elegans, Genetics 77 (1974) 95–104. J.A. Lewis, J.T. Fleming, Basic culture methods, Meth. Cell Biol. 48 (1995) 3–29. O. Shimomura, C. Wu, A. Murai, H. Nakamura, Evaluation of five imidazopyrazinone-type chemiluminescent superoxide probes and their application to the measurement of superoxide anion generated by Listeria monocytogenes, Anal. Biochem. 258 (1998) 230–235. R.L. Levine, D. Garland, C.N. Oliver, A. Amici, I. Climent, A.G. Lenz, B.W. Ahn, S. Shaltiel, E.R. Stadtman, Determination of carbonyl content in oxidatively modified proteins, Meth. Enzymol. 186 (1990) 464–478. A. Nakamura, S. Goto, Analysis of protein carbonyls with 2, 4-dinitrophenyl hydrazine and its antibodies by immunoblot in two-dimensional gel electrophoresis, J. Biochem. (Tokyo) 119 (1996) 768–774. H. Suda, T. Shouyama, K. Yasuda, N. Ishii, Direct measurement of oxygen consumption rate on the nematode Caenorhabditis elegans by using an optical technique, Biochem. Biophys. Res. Commun. 330 (2005) 839–843. H. Adachi, Y. Fujiwara, N. Ishii, Effects of oxygen on protein carbonyl and aging in Caenorhabditis elegans mutants with long (age-1) and short (mev-1) life spans, J. Gerontol. A Biol. Sci. Med. Sci. 53 (1998) B240–B244. K. Yasuda, T. Ishii, H. Suda, A. Akatsuka, P.S. Hartman, S. Goto, M. Miyazawa, N. Ishii, Age-related changes of mitochondrial structure and function in Caenorhabditis elegans, Mech. Ageing Dev. 127 (2006) 763–770. F. Simmer, C. Moorman, A.M. van der Linden, E. Kuijk, P.V. van den Berghe, R.S. Kamath, A.G. Fraser, J. Ahringer, R.H. Plasterk, Genome-wide RNAi of C. elegans using the hypersensitive rrf-3 strain reveals novel gene functions. PLoS Biol. 1 (2003) E12. B. Sonnichsen, L.B. Koski, A. Walsh, P. Marschall, B. Neumann, M. Brehm, A.M. Alleaume, J. Artelt, P. Bettencourt, E. Cassin, M. Hewitson, C. Holz, M. Khan, S. Lazik, C. Martin, B. Nitzsche, M. Ruer, J. Stamford, M. Winzi, R. Heinkel, M. Roder, J. Finell, H. Hantsch, S.J. Jones, M. Jones, F. Piano, K.C. Gunsalus, K. Oegema, P. Gonczy, A. Coulson, A.A. Hyman, C.J. Echeverri, Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans, Nature 434 (2005) 462–469. R.S. Kamath, A.G. Fraser, Y. Dong, G. Poulin, R. Durbin, M. Gotta, A. Kanapin, N. Le Bot, S. Moreno, M. Sohrmann, D.P. Welchman, P. Zipperlen, J. Ahringer, Systematic functional analysis of the Caenorhabditis elegans genome using RNAi, Nature 421 (2003) 231–237. T. Kanazawa, M.D. Zappaterra, A. Hasegawa, A.P. Wright, E.D. Newman-Smith, K.F. Buttle, K. McDonald, C.A. Mannella, A.M. van der Bliek, The C. elegans Opa1 homologue EAT-3 is essential for resistance to free radicals. PLoS Genet. 4 (2008) e1000022. F.J. Tan, M. Husain, C.M. Manlandro, M. Koppenol, A.Z. Fire, R.B. Hill, CED-9 and mitochondrial homeostasis in C. elegans muscle, J. Cell Sci. 121 (2008) 3373– 3382. R. Sugioka, S. Shimizu, Y. Tsujimoto, Fzo1, a protein involved in mitochondrial fusion, inhibits apoptosis, J. Biol. Chem. 279 (2004) 52726–52734. D.G. Breckenridge, B.H. Kang, D. Kokel, S. Mitani, L.A. Staehelin, D. Xue, Caenorhabditis elegans drp-1 and fis-2 regulate distinct cell-death execution pathways downstream of ced-3 and independent of ced-9, Mol. Cell 31 (2008) 586–597.