Mitoflash frequency in early adulthood predicts lifespan in Caenorhabditis elegans

Journal name:
Nature
Volume:
508,
Pages:
128–132
Date published:
DOI:
doi:10.1038/nature13012
Received
Accepted
Published online

It has been theorized for decades that mitochondria act as the biological clock of ageing1, but the evidence is incomplete. Here we show a strong coupling between mitochondrial function and ageing by in vivo visualization of the mitochondrial flash (mitoflash), a frequency-coded optical readout reflecting free-radical production and energy metabolism at the single-mitochondrion level2, 3. Mitoflash activity in Caenorhabditis elegans pharyngeal muscles peaked on adult day 3 during active reproduction and on day 9 when animals started to die off. A plethora of genetic mutations and environmental factors inversely modified the lifespan and the day-3 mitoflash frequency. Even within an isogenic population, the day-3 mitoflash frequency was negatively correlated with the lifespan of individual animals. Furthermore, enhanced activity of the glyoxylate cycle contributed to the decreased day-3 mitoflash frequency and the longevity of daf-2 mutant animals. These results demonstrate that the day-3 mitoflash frequency is a powerful predictor of C.elegans lifespan across genetic, environmental and stochastic factors. They also support the notion that the rate of ageing, although adjustable in later life, has been set to a considerable degree before reproduction ceases.

At a glance

Figures

  1. Mitoflash events in mt-cpYFP-transgenic C.[thinsp]elegans.
    Figure 1: Mitoflash events in mt-cpYFP-transgenic C.elegans.

    a, Space–time plots of mitoflash events in mt-cpYFP-transgenic C.elegans pharynx on adult days 1, 3, 5, 9 and 19 (D1, D3, D5, D9 and D19, respectively). The spatial localization of mitoflash events is shown by surface plots overlaying the respective confocal images of the pharynx. Numerals denote the order of occurrence when more than one mitoflash was registered in the optical section (1μm thick). Temporal diaries of these events, marked as vertical ticks, during a 200-s acquisition window are shown beneath the images. b, Mitoflash frequencies in worms with defective respiratory chain components (n = 10). Mutants were gas-1(fc21), nuo-6(qm200), mev-1(RNAi), cyc-1(RNAi), isp-1(qm150) and atp-3(RNAi). Ctrl, control. c, Mitoflash response to glucose supplementation or starvation (n = 20). d, Mitoflash response to treatment with paraquat (PQ) or H2O2, or to light exposure in worms expressing mitochondrion-targeted KillerRed (n = 5). e, Age dependence of mitoflash activity in C.elegans (n = 20). In bd, day-1 adults were used for the PQ, H2O2 and starvation experiments, and day-3 adults for the others. Data are expressed as means±s.e.m. Asterisk, 0.01P<0.05; two asterisks, 0.001P<0.01; three asterisks, P<0.001; Wilcoxon rank-sum test.

  2. Mitoflashes in lifespan mutants.
    Figure 2: Mitoflashes in lifespan mutants.

    Age-dependent changes in mitoflash activity in the long-lived insulin signalling mutants daf-2(e1370) (a) and age-1(RNAi) (b), the WT-like daf-2(e1370); daf-16(mu86) double mutant (c), and the short-lived mutants hsf-1(sy441) (d) and elo-5(RNAi) (e). Insets show the corresponding survival curves. For each mitoflash result (mean±s.e.m.), n = 9 or 10 worms except for the daf-2 mutant on day 13, age-1(RNAi) on day 17, daf-2;daf-16 on day 17, and elo-5(RNAi) on day 15, for which n = 8. For the lifespan curves in ae respectively, n = 80, 93, 85, 111 and 103 worms for the mutants, or 93, 93, 98, 93 and 94 for the WT or WT on empty RNAi control.

  3. Mitoflash frequency on day 3 predicts lifespan alterations by genetic, environmental and stochastic factors.
    Figure 3: Mitoflash frequency on day 3 predicts lifespan alterations by genetic, environmental and stochastic factors.

    a, Scatter plot showing the relationship between the average lifespan (ordinate) and the day-3 mitoflash frequency (abscissa) for WT and 29 mutant strains, all with transgenic expression of mt-cpYFP. Data are expressed as percentages relative to those of the WT or WT on control RNAi (Supplementary Table 1). For the mitoflash measurement, n = 20 for elo-5(RNAi), hsf-1(sy441) and the matched controls, 15 for sod-1(RNAi), sod-2(RNAi), sod-2o.e. and the matched controls, 9 for pha-4(RNAi) and R09B3.3(RNAi), 7 for K07C11.2(RNAi) and 10 for all others. For the sample size in lifespan assays, see Supplementary Table 3 (n>55 for each). Spearman’s correlation: −0.82 (P<0.001). b, c, Four-point cubic spline regression (b) and linear regression (c) of the data in a. The regression line (solid) and the 95% prediction interval (dashed lines) of the mean lifespan are shown. d, Scatter plot of average lifespan as a function of the day-3 mitoflash frequency in mt-cpYFP transgenic C.elegans subjected to 26 environmental conditions. Data are expressed as percentages relative to those under standard culture conditions (Supplementary Table 2). Spearman’s correlation: −0.80 (P<0.001). e, f, Four-point cubic spline regression (e) and linear regression (f) of the data in d. The solid and dashed lines are as described for b and c. For the mitoflash measurement, n = 20 for the glucose experiments and n = 10 for all others. For the sample size in lifespan assays, see Supplementary Table 3 (n>40 for each).

  4. Low mitoflash activity due to mitochondrial metabolic shift contributes to the longevity of the daf-2 mutant.
    Figure 4: Low mitoflash activity due to mitochondrial metabolic shift contributes to the longevity of the daf-2 mutant.

    a, The tricarboxylic acid cycle and the glyoxylate cycle. b, Relative ICL-1 protein abundance in isolated mitochondria determined by 15N metabolic labelling and quantitative mass spectrometry. Ratios are (unlabelled ICL-1 of indicated strain/15N-labelled ICL-1) divided by (unlabelled ICL-1 of WT/15N-labelled ICL-1) and are shown as means±s.e.m. (n = 3). c, Right: higher complexII activity (means±s.e.m., n = 3) in the mitochondria isolated from the daf-2(e1370) mutant than in those purified from WT or the daf-2(e1370);daf-16(mu86) double mutant. Units of maximum velocity are 10−3 absorbance units at 600 nm per minute per microgram of protein. Left: electron micrographs of the mitochondrial samples used in the assay. d, Effect of icl-1 RNAi on the mitoflash activity in daf-2(e1370) and the daf-2(e1370);daf-16(mu86) double mutant. Means±s.e.m., n = 25 worms. e, icl-1(RNAi) shortened the lifespan of the daf-2(e1370) mutant but not the WT or the daf-2;daf-16 double mutant. P<0.001 between daf-2 and daf-2;icl-1(RNAi), log-rank. n = 84/82, 66/79 and 80/71 for WT, daf-2 and daf-2;daf-16 animals on control/icl-1 RNAi, respectively. In three independent experiments, icl-1(RNAi) significantly shortened the lifespan of the daf-2(e1370) mutant by 27% (shown), 12% and 15% (not shown). f, A model depicting the coupling between mitochondrial activity and ageing under the influence of genetic, environmental and stochastic factors. Note that a shift towards enhanced glyoxylate cycle and complexII activity decreases mitoflash production while extending the lifespan. Asterisk, 0.01P<0.05; two asterisks, 0.001P<0.01; three asterisks, P<0.001; Student’s t-test (b, c); Wilcoxon rank-sum test (d).

  5. Targeted expression of cpYFP in the mitochondrial matrix of C.[thinsp]elegans.
    Extended Data Fig. 1: Targeted expression of cpYFP in the mitochondrial matrix of C.elegans.

    a, SDHB-1 is the iron–sulphur subunit of complex II. The promoter and mitochondrial localization sequence of SDHB-1 was used for targeted expression of cpYFP in the mitochondrial matrix. SDHA, B, C and D, succinate dehydrogenase subunit A, B, C and D; Q, coenzyme Q. b, Low-magnification images of a C.elegans worm with an integrated Psdhb-1::mtLS::cpYFP transgene at a long (left) or short (right) exposure time. The pharynx has the highest expression level of mt-cpYFP. c, High-magnification images showing that mt-cpYFP co-localized with mito-tracker red in the pharynx, body-wall muscles, intestine and germ cells. The mitoflash activity in the pharynx (about one to four mitoflash events per anterior pharynx per 200s; see Fig. 1e and the WT traces in Fig. 2) is at least tenfold that in the body-wall muscles (the average mitoflash events per cell per 200s on adult days 1, 3, 5 and 9 were 0.1, 0.1, 0.1 and 0.2, respectively; n = 16, 16, 12 and 12) or the intestine (less than 0.1 mitoflash event per cell per 200s on adult day3; n = 40).

  6. Characteristics of the C.[thinsp]elegans mitoflash.
    Extended Data Fig. 2: Characteristics of the C.elegans mitoflash.

    a, Averaged time courses of mitoflash events in adult worms under the basal condition (black, n = 19), after 6h of starvation (orange, n = 15) or in the presence of 20mgl−1 glucose (maroon, n = 25). b, Averaged time courses of mitoflash events in untreated adult worms (black, n = 19), or those treated with 100mM H2O2 (blue, n = 21) or paraquat (magenta, n = 16), or subjected to mitochondrial ROS generated by the photoactivation of KillerRed (brown, n = 15). An average mitoflash event starts with a 19±2% increase in the cpYFP fluorescence intensity (ΔFmax/F0) in 1.8±0.2s, followed by a fluorescence decay with a half-time of 5.4±0.3s, and ends with a fluorescence intensity slightly below the baseline level.

  7. The first mitoflash peak on adult days 2-3 can be suppressed by eliminating the germline.
    Extended Data Fig. 3: The first mitoflash peak on adult days 2–3 can be suppressed by eliminating the germline.

    a, Germline elimination by treating WT C.elegans with 5-fluoro-2′-deoxyuridine (FUDR) or by using the gon-2(q362ts) allele at the restrictive temperature. Differential interference contrast images of day-3 adults are shown. b, Mitoflash frequencies (means±s.e.m.) of WT, FUDR-treated WT, and gon-2(q362ts) mutant worms on adult days 1–5 (n = 15 worms). All animals were cultured at 27°C until the late larval stage 2 or early larval stage 3 before being moved to 25°C, at which point half of the WT animals were cultured on plates containing 100μgml−1 FUDR. The worms were checked for the presence or absence of a germline before imaging for mt-cpYFP.

  8. Relationship between day-3 mitoflash frequency and lifespan variation due to stochastic factors.
    Extended Data Fig. 4: Relationship between day-3 mitoflash frequency and lifespan variation due to stochastic factors.

    a, Linear regression of day-3 mitoflash frequency and the lifespan of individual animals in a population of mt-cpYFP-expressing WT, long-lived age-1(hx546), or short-lived elo-5(RNAi) worms (n = 78, 65 and 58, respectively). Superimposed data are offset by a value of 0.1 along the x axis. b, Survival curves of the high, medium and low mitoflash frequency (MF) groups in a. P<0.001 between high and low MF groups in each population; P<0.05 between any two groups within a population except for the age-1 low and medium MF groups (log-rank). The mean lifespans of the high, medium and low MF groups were, respectively: for WT, 13.7, 20.1 and 24.8 days; for age-1(hx546), 17.9, 23.7 and 27.2 days; and for elo-5(RNAi) 15.3, 17.9 and 20.7 days.

  9. No correlation between day-9 mitoflash activity and the lifespan of individual WT worms.
    Extended Data Fig. 5: No correlation between day-9 mitoflash activity and the lifespan of individual WT worms.

    Scatter plot of lifespans of individual C.elegans animals as a function of the number of mitoflash events per 200s on adult day9. The linear regression line (solid black) and the 95% prediction interval (dashed lines) are drawn. There is no statistically significant correlation between the values on the x and y axes (n = 132, R2<0.01, P = 0.46).

Videos

  1. Spontaneous mitoflash activity in a day-3 WT worm
    Video 1: Spontaneous mitoflash activity in a day-3 WT worm
  2. 4D (XYZ-T) imaging of mitoflash activity in a day-3 WT worm.
    Video 2: 4D (XYZ-T) imaging of mitoflash activity in a day-3 WT worm.
    Stacks of 3 optical sections at 0.5 μm Z intervals were obtained every 1 s. Mitoflash events are colour-coded in the time-lapse projections generated from the stacks with varying angles of view.
  3. Mitoflash activity stimulated with 100 mM H2O2 in a day-1 WT worm
    Video 3: Mitoflash activity stimulated with 100 mM H2O2 in a day-1 WT worm

References

  1. Harman, D. The biologic clock: the mitochondria? J. Am. Geriatr. Soc. 20, 145147 (1972)
  2. Wang, W. et al. Superoxide flashes in single mitochondria. Cell 134, 279290 (2008)
  3. Fang, H. et al. Imaging superoxide flash and metabolism-coupled mitochondrial permeability transition in living animals. Cell Res. 21, 12951304 (2011)
  4. Gavrilov, L. A. & Gavrilova, N. S. The quest for a general theory of aging and longevity. Science Aging Knowledge Environ.2003 (28). RE5 (2003)
  5. Szewczyk, A. & Wojtczak, L. Mitochondria as a pharmacological target. Pharmacol. Rev. 54, 101127 (2002)
  6. Alexeyev, M. F. Is there more to aging than mitochondrial DNA and reactive oxygen species? FEBS J. 276, 57685787 (2009)
  7. Lemire, B. D., Behrendt, M., DeCorby, A. & Gaskova, D. C. elegans longevity pathways converge to decrease mitochondrial membrane potential. Mech. Ageing Dev. 130, 461465 (2009)
  8. Kenyon, C. J. The genetics of ageing. Nature 464, 504512 (2010)
  9. Bulina, M. E. et al. A genetically encoded photosensitizer. Nature Biotechnol. 24, 9599 (2006)
  10. Kenyon, C. et al. A C. elegans mutant that lives twice as long as wild type. Nature 366, 461464 (1993)
  11. Yang, W. & Hekimi, S. A mitochondrial superoxide signal triggers increased longevity in Caenorhabditis elegans. PLoS Biol. 8, e1000556 (2010)
  12. Alavez, S. et al. Amyloid-binding compounds maintain protein homeostasis during ageing and extend lifespan. Nature 472, 226229 (2011)
  13. Maier, W., Adilov, B., Regenass, M. & Alcedo, J. A neuromedin U receptor acts with the sensory system to modulate food type-dependent effects on C. elegans lifespan. PLoS Biol. 8, e1000376 (2010)
  14. Cho, S. C. et al. DDS, 4,4′-diaminodiphenylsulfone, extends organismic lifespan. Proc. Natl Acad. Sci. USA 107, 1932619331 (2010)
  15. Liao, V. H. et al. Curcumin-mediated lifespan extension in Caenorhabditis elegans. Mech. Ageing Dev. 132, 480487 (2011)
  16. Robida-Stubbs, S. et al. TOR signaling and rapamycin influence longevity by regulating SKN-1/Nrf and DAF-16/FoxO. Cell Metab. 15, 713724 (2012)
  17. Choi, S. S. High glucose diets shorten lifespan of Caenorhabditis elegans via ectopic apoptosis induction. Nutr. Res. Pract. 5, 214218 (2011)
  18. Cypser, J. R., Tedesco, P. & Johnson, T. E. Hormesis and aging in Caenorhabditis elegans. Exp. Gerontol. 41, 935939 (2006)
  19. Honda, Y., Tanaka, M. & Honda, S. Trehalose extends longevity in the nematode Caenorhabditis elegans. Aging Cell 9, 558569 (2010)
  20. Panowski, S. H. et al. PHA-4/Foxa mediates diet-restriction-induced longevity of C. elegans.. Nature 447, 550555 (2007)
  21. Steinbaugh, M. J., Sun, L. Y., Bartke, A. & Miller, R. A. Activation of genes involved in xenobiotic metabolism is a shared signature of mouse models with extended lifespan. Am. J. Physiol. Endocrinol. Metab. 303, E488E495 (2012)
  22. Pincus, Z., Smith-Vikos, T. & Slack, F. J. MicroRNA predictors of longevity in Caenorhabditis elegans. PLoS Genet. 7, e1002306 (2011)
  23. Hou, Y. et al. Permeability transition pore-mediated mitochondrial superoxide flashes mediate an early inhibitory effect of Aβ1–42 on neural progenitor cell proliferation. Neurobiol. Aging http://dx.doi.org/10.1016/j.neurobiolaging.2013.11.002 (18 November 2013)
  24. Herndon, L. A. et al. Stochastic and genetic factors influence tissue-specific decline in ageing C. elegans.. Nature 419, 808814 (2002)
  25. Huang, C., Xiong, C. & Kornfeld, K. Measurements of age-related changes of physiological processes that predict lifespan of Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 101, 80848089 (2004)
  26. Hsu, A. L., Feng, Z., Hsieh, M. Y. & Xu, X. Z. Identification by machine vision of the rate of motor activity decline as a lifespan predictor in C. elegans.. Neurobiol. Aging 30, 14981503 (2009)
  27. Sanchez-Blanco, A. & Kim, S. K. Variable pathogenicity determines individual lifespan in Caenorhabditis elegans. PLoS Genet. 7, e1002047 (2011)
  28. Murphy, C. T. et al. Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature 424, 277283 (2003)
  29. Dong, M. Q. et al. Quantitative mass spectrometry identifies insulin signaling targets in C. elegans.. Science 317, 660663 (2007)
  30. Yankovskaya, V. et al. Architecture of succinate dehydrogenase and reactive oxygen species generation. Science 299, 700704 (2003)
  31. Kamath, R. S. et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231237 (2003)
  32. Cabreiro, F. et al. Increased life span from overexpression of superoxide dismutase in Caenorhabditis elegans is not caused by decreased oxidative damage. Free Radic. Biol. Med. 51, 15751582 (2011)
  33. Li, J. et al. Proteomic analysis of mitochondria from Caenorhabditis elegans. Proteomics 9, 45394553 (2009)
  34. Birch-Machin, M. A. & Turnbull, D. M. Assaying mitochondrial respiratory complex activity in mitochondria isolated from human cells and tissues. Methods Cell Biol. 65, 97117 (2001)

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Author information

  1. These authors contributed equally to this work.

    • En-Zhi Shen,
    • Chun-Qing Song &
    • Yuan Lin

Affiliations

  1. College of Biological Sciences, China Agricultural University, Beijing 100094, China

    • En-Zhi Shen &
    • Chun-Qing Song
  2. National Institute of Biological Sciences, Beijing, Beijing 102206, China

    • En-Zhi Shen,
    • Chun-Qing Song,
    • Wen-Hong Zhang,
    • Wen-Yuan Liu,
    • Pan Zhang,
    • Cheng Zhan &
    • Meng-Qiu Dong
  3. State Key Laboratory of Biomembrane and Membrane Biotechnology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China

    • Yuan Lin,
    • Jiejia Xu,
    • Na Lin,
    • Xianhua Wang &
    • Heping Cheng
  4. Department of Statistics, National Cheng Kung University, Tainan 70101, Taiwan

    • Pei-Fang Su
  5. Vanderbilt Centre for Quantitative Sciences, Vanderbilt University, Nashville, Tennessee 37232, USA

    • Yu Shyr

Contributions

E.-Z.S. and C.-Q.S. designed experiments, generated strains, collected and analysed data and prepared figures. Y.L. collected and analysed data. W.-H.Z. and P.Z. performed lifespan analyses. P.-F.S. and Y.S. performed statistical analyses. W.-Y.L., J.X. and C.Z. processed image data and prepared figures or video files. N.L. helped to characterize the C.elegans mitoflash. X.W. was involved in the study design and manuscript writing. H.C. and M.-Q.D. designed the study, interpreted the data and wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

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Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Targeted expression of cpYFP in the mitochondrial matrix of C.elegans. (221 KB)

    a, SDHB-1 is the iron–sulphur subunit of complex II. The promoter and mitochondrial localization sequence of SDHB-1 was used for targeted expression of cpYFP in the mitochondrial matrix. SDHA, B, C and D, succinate dehydrogenase subunit A, B, C and D; Q, coenzyme Q. b, Low-magnification images of a C.elegans worm with an integrated Psdhb-1::mtLS::cpYFP transgene at a long (left) or short (right) exposure time. The pharynx has the highest expression level of mt-cpYFP. c, High-magnification images showing that mt-cpYFP co-localized with mito-tracker red in the pharynx, body-wall muscles, intestine and germ cells. The mitoflash activity in the pharynx (about one to four mitoflash events per anterior pharynx per 200s; see Fig. 1e and the WT traces in Fig. 2) is at least tenfold that in the body-wall muscles (the average mitoflash events per cell per 200s on adult days 1, 3, 5 and 9 were 0.1, 0.1, 0.1 and 0.2, respectively; n = 16, 16, 12 and 12) or the intestine (less than 0.1 mitoflash event per cell per 200s on adult day3; n = 40).

  2. Extended Data Figure 2: Characteristics of the C.elegans mitoflash. (70 KB)

    a, Averaged time courses of mitoflash events in adult worms under the basal condition (black, n = 19), after 6h of starvation (orange, n = 15) or in the presence of 20mgl−1 glucose (maroon, n = 25). b, Averaged time courses of mitoflash events in untreated adult worms (black, n = 19), or those treated with 100mM H2O2 (blue, n = 21) or paraquat (magenta, n = 16), or subjected to mitochondrial ROS generated by the photoactivation of KillerRed (brown, n = 15). An average mitoflash event starts with a 19±2% increase in the cpYFP fluorescence intensity (ΔFmax/F0) in 1.8±0.2s, followed by a fluorescence decay with a half-time of 5.4±0.3s, and ends with a fluorescence intensity slightly below the baseline level.

  3. Extended Data Figure 3: The first mitoflash peak on adult days 2–3 can be suppressed by eliminating the germline. (197 KB)

    a, Germline elimination by treating WT C.elegans with 5-fluoro-2′-deoxyuridine (FUDR) or by using the gon-2(q362ts) allele at the restrictive temperature. Differential interference contrast images of day-3 adults are shown. b, Mitoflash frequencies (means±s.e.m.) of WT, FUDR-treated WT, and gon-2(q362ts) mutant worms on adult days 1–5 (n = 15 worms). All animals were cultured at 27°C until the late larval stage 2 or early larval stage 3 before being moved to 25°C, at which point half of the WT animals were cultured on plates containing 100μgml−1 FUDR. The worms were checked for the presence or absence of a germline before imaging for mt-cpYFP.

  4. Extended Data Figure 4: Relationship between day-3 mitoflash frequency and lifespan variation due to stochastic factors. (265 KB)

    a, Linear regression of day-3 mitoflash frequency and the lifespan of individual animals in a population of mt-cpYFP-expressing WT, long-lived age-1(hx546), or short-lived elo-5(RNAi) worms (n = 78, 65 and 58, respectively). Superimposed data are offset by a value of 0.1 along the x axis. b, Survival curves of the high, medium and low mitoflash frequency (MF) groups in a. P<0.001 between high and low MF groups in each population; P<0.05 between any two groups within a population except for the age-1 low and medium MF groups (log-rank). The mean lifespans of the high, medium and low MF groups were, respectively: for WT, 13.7, 20.1 and 24.8 days; for age-1(hx546), 17.9, 23.7 and 27.2 days; and for elo-5(RNAi) 15.3, 17.9 and 20.7 days.

  5. Extended Data Figure 5: No correlation between day-9 mitoflash activity and the lifespan of individual WT worms. (95 KB)

    Scatter plot of lifespans of individual C.elegans animals as a function of the number of mitoflash events per 200s on adult day9. The linear regression line (solid black) and the 95% prediction interval (dashed lines) are drawn. There is no statistically significant correlation between the values on the x and y axes (n = 132, R2<0.01, P = 0.46).

Supplementary information

Video

  1. Video 1: Spontaneous mitoflash activity in a day-3 WT worm (5.45 MB, Download)
  2. Video 2: 4D (XYZ-T) imaging of mitoflash activity in a day-3 WT worm. (6.63 MB, Download)
    Stacks of 3 optical sections at 0.5 μm Z intervals were obtained every 1 s. Mitoflash events are colour-coded in the time-lapse projections generated from the stacks with varying angles of view.
  3. Video 3: Mitoflash activity stimulated with 100 mM H2O2 in a day-1 WT worm (12.88 MB, Download)

PDF files

  1. Supplementary Information (361 KB)

    This file contains Supplementary Tables 1-3 and additional references.

Comments

  1. Report this comment #63019

    Vsevolod Belousov said:

    Although it is great to see some flashes in the mitochondria that are correlative with the lifespan, it would be much greater to proof that the flashes are really due to the ROS production, but not the pH oscillations. cpYFP is much better pH sensor than superoxide sensor. As no adequate control for pH was done in this paper and in previous mitoflashes papers (as checking flashes with the pH sensor of the same pKa and similar dynamic range, like SypHer), the flashes probably reflect pH oscillations due to, for example, Ca2+ signaling imbalance.
    Another problem with cpYFP as superoxide sensor is that it was never ever used for superoxide detection in models other than mitoflashes. To better understand the issue, I'd recommend the following literatture:
    1. Wang et al, Superoxide flashes in single mitochondria, Cell 134 (2008) 279?290.
    2. F.L. Muller, A critical evaluation of cpYFP as a probe for superoxide, Free Radic. Biol. Med. 47 (2009) 1779?1780.
    3. Schwarzlander et al, The circularly permuted yellow fluorescent protein cpYFP that has been used as a superoxide probe is highly responsive to pH but not superoxide in mitochondria: implications for the existence of superoxide ?flashes?, Biochem. J. 437 (2011) 381?387.
    4. Schwarzlander et al, Mitochondrial ?flashes?: a radical concept repHined, Trends Cell Biol. 22 (2012) 503?508.
    5. E. Quatresous, C. Legrand, S. Pouvreau, Mitochondria-targeted cpYFP: pH or superoxide sensor? J. Gen. Physiol. 140 (2012) 567?570.

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