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. 2020 Jun;34(6):8475-8492.
doi: 10.1096/fj.201903235R. Epub 2020 May 8.

Disruption of mitochondrial dynamics increases stress resistance through activation of multiple stress response pathways

Emily Machiela  1 , Thomas Liontis  2   3 , Dylan J Dues  1 , Paige D Rudich  2   3 , Annika Traa  2   3 , Leslie Wyman  1 , Corah Kaufman  1 , Jason F Cooper  1 , Leira Lew  1 , Saravanapriah Nadarajan  4 , Megan M Senchuk  1 , Jeremy M Van Raamsdonk  1   2   3   4   5
Affiliations

Disruption of mitochondrial dynamics increases stress resistance through activation of multiple stress response pathways

Emily Machiela et al. FASEB J. 2020 Jun.

Abstract

Mitochondria are dynamic organelles that can change shape and size depending on the needs of the cell through the processes of mitochondrial fission and fusion. In this work, we investigated the role of mitochondrial dynamics in organismal stress response. By using C. elegans as a genetic model, we could visualize mitochondrial morphology in a live organism with well-established stress assays and well-characterized stress response pathways. We found that disrupting mitochondrial fission (DRP1/drp-1) or fusion (OPA1/eat-3, MFN/fzo-1) genes caused alterations in mitochondrial morphology that impacted both mitochondrial function and physiologic rates. While both mitochondrial fission and mitochondrial fusion mutants showed increased sensitivity to osmotic stress and anoxia, surprisingly we found that the mitochondrial fusion mutants eat-3 and fzo-1 are more resistant to both heat stress and oxidative stress. In exploring the mechanism of increased stress resistance, we found that disruption of mitochondrial fusion genes resulted in the upregulation of multiple stress response pathways. Overall, this work demonstrates that disrupting mitochondrial dynamics can have opposite effects on resistance to different types of stress. Our results suggest that disruption of mitochondrial fusion activates multiple stress response pathways that enhance resistance to specific stresses.

Keywords: C. elegans; genetics; mitochondria; mitochondrial dynamics; stress resistance.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

FIGURE 1
FIGURE 1
Disruption of mitochondrial fission and fusion genes causes alterations in mitochondrial morphology and function at day 1 of adulthood. A, Mitochondrial fusion mutants eat‐3 and fzo‐1 exhibit increased mitochondrial fragmentation compared to wild‐type worms. As a result, these worms have decreased mitochondrial area (B), increased number of mitochondria (C), and increased mitochondrial circularity (D) compared to wild‐type worms. eat‐3 and fzo‐1 mutants also have decreased oxygen consumption at day 1 of adulthood compared to drp‐1 or wild‐type worms (E). ATP production is decreased in mitochondrial fission and fusion mutants at day 1 of adulthood compared to wild‐type worms (F). Error bars represent SEM. Significance indicates difference from wild‐type worms. *P < .05, **P < .01, ***P < .001
FIGURE 2
FIGURE 2
Disruption of mitochondrial fission and fusion genes causes alterations in mitochondrial morphology and function at day 7 of adulthood. A, Mitochondrial fragmentation increases with age in all strains. At day 7, eat‐3 mutants still have decreased mitochondrial size (B), increased mitochondrial number (C), increased mitochondrial circularity (D), decreased oxygen consumption (E), and decreased levels of ATP (F). fzo‐1 mutants show a trend toward similar changes but only the deficit in ATP levels is significant. Quantification of mitochondrial morphology in drp‐1 mutants shows no significant differences from wild‐type worms, although there is a trend toward increased mitochondrial area. Error bars represent SEM. Significance indicates difference from wild‐type worms. *P < .05, **P < .01, ***P < .001
FIGURE 3
FIGURE 3
Disruption of mitochondrial fission and fusion genes causes a slowing of physiologic rates. A, Mitochondrial fission and fusion mutants show an increase in embryonic lethality compared to wild‐type worms. B, After hatching, a significantly increased proportion of eat‐3 mutants fail to develop to adulthood. C, The total number of progeny produced from drp‐1, eat‐3, and fzo‐1 worms is markedly decreased compared to wild‐type worms. D, The mitochondrial fission and fusion mutant strains also exhibit slow postembryonic development. E, The rate of movement (thrashing rate) is decreased in drp‐1 and eat‐3 mutants compared to wild‐type worms. F, Defecation cycle is significantly elongated in mitochondrial fission and fusion mutants. Overall, both the mitochondrial fission and fusion mutant strains exhibit slow physiologic rates. Error bars represent SEM. Significance indicates difference from wild‐type worms. *P < .05, **P < .01, ***P < .001
FIGURE 4
FIGURE 4
Exposure to multiple types of stress causes mitochondrial fragmentation. Worms were exposed to four different stresses and the morphology of the mitochondria was immediately quantified. Oxidative stress was induced by exposing worms to 2 mM paraquat for 24 hours. Heat stress was induced at 37°C for 2 hours, osmotic stress was induced at 400 mM NaCl for 24 hours. Anoxia was induced for 24 hours. A. All four types of stress caused mitochondrial fragmentation. Quantification of mitochondrial morphology revealed that exposure to exogenous stress can cause an increase in mitochondrial number (B), a decrease in mitochondrial size (C), and an increase in mitochondrial circularity (D). Error bars represent SEM. Significance indicates difference from wild‐type worms. *P < .05, **P < .01, ***P < .001
FIGURE 5
FIGURE 5
Disruption of mitochondrial fission and fusion genes alters resistance to stress. A. Mitochondrial fission and fusion mutants exhibit increased resistance to oxidative stress compared to wild‐type worms in acute (A, 300 µM juglone) and chronic (B, 4 mM paraquat) assays during adulthood. C. In contrast, eat‐3 worms fail to develop to adulthood under conditions of oxidative stress (0.4 mM paraquat) that are well tolerated by wild‐type worms. D. While mitochondrial fusion mutants show increased resistance to heat stress, mitochondrial fission mutants are markedly more sensitive than wild‐type worms. Both mitochondrial fission and fusion mutants are more sensitive to osmotic stress (E, 500 mM NaCl) and anoxia (F, 0% oxygen, 48 hours) than wild‐type worms. Error bars represent SEM. Significance indicates difference from wild‐type worms. *P < .05, **P < .01, ***P < .001
FIGURE 6
FIGURE 6
Mutants of fission accessory genes have altered resistance to stress. As with the mitochondrial fission gene drp‐1, eletion of mitochondrial fission accessory genes results in increased resistance to oxidative (A,B) stress, but increase sensitivity to heat stress (C), osmotic stress (D), and anoxia (E). Error bars represent SEM. Significance indicates difference from wild‐type worms. *P < .05, **P < .01, ***P < .001
FIGURE 7
FIGURE 7
Disruption of mitochondrial fission and fusion genes activates stress response pathways. To examine the activation of stress response pathways, mitochondrial fission and fusion mutants were crossed to fluorescent reporter strains for the mitochondrial unfolded protein response (A, Phsp‐6::GFP), the SKN‐1‐mediated oxidative stress response (B, Pgst‐4::GFP), the cytosolic unfolded protein response (C, Phsp‐16.2::GFP), the HIF‐1‐mediated hypoxia response (D, Pnhr‐57::GFP), and the DAF‐16‐mediated stress response (E, Pmtl‐1::RFP). For all of these stress response pathways, both mitochondrial fusion mutants, eat‐3 and fzo‐1, showed increased activation compared to wild‐type worms. Note that the Phsp‐16.2::GFP reporter was induced by a mild 35°C heat stress. Error bars represent SEM. Significance indicates difference from wild‐type worms. **P < .01, ***P < .001
FIGURE 8
FIGURE 8
Increased resistance to oxidative stress in mitochondrial fission and fusion mutants requires stress‐responsive transcription factors. To assess the contribution of different stress response pathways to the enhanced oxidative stress resistance in drp‐1, eat‐3, and fzo‐1 worms, worms were treated with RNAi targeting the transcription factor response for mediating each pathway. atfs‐1 RNAi decreased oxidative stress resistance in drp‐1, eat‐3, and fzo‐1 worms. RNAi against skn‐1 increased oxidative stress resistance in wild‐type, drp‐1, and fzo‐1 worms but decreased it in eat‐3 worms. hsf‐1 RNAi decreased resistance to oxidative stress in all strains, except fzo‐1, which only exhibited a trend toward decreased resistance. RNAi against hif‐1 significantly decreased resistance to oxidative stress in fzo‐1 worms. daf‐16 RNAi only significantly decreased oxidative stress resistance in fzo‐1 worms. Combined this indicates that there are multiple stress response pathways contributing to the enhanced oxidative stress resistance in the mitochondrial fission and fusion mutants. Error bars indicate SEM. Black line indicates empty vector (EV), green line indicates atfs‐1 RNAi, purple line indicates skn‐1 RNAi, blue line indicates hsf‐1 RNAi, red line indicates hif‐1 RNAi, and orange line indicates daf‐16 RNAi
FIGURE 9
FIGURE 9
Increased resistance to heat stress in mitochondrial fusion mutants requires stress‐responsive transcription factors. To assess the contribution of different stress response pathways to the enhanced heat stress resistance in eat‐3 and fzo‐1 worms, worms were treated with RNAi targeting the transcription factor response for mediating each pathway. atfs‐1 RNAi decreased heat stress resistance in fzo‐1 worms. skn‐1 RNAi increased resistance to heat stress in drp‐1 mutants. RNAi against hsf‐1 decreased heat stress resistance in wild‐type, eat‐3, and fzo‐1 worms but did not affect drp‐1 worms. Neither hif‐1 RNAi nor daf‐16 RNAi decreased resistance to heat stress in any strain. Combined this indicates that there are multiple stress response pathways contributing to the enhanced heat stress resistance in the mitochondrial fusion mutants. Error bars indicate SEM. Black line indicates empty vector (EV), green line indicates atfs‐1 RNAi, purple line indicates skn‐1 RNAi, blue line indicates hsf‐1 RNAi, red line indicates hif‐1 RNAi, and orange line indicates daf‐16 RNAi
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