Active control of mitochondrial network morphology by metabolism-driven redox state

Abstract

Mitochondria are dynamic organelles that constantly change morphology. What controls mitochondrial morphology however remains unresolved. Using actively respiring yeast cells growing in distinct carbon sources, we find that mitochondrial morphology and activity are unrelated. Cells can exhibit fragmented or networked mitochondrial morphology in different nutrient environments independent of mitochondrial activity. Instead, mitochondrial morphology is controlled by the intracellular redox state, which itself depends on the nature of electron entry into the electron transport chain (ETC)-through complex I/II or directly to coenzyme Q/cytochrome c. In metabolic conditions where direct electron entry is high, reactive oxygen species (ROS) increase, resulting in an oxidized cytosolic environment and rapid mitochondrial fragmentation. Decreasing direct electron entry into the ETC by genetic or chemical means, or reducing the cytosolic environment rapidly restores networked morphologies. Using controlled disruptions of electron flow to alter ROS and redox state, we demonstrate minute-scale, reversible control between networked and fragmented forms in an activity-independent manner. Mechanistically, the fission machinery through Dnm1 responds in minute-scale to redox state changes, preceding the change in mitochondrial form. Thus, the metabolic state of the cell and its consequent cellular redox state actively control mitochondrial form.

Keywords: electron transport chain; mitochondrial network; reactive oxygen species; redox state.

Fig. 1.

Mitochondrial morphology does not correlate with activity in different nutrient environments. (A) Schematic illustrating whether mitochondrial network morphologies and activities are interconnected. (B) Experiment design to investigate the morphology–activity relationship, which involves growing cells in five different carbon sources as indicated, measuring mitochondrial activity using standardized assays (C–E), and quantifying mitochondrial morphology (G–J). Measurement of multiple measures of mitochondrial activity in different carbon sources: (C) average Mitotracker fluorescence that reports on mitochondrial membrane potential, measured by single-cell imaging. Data represent average intensity per pixel for >100 cells from three independent experiments (mean ±SEM). (D) ETC complex IV subunit Cox2 protein levels measured by western blot using an anti-Cox2 antibody. A representative blot (out of three biological replicates, n = 3) and their quantifications are shown. (E) Basal oxygen consumption rate (OCR) corresponding to ~3 × 10⁵ cells, from three independent experiments (n = 3), normalized to the OD₆₀₀. (F) Schematic summary of mitochondrial activity in cells grown in different carbon sources. All respiratory carbon sources (glycerol, glycerol ethanol, ethanol, and lactate) show higher baseline mitochondrial activity as compared to cells grown in glucose. However, there are considerable differences in morphology and mitochondrial activities even between various respiratory carbon sources. (G) Representative microscopy images of cells with mitochondria targeted with mNeonGreen. The Top panel shows the maximum intensity projection of raw 3D z-stack, and the Bottom panel shows 3D renderings of the z-stack after deconvolution. (H–J) Quantitative 3D analysis and comparison of mitochondrial network morphology across different carbon sources. Data represent analysis of more than 200 cells from three independent experiments (mean ± SEM). (Scale bar, 2 µm.) (K) Schematic summary of mitochondrial activity versus respective mitochondrial morphology for cells grown in different carbon sources. Data represent mean ± SD unless otherwise stated. *P < 0.05, **P < 0.01, and ***P < 0.001; n.s., nonsignificant difference, calculated using unpaired Student’s t tests.

Fig. 2.

Relationship of cellular oxidative state and mitochondrial morphology. (A) Schematic showing whether respiration-dependent changes in cellular redox state can explain changes in mitochondrial morphology. (B) The total cellular redox state (measured by DCFDA fluorescence) in cells grown in different carbon sources. The relative DCFDA fluorescence was measured in cells in different media as described earlier (Materials and Methods). (C) The relative GSH levels in cells grown in different carbon sources. Cells were grown in different media as described and GSH levels were measured by LC–MS/MS. (D) Schematic showing maleimide-PEG (mPEG) assay for protein redox status estimation. (E) Cytosolic oxidative state in different carbon sources. Cells were grown in different media as described, and oxidation state of the cytosol was measured by electrophoretic mobility shift of mPEG bound to cytosolic facing Tom70, by western blotting (using anti-FLAG antibody). (F) Mitochondrial oxidative state in different carbon sources. Cells were grown in different media as described, and oxidation state of mitochondria was measured by electrophoretic mobility shift of mPEG bound to mitochondrial Idh2 by western blotting (using anti-FLAG antibody). Data represent mean ± SD from three biological replicates (n = 3) for all the experiments. (G) Schematic summary of how mitochondrial morphology correlates with cellular redox state in cells grown in different carbon sources with varied mitochondrial activities. *P < 0.05, **P < 0.01, and ***P < 0.001; n.s., nonsignificant difference, calculated using unpaired Student’s t tests for data in (A) and (B) and paired Student’s t tests for data in (E) and (F).

Fig. 3.

Mitochondrial network morphology changes in response to redox perturbations. (A) Schematic showing whether mitochondrial morphology can be reversibly modulated by changing redox state upon addition of oxidants or antioxidants. (B) Cells grown in lactate upon incubation with Glutathione (GSH) for 30 min show a reversal in mitochondrial morphology from fragmented to networked [quantified in (C)]. (D) Cells grown in ethanol are first incubated with H₂O₂ for 30 min and then further incubated with GSH for 30 min. Addition of H₂O₂ fragments mitochondria that is then restored upon further addition of GSH. For data shown in panels (B) and (D), changes in mitochondrial morphology, redox state, and activity are quantified in (C) and (E), respectively. Mitochondrial morphology is assessed by quantifying average mitochondrial length per mitochondria, redox state is measured by evaluating changes in DCFDA or DHE fluorescence and Mitotracker fluorescence and OCR are used as a proxy for measuring changes in mitochondrial activity. Data represent mean ± SEM (see Materials and Methods for details). (Scale bar, 2 µm.) *P < 0.05, **P < 0.01, and ***P < 0.001; n.s., nonsignificant difference, calculated using unpaired Student’s t tests.

Fig. 4.

Networked mitochondria and reduced environments are restored in LDH and G3PDH mutants. (A) Schematic showing differences in electron injection into ETC when cells are grown on different carbon sources. Panel (B) shows classification of the mode of electron entry into ETC for different carbon sources. (C) Quantification of mitochondrial network morphology in L-lactate dehydrogenase (Cyb2) deletion mutants grown in L-Lactate medium. (D) Quantification of mitochondrial network morphology in glycerol-3-phosphate dehydrogenase (Gut2) deletion mutants grown in glycerol medium. Data represent mean ± SEM. See SI Appendix, Supplementary Methods for details. (E) The redox state in Cyb2 and Gut2 deletion mutants (Δcyb2 and Δgut2) as estimated by changes in DCFDA fluorescence intensity. (F) OCR measured using a Seahorse analyzer, in order to estimate changes in mitochondrial respiration. (G) Schematic summary illustrating how direct electron transfer might lead to increased ROS and fragmented mitochondria. Data represent mean ± SD from three biological replicates (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001; n.s., nonsignificant difference, calculated using unpaired Student’s t tests.

Fig. 5.

Effect of altering the nature of electron flow on mitochondrial morphology. (A) Schematic showing how different ETC inhibitors might result in ROS generation. (B–D) Changes in mitochondrial morphology for cells grown in ethanol after the addition of sodium azide (complex IV inhibitor), myxothiazol (complex III inhibitor), and carboxin (complex II inhibitor), respectively. Mitochondrial morphology is restored upon incubation of cells with an antioxidant cocktail of ascorbic acid (Vit C) and mitoTEMPO (mitochondria-targeted superoxide scavenger). Mitochondrial morphology is assessed by quantifying average mitochondrial length per mitochondria, redox state is measured by evaluating changes in DCFDA or DHE fluorescence (SI Appendix, Fig. S5A), and OCR is used for measuring changes in mitochondrial respiration activity. Data represent mean ± SEM. (Scale bar, 2 µm.) *P < 0.05, **P < 0.01, and ***P < 0.001; n.s., nonsignificant difference, calculated using unpaired Student’s t tests.

Fig. 6.

The fission GTPase Dnm1 rapidly and reversibly responds to redox state. (A) Maximum intensity projected images showing Dnm1 puncta (green) colocalized on mitochondrial tubules (red) in different carbon sources, with (B) showing the quantification of the number of Dnm1 puncta in these carbon sources. (Scale bar, 2 µm.) Cells grown in glycerol and lactate that show fragmented mitochondria also have a higher number of Dnm1 puncta per mitochondrial length. Also see Materials and Methods for details of Dnm1 visualization. (C) Real-time tracking of Dnm1 puncta (green) in cells grown in ethanol. The number of Dnm1 puncta increases upon adding myxothiazol (complex III inhibitor that increases ROS and induces mitochondrial fragmentation as shown earlier) within 5 min. Upon adding an antioxidant cocktail of ascorbic acid (Vit C) and mitoTEMPO (mitochondria-targeted superoxide scavenger), Dnm1 puncta reduces within ~5 min, with almost complete reduction in 30 to 60 min. (D) Quantification of Dnm1 puncta as a function of time (data include time-lapse imaging for 87 cells). (E) An illustrative model proposing redox state as an integrated systems-level biochemical signal to regulate mitochondrial network morphology. *P < 0.05, **P < 0.01, and ***P < 0.001; n.s., nonsignificant difference, calculated using unpaired Student’s t tests.