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Multiple dynamin family members collaborate to drive mitochondrial division

Nature volume 540, pages 139–143 (2016)Cite this article

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Matters Arising to this article was published on 19 June 2019

Abstract

Mitochondria cannot be generated de novo; they must grow, replicate their genome, and divide in order to be inherited by each daughter cell during mitosis. Mitochondrial division is a structural challenge that requires the substantial remodelling of membrane morphology1,2,3. Although division factors differ across organisms, the need for multiple constriction steps and a dynamin-related protein (Drp1, Dnm1 in yeast) has been conserved4,5,6. In mammalian cells, mitochondrial division has been shown to proceed with at least two sequential constriction steps: the endoplasmic reticulum and actin must first collaborate to generate constrictions suitable for Drp1 assembly on the mitochondrial outer membrane; Drp1 then further constricts membranes until mitochondrial fission occurs2,7,8,9. In vitro experiments, however, indicate that Drp1 does not have the dynamic range to complete membrane fission7. In contrast to Drp1, the neuron-specific classical dynamin dynamin-1 (Dyn1) has been shown to assemble on narrower lipid profiles and facilitate spontaneous membrane fission upon GTP hydrolysis10,11. Here we report that the ubiquitously expressed classical dynamin-2 (Dyn2) is a fundamental component of the mitochondrial division machinery. A combination of live-cell and electron microscopy in three different mammalian cell lines reveals that Dyn2 works in concert with Drp1 to orchestrate sequential constriction events that build up to division. Our work underscores the biophysical limitations of Drp1 and positions Dyn2, which has intrinsic membrane fission properties, at the final step of mitochondrial division.

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Figure 1: Dyn2 is required for mitochondrial division.
Figure 2: Dynamics of Dyn2 recruitment during mitochondrial division.
Figure 3: Dyn2-depleted cells reveal dynamic mitochondrial super-constrictions.
Figure 4: Dyn2 is required for STS-induced mitochondrial fragmentation.

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Acknowledgements

We thank C. Ozzello for sectioning cells for electron microscopy analysis; A. Cheng, J. Ryoo and D. G. Drubin for providing the genome-edited Dyn2–GFP Sk-Mel2 cell line; J. R. Friedman, D. Dambournet, and K. C. Cook for critical reading of the manuscript; and A. Hoenger and M. West for helpful discussions. Electron microscopy was performed at the University of Colorado, Boulder electron microscopy services core facility. Super-resolution microscopy in the Department of Molecular, Cellular, and Developmental Biology was made possible by equipment supplements R01 GM79097 (D. Xue) and P01 GM105537 (M. Winey). This work is supported by grants from the National Institutes of Health to G.K.V. (GM083977), J.E.L. (F32CA174158), L.M.W. (F32GM116371) and to H.W. (T32GM08759).

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Authors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, 80309, Colorado, USA

    Jason E. Lee, Laura M. Westrate, Haoxi Wu, Cynthia Page & Gia K. Voeltz

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  1. Jason E. Lee
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Contributions

J.E.L. and G.K.V. contributed to experimental design. L.M.W. and C.P. performed the experiments in Fig. 3f–h; H.W. performed the experiments in Extended Data Fig. 4d–f; J.E.L. conducted the remainder of the experiments, data analysis, and figure composition. J.E.L. and G.K.V. wrote the manuscript.

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Correspondence to Gia K. Voeltz.

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Extended data figures and tables

Extended Data Figure 1 Dynamin-2 is required for mitochondrial division.

a, Representative images of scrambled-, Drp1- or Dyn2-siRNA-transfected HeLa cells expressing mito–BFP. n = 36 cells for each siRNA treatment. Scale bars, 10 μm. b, Immunoblots with antibodies against Dyn2, Drp1, and GAPDH in cells treated with the indicated siRNA. c, The effect on mitochondrial morphology was quantified within a 230 μm2 region of interest (ROI) for mean mitochondrial area per mitochondrion (left graph), and mean mitochondria per ROI (right graph). As in COS-7 cells, Drp1- or Dyn2-depleted cells had larger mitochondria and fewer mitochondria per ROI when compared to control cells. Data were obtained from three biological replicate experiments for each of scrambled siRNA, Drp1 siRNA and Dyn2 siRNA treatments. Error bars represent s.e.m.; *P < 0.01 by ANOVA. d, Immunoblot analyses were performed on scrambled- and Dyn2-siRNA-treated cell lysates with antibodies against Dyn2, GAPDH, and mitochondrial fission (Drp1 and Mff) and fusion (Mfn2 and Opa1) machineries.

Extended Data Figure 2 Inhibition of clathrin-mediated endocytosis does not affect mitochondrial morphology.

a, Representative images of TOM20 immunofluorescence in COS-7 cells transfected with scrambled control (n = 50), AP-2 (n = 50), or Dyn2 siRNA (n = 52). Scale bars = 10 μm. b, Immunoblots with antibodies against AP-2, Dyn2, and GAPDH in siRNA-treated cells. c, The effect on mitochondrial morphology was quantitated within a 230 μm2 ROI for mean mitochondrial area per mitochondrion (left) and mean mitochondria per ROI (right). Dyn2-depleted cells had larger mitochondria and less mitochondrion per ROI compared to control cells, as in Fig. 1; however, AP-2-depleted cells displayed mitochondrial morphology that was qualitatively and quantitatively similar to control cells. These data were obtained from three biological replicate experiments. Error bars represent the s.e.m. *P < 0.01 by ANOVA.

Extended Data Figure 3 Dyn2 depletion does not phenocopy starvation-induced inhibition of Drp1.

a, b, The phosphorylation status of Drp1 was evaluated by immunoblot on whole-cell lysates in scrambled- and Dyn2-siRNA-treated cells using antibodies against phospho-Ser637-Drp1 (a) and phosphor-Ser616-Drp1 (b). Antibodies against Drp1 and Dyn2 were used to measure total Drp1 and Dyn2 levels, respectively, and anti-GAPDH was used as a loading control. The optical densities of phosphorylated-serine Drp1 were normalized to their corresponding GAPDH signal (graphs in a, b). The data represented in graphs in both a and b were obtained from three biological replicate experiments. Error bars represent the s.e.m. *P < 0.01 by paired t-test.

Extended Data Figure 4 Live-cell imaging of mitochondrial division machinery before, during and after division.

a, Representative example of mitochondrial division (6 events from 108 cells) in COS-7 cells expressing mito–BFP (grey), mCherry–Drp1 (red), and Dyn2–mNeon (green) (Supplementary Video 2). Insets show the temporal and spatial dynamics of Drp1 (arrows) and Dyn2 (arrowheads) before and during mitochondrial division. b, A schematic identifying two temporal moments of interest with respect to Drp1, Dyn2 and mitochondrial dynamics—the frame before and the frame after division—with a dashed line that identifies the region that was analysed by line scan. c, Line-scan analysis of Drp1 and Dyn2 leading up to, and following, mitochondrial division. d, Representative example mitochondrial division (27 events from 49 cells) in PtK1 cells expressing mito–BFP (grey), mCherry–Drp1 (red), and GFP–Mff (green). Insets show the temporal and spatial dynamics of Drp1 (arrows) and Mff (arrowheads) before and during mitochondrial division. e, Schematic identifying the two temporal moments of interest with respect to Mff, Drp1 and mitochondrial dynamics. f, Line-scan analysis of Mff and Drp1 leading up to and following mitochondrial division. Note, that the interaction between Drp1 and its adaptor, Mff, is maintained throughout the process of mitochondrial division; by contrast, Dyn2 associates with only one daughter mitochondrion. Scale bars for whole-cell panels and inset panels are 10 μm and 1 μm, respectively (a, d).

Extended Data Figure 5 STS treatment stalls division factors at mitochondrial constrictions.

a–d, Structure illumination microscopy was used to capture images of PtK1 cells expressing mCherry–Drp1 (red), mito–BFP (grey), and either GFP–Mff (a and b; green, n = 10 and 12 cells, respectively) or Dyn2–mNeon (c and d, green, n = 13 cells for each) that were untreated or treated with 1 μM staurosporine (STS). The effect of STS treatment on division machinery localization was scored by line-scan analyses. Line-scan analysis verified the co-localization of Drp1 at Mff-marked constrictions (a, b) and Dyn2 at Drp1-marked constrictions (c, d). Under steady-state conditions, 66.5% of Mff-marked constrictions co-labelled with Drp1 (336 out of 505 Mff-marked constrictions), whereas STS treatment increased the co-localization of Drp1 with Mff-marked constrictions to 85.1% (538 out of 632 Mff-marked constrictions). e, 24.9% of Drp1-marked constrictions were co-labelled with Dyn2 in untreated cells (128 out of 514 constrictions). The co-localization of Dyn2 to Drp1-marked constrictions increased to 39.7% following STS treatment (140 out of 353 Drp1-marked constrictions). f, STS treatment results in an increase of Drp1 at Mff-marked constrictions as well as an increase in Dyn2 localization to Drp1-marked constrictions. Scale bars for whole cell panels and the inset panel are 10 μm and 1 μm, respectively (a–d).

Extended Data Figure 6 Drp1 and Dyn2 depletion delays cytochrome c release from mitochondria after STS treatment.

a, b, Scrambled-, Drp1-, and Dyn2-siRNA-treated cells were first treated with 75 μM zVAD-fmk for 4 h, then either left untreated or treated with STS for 1.5 or 5 h. Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and immuno-labelled for cytochrome c (green) and TOM20 (red). a, The percentage of cells displaying cytochrome c release was scored. n values are shown on each graph and depict the total number of cells scored. Error bars represent s.e.m. b, Spatial resolution of the subcellular localization of cytochrome c. The main panel displays a merged image of cytochrome c/TOM20 with a single-channel image of cytochrome c (inset). The co-localization of cytochrome c with mitochondria was analysed using the ‘coloc2’ ImageJ plugin and the mean Pearson’s R coefficient is displayed in the bottom-right corner of each image. n = 40 cells per condition. Data were obtained from three biological replicates. Scale bars, 50 μm (a) or 10 μm (b).

Extended Data Figure 7 Bax activation is accelerated in Drp1- and Dyn2-depleted cells following STS treatment.

Scrambled-, Drp1-, and Dyn2-siRNA-treated cells were fixed as in Extended Data Fig. 6 and immuno-labelled with an antibody targeting activated Bax (Bax6A7; green), and TOM20 to stain mitochondria (red). a, The percentage of cells displaying Bax activation from the images taken (representative images shown on the left) was scored and plotted (right). n values represent the total number of cells scored. Error bars represent the s.e.m. b, Spatial resolution of the subcellular localization of activated Bax. The main panel displays a merged image of activated Bax/TOM20, a single-channel image of activated Bax is shown in the inset. The co-localization of activated Bax with mitochondria was analysed using the ‘coloc2’ ImageJ plugin. The mean Pearson’s R coefficient is displayed in the bottom-right corner of each image. n = 40 cells per condition. Data were obtained from three biological replicates. Scale bars, 50 μm (a) or 10 μm (b).

Supplementary information

Supplementary Information

This file contains the uncropped blots. (PDF 231 kb)

Dyn2 co-localized to a Drp1-marked mitochondrial division site in Sk-Mel2 cells

Sk-Mel2 cells endogenously expressing Dyn2-GFP (green) were transfected with mCh-Drp1 (red) and mito-BFP (gray). Time-lapsed images were captured every 5 sec. Line-scan analysis was conducted at each time-point to show the localization of division factors to a constriction that is about to undergo division, as well as, the continuity of the inner mitochondria membrane until membrane fission occurs. This video is represented in Fig. 2a,b. (MOV 26 kb)

Dyn2 co-localized to a Drp1-marked mitochondrial division site in COS-7 cells

COS-7 cells were transfected with Dyn2-mNeon (green), mCh-Drp1 (red) and mito-BFP (gray). Time-lapsed images were captured every 5 sec. Line-scan analysis was conducted at each time-point to show the localization of division factors to a constriction that is about to undergo division, as well as, the continuity of the inner mitochondria membrane until membrane fission occurs. This video is represented in ED Fig. 4a-c. (MOV 30 kb)

Dyn2 co-localized to a Drp1-marked mitochondrial division site in PtK1 cells

PtK1 cells were transfected with Dyn2-mNeon (green), mCh-Drp1 (red) and mito-BFP (gray). Time-lapsed images were captured every 5 sec. Line-scan analysis was conducted at each time-point to show the localization of division factors to a constriction that is about to undergo division, as well as, the continuity of the inner mitochondria membrane until membrane fission occurs. This video is represented in Fig. 2c,d. (MOV 45 kb)

BAPTA-induced Drp1-marked mitochondrial division in scrambled siRNA-treated COS-7 cells

COS-7 cells were transfected with Scrambled siRNA, and plasmids encoding mCh-Drp1 (cyan) and mito-BFP (red). Cells were treated with 10 μM BAPTA-AM for 5 minutes followed by live-cell imaging. Time-lapsed images were captured every 5 sec. Line-scan analysis was conducted at each time-point to show the localization of Drp1 to a constriction that is about to undergo division, as well as, the continuity of the inner mitochondria membrane until membrane fission occurs. Drp1 puncta can be observed accumulating at the mitochondrial constriction leading up to division. Upon division, Drp1 puncta splits into two puncta that remain co-localized to the ends of each newly created mitochondrion. This video is represented in Fig. 3d. (MOV 81 kb)

Dyn2 depletion blocked BAPTA-induced Drp1-marked mitochondria division

COS-7 cells were transfected with Dyn2 siRNA, and plasmids encoding mCh-Drp1 (cyan) and mito-BFP (red). Cells were treated with 10 μM BAPTA-AM for 5 minutes followed by live-cell imaging. Time-lapsed images were captured every 5 sec. Line-scan analysis was conducted at each time-point to show the localization of Drp1 to a constriction that is about to undergo division, as well as, the continuity of the inner mitochondria membrane until membrane fission occurs. The constriction exhibits the dynamics of a division event without the final act of membrane fission. Drp1 puncta separates into two distinct puncta, which coincides with an increase in the width of constriction. The separation of Drp1 puncta and the super-constriction region are transient because the both collapse in a fail division attempt. This video is represented in Fig. 3e. (MOV 114 kb)

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Lee, J., Westrate, L., Wu, H. et al. Multiple dynamin family members collaborate to drive mitochondrial division. Nature 540, 139–143 (2016). https://doi.org/10.1038/nature20555

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