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Distinct fission signatures predict mitochondrial degradation or biogenesis

Abstract

Mitochondrial fission is a highly regulated process that, when disrupted, can alter metabolism, proliferation and apoptosis1,2,3. Dysregulation has been linked to neurodegeneration3,4, cardiovascular disease3 and cancer5. Key components of the fission machinery include the endoplasmic reticulum6 and actin7, which initiate constriction before dynamin-related protein 1 (DRP1)8 binds to the outer mitochondrial membrane via adaptor proteins9,10,11, to drive scission12. In the mitochondrial life cycle, fission enables both biogenesis of new mitochondria and clearance of dysfunctional mitochondria through mitophagy1,13. Current models of fission regulation cannot explain how those dual fates are decided. However, uncovering fate determinants is challenging, as fission is unpredictable, and mitochondrial morphology is heterogeneous, with ultrastructural features that are below the diffraction limit. Here, we used live-cell structured illumination microscopy to capture mitochondrial dynamics. By analysing hundreds of fissions in African green monkey Cos-7 cells and mouse cardiomyocytes, we discovered two functionally and mechanistically distinct types of fission. Division at the periphery enables damaged material to be shed into smaller mitochondria destined for mitophagy, whereas division at the midzone leads to the proliferation of mitochondria. Both types are mediated by DRP1, but endoplasmic reticulum- and actin-mediated pre-constriction and the adaptor MFF govern only midzone fission. Peripheral fission is preceded by lysosomal contact and is regulated by the mitochondrial outer membrane protein FIS1. These distinct molecular mechanisms explain how cells independently regulate fission, leading to distinct mitochondrial fates.

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Fig. 1: Mitochondrial fissions are bimodally positioned and linked to distinct physiologies.
Fig. 2: Midzone and peripheral fissions differ in mitochondrial DNA content and fates.
Fig. 3: Midzone and peripheral fissions are independently regulated by distinct molecular machineries.

Data availability

All imaging as well as numerical data relevant to this study are publicly available on the online repository Zenodo (https://doi.org/10.5281/zenodo.3550643). The data are organized according to their appearance in the main figures and Extended Data figures. The unprocessed western blot gels are provided in Supplementary Fig. 1. Plasmids and cell lines are available from the corresponding authors on request. Source data are provided with this paper.

Code availability

The custom-written Fiji-macro script for BrU quantification is available at https://github.com/TimoHenry/MitochondrialRNAgranules and the custom written Fiji-macro for caspase staining is available at https://github.com/jutziw/mitochondrial_division.

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Acknowledgements

We thank C. Cottiny and M. Colomer for experimental and technical assistance, M.-C. Croisier and G. Knott from the BioEM (EPFL) for carrying out the electron microscopy, T. Laroche (BIOP, EPFL) for imaging support and M. Ryan for the MEF lines. We thank T. Misgeld, J.-C. Martinou and P. Ramdya for feedback on the manuscript. Research in S.M.’s laboratory is supported by the National Centre of Competence in Research Chemical Biology, the European Research Council (CoG 819823, Piko) and the Swiss National Science Foundation (182429). T.K. received funding from the European Molecular Biology Organization (ALTF-739-2016) and the Munich Cluster for Systems Neurology (SyNergy). This work is supported in part by grants from the Swiss National Science Foundation to T.P. (no. CRSII5_173738 and no. 31003A_182322).

Author information

Affiliations

Authors

Contributions

T.K. and S.M. conceived the project and designed experiments. T.K. performed imaging experiments and analysis. T.R. performed the TWINKLE, FASTKD2, BrU and MitoSOX imaging and contributed to the analysis. T.K. and J.W. performed the caspase, DRP1 and LC3 imaging. D.M. developed and adjusted the iSIM set-up. T.K., T.R. and D.M. performed the transmission electron microscopy (TEM) experiments. S.Z. performed the western blots. F.P.R., M.N. and T.P. designed and performed culturing of mouse cardiomyocytes and the proliferation assay. T.W., S.Z. and H.P.L. designed and cloned the CRISPR–Cas9 transgenic lines. T.K. and S.M. designed figures and wrote the manuscript, with input from all authors.

Corresponding authors

Correspondence to Tatjana Kleele or Suliana Manley.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature thanks Henry Higgs and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Distribution of mitochondrial fission sites.

a, Histogram of the relative position of constriction/fission measured by mitochondrial volume (n = 190 fissions). The two peaks are coloured in orange (0–25 bin; ‘peripheral position’) and green (25–50 bin; ‘midzone position’). b, Relative position of mitochondrial fission measured by length versus measured by area (n = 190 fissions). c, Stacked histogram of the relative position of fission for different bins grouped by the total length of the dividing mitochondria (n = 1393). d, Scatter plot of the total length of dividing mitochondria versus the relative position of the fission site along the length axis with peripheral fissions (0–25% bin) coloured in orange and midzone fissions (25–50% bin) coloured in green (n = 1393 fissions). e, Length distribution of the smaller (light colour) and larger (dark colour) daughter mitochondria arising from peripheral left, orange) and midzone (right, green) fissions. f, Histogram of relative position of fission in datasets acquired with a mitochondrial inner membrane marker (left, COX8 targeting domain; n = 510 fissions) and a mitochondrial outer membrane marker (right, TOM20; n = 368 fissions).

Source data

Extended Data Fig. 2 Physiological changes preceding fission are independent of volume and absolute length.

a, Normalized mito–GFP intensity depending on the relative position of fission measured in mitochondria immediately before fission. (n = 50 fissions). b, Dependence on the length of the daughter mitochondria of normalized mito–GFP intensity immediately before peripheral (orange) or midzone (green) fissions (n = 50 fissions). c, Dependence on the length of the daughter mitochondria of normalized TMRE intensity immediately before peripheral (orange) or midzone (green) fissions (n = 56 fissions). d, Dependence on the length of the daughter mitochondria of normalized mito–SypHer intensity before fission (n = 53 fissions). e, Dependence of the length of the daughter mitochondria on normalized MitoSOX intensity before fission (n = 52 fissions). f, Rates of peripheral and midzone fissions in control Cos-7 cells (n = 10 fields of view) versus cells treated with 500 nM ROS scavenger MitoQ (n = 10 fields of view). g, Dependence on the length of the daughter mitochondria of ratiometric intensity of mito–GRX1–roGFP immediately before fission (n = 52 fissions). h, Dependence on the length of the daughter mitochondria of normalized mito–R-GECO1 intensity before fission (n = 50 fissions). i, Dependence on the length of the daughter mitochondria of normalized CEPIA3–mt intensity before fission (n = 61 fissions). j, Mitochondrial pH before and after a peripheral or midzone fission from SIM movies of mito–SypHer transfected Cos-7 cells. k, Normalized mito–SypHer intensity as a function of relative position of fission, measured immediately before fission (n = 53 fissions). l, Mitochondrial ROS before and after a peripheral or midzone fission from ratiometric images of mito–GRX1–roGFP transfected Cos-7 cells. m, Ratiometric mito–GRX1–roGFP intensity as a function of relative position of fission, measured immediately before fission (n = 52 fissions). n, Mitochondrial matrix Ca2+ before and after a peripheral or midzone fission from SIM movies of mito–R-GECO1 transfected Cos-7 cells. o, Normalized mito–R-GECO1 intensity as a function of relative position of fission, measured immediately before fission (n = 50 fissions). p, Mitochondrial matrix Ca2+ before and after a peripheral or midzone fission from SIM movies of CEPIA3–mt transfected Cos-7 cells. q, Normalized CEPIA3–mt intensity as a function of relative position of fission, measured immediately before fission (n = 61 fissions). In a, k, m, o and q circles indicate individual measurements; values of binned groups represented as box plots (line, mean; bounds of box: 25th and 75th percentiles; whiskers, min/max values). Light blue areas indicate mean intensity in non-dividing mitochondria (± s.d.). n.s. >0.05, ***P < 0.001. Number of experiments, statistical tests and exact P values are provided in Supplementary Table 1. Scale bars, 0.5 μm. Fission sites are indicated by arrowheads.

Source data

Extended Data Fig. 3 Redistribution of mitochondrial DNA and RNA granules.

a, Distribution of PicoGreen foci in the small and large daughter mitochondrion derived from peripheral and midzone fission (n = 78 fissions). b, Normalized TMRE intensity in the small daughter mitochondria from peripheral fissions that contain 0, 1 or 2 nucleoids (n = 20 fissions). c, SIM images of mitochondrial RNA granules (MRGs) (FASTKD2) before and after fission. d, Number of MRGs per μm length as a function of fission position (n = 84 fissions). Blue line shows average MRG per length in non-dividing mitochondria (n = 41). e, Distribution of the number of MRGs (FASTKD2; n = 84 fissions). f, Number of replicating nucleoids (TWINKLE; n = 74 fissions) in smaller (light) and larger (dark) daughter mitochondria from peripheral (orange) and midzone (green). g, Number of BrU-positive foci per cell in control Cos-7 cells (n = 39) and cells exposed to UV light for 3 min before measurement (n = 98). h, Time-lapse SIM sequence of Cos-7 mitochondria (mito–RFP, grey) and autophagosomes (EGFP−LC3, green), where the small daughter mitochondrion from a peripheral fission is being taken up by an autophagosome (asterisk). In b and g circles indicate individual measurements; line, mean; bounds of box, 25th and 75th percentiles; whiskers, min/max values. *P < 0.05, ***P < 0.001. Number of experiments, statistical tests and exact P values are provided in Supplementary Table 1. Scale bars, 0.5 μm.

Source data

Extended Data Fig. 4 Time course of physiological changes and recruitment of fission regulators.

a, b, Time course of fluorescent signals in four examples of Cos-7 mitochondria displaying normalized TMRE intensity (a) and mito–R-GECO1 intensity (b) with corresponding SIM images in the mitochondrial compartment giving rise to the smaller daughter mitochondria before a peripheral division. c, Average inner membrane diameter at the constriction site at several time points before fission, measured in mito–R-GECO1 transfected Cos-7 cells during the time window where Ca2+ is elevated (green box in b, n = 10 fission events). d, e, Time course of lysosome co-localization (d) and autophagosome co-localization (e) at constriction sites for peripheral fissions, by measuring LAMP1−mEGFP and EGFP−LC3 intensity, respectively. For EGFP−LC3 measurements, cells were pre-treated with 10 μM CCCP to increase LC3 signals. f, Normalized DRP1 intensity at the constriction sites before peripheral fission in four examples of Cos-7 mitochondria with corresponding SIM images. Blue dotted lines (t = 0 s) mark the time point of fission.

Source data

Extended Data Fig. 5 Peripheral and midzone fissions interact differently with the mitochondrial network, and the distribution of the fission positions is regulated independently.

a, Schematic diagram depicting the fate (‛no event’, another ‛fission’ or ‛fusion’) of each daughter mitochondrion from peripheral and midzone fissions after the initial division in postnatal cardiomyocytes. Only mitochondria that could be traced for more than 100 s after fission were included in the analysis. b, Distribution of the relative position of fission in starved Cos-7 cells, with peripheral (1–25%) fission labelled in orange and midzone fissions (25–50%) in green (n = 212 fissions). The frequency distribution of Cos-7 control samples is superimposed in grey (replotted from Fig. 1c). c, d, Distribution of the relative position of constriction/fission along the length axis of isoproterenol-treated mouse cardiomyocyte mitochondria (cn = 356 fissions) and miR-199 treated cardiomyocytes (dn = 225 fissions) respectively. The frequency distribution of untreated mouse cardiomyocyte samples is superimposed in grey (replotted from Fig. 1e).

Source data

Extended Data Fig. 6 Peripheral and midzone fissions are both DRP1 mediated but involve distinct upstream mechanisms.

a, Two-colour SIM images of mitochondria (mito–GFP, greyscale) and DRP1 (mCherry–DRP1, red) undergoing peripheral or midzone fission. b, Normalized DRP1 intensity on the constriction sites of peripheral and midzone divisions. The threshold for a DRP1 accumulation (blue dotted line) was set at a signal >3× over background (n = 107 fissions). c, Time-lapse sequence of a SIM movie, in which both mitochondrial outer membrane (TOM20–GFP) and inner membrane (mito–RFP) were labelled to detect mitochondria-derived vesicle (MDV) formation (arrowhead). d, Quantification of the fission positions for mitochondria undergoing MDV formation or not before or after division (n = 41 fissions). e, SIM images of peripheral and midzone constrictions in fixed Cos-7 cells labelled with anti-PDZD8 (red). f, Distribution of normalized fluorescence intensities of anti-PDZD8 staining in fixed Cos-7 cells for peripheral (orange) and midzone (green) fissions (n = 38 fissions). g, Rate of peripheral and midzone fissions in control cells (n = 10 FOV) versus cells treated with INF2 siRNA (n = 10 FOV). h, Correlated confocal and transmission electron microscopy (CLEM) of mitochondria in Cos-7 cells labelled with mito–GFP, fixed 24 h after expression. Zoom-in of two individual mitochondria with a peripheral (orange frame) and a midzone (green frame) constriction in the TEM plane containing the widest diameter of the constriction site. Pseudo-colouring of three consecutive TEM z-sections recombined into a single rendering shows mitochondria (green) and ER (magenta). Scale bar represents 2 μm in confocal and 200 nm in TEM images. i, k, m, SIM images of peripheral and midzone constrictions in fixed Cos-7 cells labelled with anti-TOM20 (grey) and anti-MFF (green) (i), anti-FIS1 (red) (k) and anti-MiD49 (green) (m). j, l, n, Distribution of normalized fluorescent intensities of anti-MFF (n = 92 fissions) (j), anti-FIS1 (n = 59 fissions) (l) and anti-MiD49 (n = 29 fissions) (n) staining in fixed Cos-7 cells for peripheral (orange) and midzone (green) fissions. o, Normalized FIS1–GFP intensity at the fission site of peripheral (left) and midzone (right) divisions (orange dots: peripheral, green dots: midzone; n = 35 fissions). p, Quantification of the peripheral (orange) and midzone (green) fission rates in wild-type (n = 16 FOV) and Mff−/−Mid49−/−Mid51−/− triple-knockout (n = 16 FOV) MEFs stained with Mitotracker Green. q, Quantification of the peripheral (orange) and midzone (green) fission rates in control Cos-7 cells and cells treated with siRNAs against MFF or FIS1 (n ≥ 13 FOV per group). n.s. P > 0.05, **P < 0.01, ***P < 0.001. Number of experiments, statistical tests and exact P values are provided in Supplementary Table 1. Scale bars, 0.5 μm. Fission sites are indicated by arrowheads.

Source data

Extended Data Fig. 7 Silencing efficiency of siRNA against FIS1, MFF and INF2 in Cos-7 cells.

a, Western blot analysis of protein levels for Cos-7 cells 72 h after transfection with two siRNAs against INF2 at two different quantities (pmol). Molecular mass is in kilodaltons. b, Western blot analysis of protein levels for Cos-7 cells 72 h after transfection with 12.5 pmol of siRNA against FIS1 or MFF. c, d, Mean fluorescence intensity in U2OS cells endogenously expressing MFF–GFP (c) or FIS1–GFP (d) in control conditions and 72 h after transfection with siRNA against FIS1 or MFF (n > 39 cells per group). e, Relative Mff RNA expression levels in control cardiomyocytes and in cardiomyocytes 48 h after transfection with siMff and with or without isoproterenol (Iso) treatment. f, Relative Fis1 RNA expression levels in control cardiomyocytes and in cardiomyocytes 48 h after transfection with siFis1 and with or without isoproterenol (Iso) treatment. *P < 0.05, **P < 0.01, ***P < 0.00. Number of experiments, statistical tests and exact P values are provided in Supplementary Table 1.

Source data

Supplementary information

Supplementary Figure 1

Uncropped western blot images shown in Extended Data Fig. 7 a b. Black dashed boxes correspond to the regions cropped for the Extended Data Figure.

Reporting Summary

Supplementary Table 1

| Statistics and Reproducibility. Detailed information about sample sizes, biological replicates, statistical test and exact P-values and confidence intervals for all numerical data.

Supplementary Video 1 Live imaging of mitochondrial fissions.

Live-cell SIM imaging of peripheral and midzone mitochondrial divisions in Cos-7 cells labelled with Mitotracker green. Video was acquired at 1 frame/sec for 3 minutes and corresponds to Fig. 1 b.

Supplementary Video 2 Live imaging of mouse cardiomyocyte.

Live-cell iSIM imaging of a post-natal mouse cardiomyocyte labelled with Mitotracker green showing peripheral (orange arrowhead) and midzone (green arrowhead) fissions. Video was acquired at 1 frame/5 sec for 6 minutes. Video corresponds to Fig. 1 f.

Supplementary Video 3 Live imaging of mitochondrial membrane potential (TMRE).

Live-cell SIM imaging of TMRE stained Cos-7 cells showing a peripheral mitochondrial divisions. Video was acquired at 1 frame/3 sec. To highlight differences in fluorescent intensities, a heat-map look-up table was chosen.

Supplementary Video 4 Live imaging of mitochondrial matrix pH (Mito-SypHer).

Live-cell SIM imaging of Cos-7 transfected with mito–SypHer showing a peripheral mitochondrial divisions. Video was acquired at 1 frame/1.5 sec. To highlight differences in fluorescent intensities, a heat-map look-up table was chosen.

Supplementary Video 5 Live imaging of mitochondrial Ca2+ (mito–R-GECO1).

Live-cell SIM imaging of Cos-7 transfected with mito–R-GECO1 showing a peripheral mitochondrial divisions. Video was acquired at 1 frame/3 sec. To highlight differences in fluorescent intensities, a heat-map look-up table was chosen.

Supplementary Video 6 Live imaging of mitochondria-lysosome contact during fission.

Live-cell iSIM imaging of lysosomes (LAMP1–mEGFP, green) and mitochondria (mito–TagRFP, grey) in Cos-7 cells. During peripheral fission (left) but not during midzone fission (right), lysosomes contact the mitochondrial constriction site. Video was acquired at 1 frame/3 sec for 3 minutes. Video corresponds to Fig. 3 d.

Supplementary Video 7 Live imaging of mitochondria-autophagosome contact after fission.

Live-cell iSIM imaging of Cos-7 cells where autophagosomes (LC3–GFP, green) and mitochondria (mito–TagRFP, grey) are labelled. After peripheral fission, the small daughter mitochondrion is subsequently engulfed by an autophagosome. Video was acquired at 1 frame/5 sec for 6.5 minutes. Video corresponds to Fig. 3 h.

Supplementary Video 8 Live imaging of mitochondria-ER contacts during fission.

Live-cell iSIM imaging of Cos-7 cells transfected with KDEL–RFP (ER) and mito–GFP (mitochondria) showing a midzone fission (left, green arrowhead) and a peripheral fission (right, orange arrowhead). Video was acquired at 1 frame/14 sec for 5 minutes.

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Kleele, T., Rey, T., Winter, J. et al. Distinct fission signatures predict mitochondrial degradation or biogenesis. Nature 593, 435–439 (2021). https://doi.org/10.1038/s41586-021-03510-6

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