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Unrestrained ESCRT-III drives micronuclear catastrophe and chromosome fragmentation

Nature Cell Biology volume 22, pages 856–867 (2020)Cite this article

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Abstract

The ESCRT-III membrane fission machinery maintains the integrity of the nuclear envelope. Although primary nuclei resealing takes minutes, micronuclear envelope ruptures seem to be irreversible. Instead, micronuclear ruptures result in catastrophic membrane collapse and are associated with chromosome fragmentation and chromothripsis, complex chromosome rearrangements thought to be a major driving force in cancer development. Here we use a combination of live microscopy and electron tomography, as well as computer simulations, to uncover the mechanism underlying micronuclear collapse. We show that, due to their small size, micronuclei inherently lack the capacity of primary nuclei to restrict the accumulation of CHMP7–LEMD2, a compartmentalization sensor that detects loss of nuclear integrity. This causes unrestrained ESCRT-III accumulation, which drives extensive membrane deformation, DNA damage and chromosome fragmentation. Thus, the nuclear-integrity surveillance machinery is a double-edged sword, as its sensitivity ensures rapid repair at primary nuclei while causing unrestrained activity at ruptured micronuclei, with catastrophic consequences for genome stability.

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Fig. 1: CHMP7 localization and its interplay with LEMD2 controls nuclear ESCRT-III dynamics.
Fig. 2: Unrestrained nuclear CHMP7 drives deformation and rupture of the nuclear envelope.
Fig. 3: Ruptured micronuclei recruit ESCRT-III and undergo ESCRT-III-driven catastrophe.
Fig. 4: Ruptured micronuclei lack the ability to restrain ESCRT-III activity.
Fig. 5: The balance between CHMP7 and LEMD2 determines micronuclear repair ability.
Fig. 6: Unrestrained CHMP7 drives DNA damage, torsional stress and chromosome fragmentation.
Fig. 7: Unrestrained ESCRT-III causes DNA damage in ruptured micronuclei.

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Data availability

The datasets generated and/or analysed during the current study are available from the corresponding authors on reasonable requests. Source data are provided with this paper.

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Acknowledgements

We thank U. Dahl Brinch, M. Smestad and L. Kymre for their assistance with the electron microscopy; K. Schink for cell lines; S. Eikvar, E. Rønning, A. Bergersen, E. Munthe and A. Baumeister for technical support; A. Engen for assistance with cell culture; C. Dillard for the artwork and C. Bassols for the IT support. We also thank V. Nähse, T. Stokke, R. Syljuåsen, T. Liyakat Ali for their valuable discussions. We thank the National Core Facility for Human Pluripotent Stem Cells, Advanced Light Microscopy Core Facility (Oslo University Hospital), Advanced Electron Microscopy Core Facility (Oslo University Hospital), Norwegian Advanced Light Microscopy Imaging Network (University of Oslo) and Section for Cancer Cytogenetics (Oslo University Hospital) for providing facilities. This study was primarily supported by grants to C.C. (Research Council of Norway (grant no. 262375) and South-Eastern Norway Regional Health Authority (grant no. 2018082)), H.S. (Norwegian Cancer Society (grant no. 182698), South-Eastern Norway Regional Health Authority (grant no. 2016087) and Research Council of Norway through its Centres of Excellence funding scheme (grant no. 262652)) and M.V. (South-Eastern Norway Regional Health Authority (grant no. 2018043) and the Radium Hospital Foundation) in addition to funding for F.M. (Radium Hospital Foundation) and S.N.G. (Trond Mohn Foundation (grant no. BFS2017TMT01) and the Royal Society (grant no. RG150696)).

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Author notes

  1. These authors contributed equally: Sebastian W. Schultz, Aurélie Bellanger.

Authors and Affiliations

  1. Centre for Cancer Cell Reprogramming, Faculty of Medicine, University of Oslo, Oslo, Norway

    Marina Vietri, Sebastian W. Schultz, Camilla Raiborg, Ellen Skarpen, Ingrid Kjos, Eline Kip, Ashish Jain, Andreas Brech & Harald Stenmark

  2. Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway

    Marina Vietri, Sebastian W. Schultz, Camilla Raiborg, Ellen Skarpen, Ingrid Kjos, Eline Kip, Ashish Jain, Andreas Brech & Harald Stenmark

  3. Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway

    Aurélie Bellanger, Louise I. Petersen, Romy Timmer, Philippe Collas & Coen Campsteijn

  4. Department of Biological Sciences, Durham University, Durham, UK

    Carl M. Jones & Sushma N. Grellscheid

  5. Computational Biology Unit, Department of Biological Sciences, University of Bergen, Bergen, Norway

    Carl M. Jones & Sushma N. Grellscheid

  6. Section for Cancer Cytogenetics, Institute for Cancer Genetics and Informatics, Oslo University Hospital, Oslo, Norway

    Christeen Ramane J. Pedurupillay & Francesca Micci

  7. Department of Immunology and Transfusion Medicine, Oslo University Hospital, Oslo, Norway

    Philippe Collas

  8. Max Planck Institute of Colloids and Interfaces, Department of Theory and Bio-Systems, Potsdam, Germany

    Roland L. Knorr

  9. Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, University of Tokyo, Tokyo, Japan

    Roland L. Knorr

  10. Department of Physics, Durham University, Durham, UK

    Halim Kusumaatmaja

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Contributions

C.C. and M.V. conceived and designed the study with contributions from S.W.S. M.V. generated cell lines, performed light microscopy, analysed data and prepared figures. S.W.S. performed and analysed electron microscopy experiments and prepared figures. A.Bellanger generated cell lines, performed light microscopy, analysed data and prepared figures. C.M.J. performed and analysed the computer simulation experiments and prepared figures. L.I.P. generated cell lines, performed light microscopy, analysed data and prepared figures. I.K. performed immunogold electron microscopy experiments together with M.V. C.R., E.S., E.K., R.T. and A.J. performed light microscopy and analysed data. C.R.J.P. performed the metaphase spread experiments. P.C. provided infrastructure. R.L.K. provided comments and contributed to the computer simulation set-up. S.N.G. and H.K. supervised the computer simulation experiments. A.Brech supervised and performed electron microscopy experiments and provided conceptual input. F.M. supervised and analysed the metaphase spread experiments. H.S. provided infrastructure and co-supervised parts of the study. C.C. supervised the study, generated constructs and cell lines, performed light microscopy, analysed data and prepared figures. C.C. and M.V. wrote the paper with contributions from all authors.

Corresponding authors

Correspondence to Harald Stenmark or Coen Campsteijn.

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Extended data

Extended Data Fig. 1 Nuclear localization of CHMP7 regulates ESCRT-III activation at the nuclear envelope.

a, Quantification of subcellular localization of REV(1.4)-eGFP fusions as shown in Fig. 1c. LMB treatment where indicated. Bars, SEM, dots represent the mean of each independent experiment. n = 3 independent experiments. In total, 218, 149, 138, 153 cells were analysed for groups from left to right. ****P < 0.0001, n.s. P = 0.3739 (NES*), P = 0.3590 (NES + LMB), df=4. b, RPE1 CHMP4B-mNG-3HA, mRuby3-NES, CHMP7NES*-FLAG cells treated with DOX to induce CHMP7NES*-FLAG overexpression. with quantification of CHMP4B foci at indicated time points after DOX addition. Bars, mean and SEM from n = 30 cells each time point across 3 independent experiments.***P < 0.0001, **P = 0.056, two-tailed unpaired t-test with Welch correction. c, as in b, but immunoblot of whole-cell lysates shows CHMP7NES*-FLAG levels at indicated time points after DOX addition. d, RPE1 CHMP4B-mNG-3HA, mRuby3-NES, CHMP7NES*-FLAG cells were treated with DOX to induce CHMP7NES*-FLAG overexpression and CHMP7NES*-FLAG mRNA and CHMP7NES*-FLAG protein levels were scored as well as CHMP4B-eGFP foci before DOX induction (PRE) and following DOX wash-out. Bars, SEM. Dots for bar graphs represent the mean of each of 3 independent experiment; dots for dot plot represent individual measurements. In total, n = 65, 73, 65, 74, 64 cells were analysed for groups from left to right. ****P < 0.0001, ***P < 0.001, **P = 0.0012, *P = 0.0408, n.s. P = 0.6379 (mRNA 24 h), P = 0.9959 (mRNA 36 h), P = 0.0998 (protein 36 h). Dunnett’s multiple comparison test compares time points of each parameter to the corresponding pre-DOX. e, As in d, representative confocal images showing CHMP4B-eGFP nuclear envelope foci. Scale bars, 10 µm f, as in d, immunoblot of whole-cell lysates shows CHMP7NES*-FLAG levels at indicated time points after DOX wash-out. g. Immunoblot of whole-cell lysates showing depletion of LEMD2 and LEMD3 upon treatment with indicated siRNAs. All microscopy images are representative of 3 independent experiments. Unprocessed western blots and statistical source data are shown in Source Data Extended Data Fig. 1.

Source data

Extended Data Fig. 2 Interplay between CHMP7 and XPO1 activity.

a, Inactivation of XPO1 drives the formation of nuclear CHMP4B foci. HeLaK CHMP4B-eGFP, mRuby3-NES, LEMD2-SNAP cells were treated with LMB and CHMP4B localization was monitored by live-cell imaging, with time after start of imaging indicated. Scale bar, 5 µm. b, ESCRT-III nuclear envelope foci induced by LMB treatment were monitored every 30 min by live-cell imaging in HeLaK CHMP4B-eGFP, mRuby3-NES, LEMD2-SNAP cells after LMB wash-out. Mean and 95% confidence interval are plotted. n = 23 cells from 16 movies. c, CHMP4B foci formation depend on CHMP7. Formation of CHMP4B nuclear envelope foci upon XPO1 inactivation was monitored in live HeLaK CHMP4B-eGFP, mRuby3-NES, LEMD2-SNAP cells, treated with indicated siRNAs and LMB where indicated. Scale bar, 5 μm. Quantification in Fig. 1e. d, Immunoblot of whole-cell lysate showing depletion of endogenous CHMP7 upon siRNA treatment. Unprocessed western blots and statistical source data are shown in Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 Nuclear localization of CHMP7 stabilizes LEMD2–CHMP7–CHMP4B complexes at the nuclear envelope.

a, LEMD2 overexpression selectively depletes CHMP4B levels. HeLaK CHMP4B-eGFP, LEMD2-mCherry cells were treated with DOX to induce overexpression of LEMD2 and were subsequently monitored by live-cell imaging. Scale bar, 5 μm. b, LEMD2 overexpression dependent depletion of CHMP4B is countered by co-overexpression of CHMP7. Widefield images of live HeLaK CHMP4B-eGFP cells transiently transfected with the indicated alleles. Transfected cells are outlined. Scale bars, 5 μm. c, Schematic representation of LEMD2 functional domains and deletions used in this study. LEM, LAP2 Emerin MAN1 domain. TM, transmembrane domain; MSC, MAN1-Src1p C-terminal domain. d, Interplay between LEMD2, CHMP4B and CHMP7 at the nuclear envelope. HeLaK CHMP4B-eGFP cells were transfected with mCherry-fusions of LEMD2 alleles and with CHMP7NES*-FLAG where indicated, fixed, stained and processed for confocal imaging. Outlines are drawn around mCherry positive cells, and the inset is show in the right-hand panel. Scale bar, 5 µm. All microscopy images are representative of 3 independent experiments.

Extended Data Fig. 4 Unrestrained nuclear CHMP7 drives nuclear envelope deformation and rupture.

a, Persistent CHMP4B foci colocalize with inner nuclear membrane proteins. Widefield images of live HeLaK CHMP4B-eGFP, mCherry-LAP2β cells transfected with CHMP7NES*-FLAG, projection of 3 z-planes. Scale bar, 5 µm. b, Formation of CHMP4B foci results in extensive membrane deformation. CLEM of HeLaK CHMP4B-eGFP, mRuby3-NES cells treated with DOX (6 h) to induce CHMP7NES*-FLAG. c, Overexpression of nuclear CHMP7 causes membrane rupture. RPE1 CHMP4B-mNG-3HA, mRuby3-NES cells were treated with DOX to induce the indicated CHMP7 allele and monitored in live-cell imaging. Nucleocytoplasmic compartmentalization was measured after 24 h. Bars, mean and SEM from n = 106, 108, 126, 145, 129, 114 cells. **P from left to right =0.0021, 0.0047, **P = 0.0062, n.s. P = 0.6298, df=4. d, Related to c, immunoblot from whole-cell lysates showing expression of endogenous CHMP7 and DOX-induced CHMP7 alleles. e, Related to Extended Data Fig. 1b,c, with quantification of nucleocytoplasmic compartmentalization in RPE1 CHMP4B-mNG-3HA, mRuby3-NES, CHMP7NES*-FLAG cells treated with DOX to induce CHMP7NES*-FLAG overexpression. Bars, mean and SEM from n = 30 cells each time point.***P < 0.0001, **P = 0.0073, two-tailed unpaired t-test with Welch correction, df=29.61, 29.97, 29.25. f, Overexpression of nuclear CHMP7 results in irreversible loss of nucleocytoplasmic compartmentalization. Live-cell imaging of DOX treated RPE1 CHMP4B-mNG-3HA, mRuby3-NES, inducible CHMP7NES*-FLAG cells. Time, minutes after DOX addition. Related to Supplementary Video 3. Scale bar, 5 µm. g, Overexpression of nuclear CHMP7 drives formation of LEMD2-CHMP4B nuclear envelope foci. Images of live HeLaK CHMP4B-eGFP, mRuby3-NES and LEMD2-SNAP cells transfected with the indicated CHMP7 alleles. Nucleus perimeter outlined. Scale bar, 5 µm. h, ESCRT-III-driven nuclear rupture relies on LEMD2 and LEMD3. RPE1 CHMP4B-mNG-3HA, mRuby3-NES cells were treated with DOX to induce CHMP7NES*-FLAG and siRNAs. Nucleocytoplasmic compartmentalization was monitored in live-cell microscopy. Related to Fig. 1d. Bars, mean and 95% confidence interval. n = 30 each sample. **P = 0.0025, *P = 0.024, n.s. P = 0.069, df=4. i, As in Fig. 2c, tilted representation with modelling of 3 isolated sections of membrane. j, As in Fig. 2d, immunogold labelled transmission EM of DOX treated RPE1 CHMP4B-mNG-3HA, inducible CHMP7NES*-FLAG.Arrowheads, gold particles labelling CHMP4B-mNG-3HA. All microscopy images and statistical analysis are derived from3 independent experiments. Unprocessed western and statistical source data are shown in Source Data Extended Data Fig. 4.

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Extended Data Fig. 5 ESCRT-III is recruited to micronuclei following rupture.

a, CHMP4B localization at ruptured micronuclei in different cell lines. Cells were treated with AZ3146, fixed and stained for endogenous CHMP4B and the nuclear envelope marker Emerin. Scale bars, 5 µm. b, ESCRT-III components and effectors localize at micronuclei. Confocal images of micronuclei from HeLaK cells fixed and immunolabelled for the indicated endogenous factors and DNA (blue) Scale bars, 1 µm. c, CHMP4B persistently accumulates at micronuclei upon rupture. HeLaK CHMP4B-eGFP, mCherry-NLS cells were treated with AZ3146 to induce formation of micronuclei, stained with SiR-Hoechst, and events at micronuclei were monitored by live-cell imaging. Scale bar, 5 µm. d, HeLaK CHMP4B-eGFP, mRuby3-NES cells were treated with AZ3146 to induce formation of micronuclei, stained with SiR-Hoechst, and events at micronuclei were monitored by live-cell imaging. Arrowheads indicate a rupturing micronucleus. Scale bar, 5 µm. e, as in Fig. 3b, Micronuclear GFP fluorescence HeLaK cells stably expressing siRNA-resistant CHMP4B-eGFP or the membrane-binding defective mutant CHMP4B4DE-eGFP, treated with CHMP4B siRNA to deplete endogenous CHMP4B and incubated with AZ3146. Insets show micronuclei. Scale bars, 5 µm. f, As in e, immunoblot of whole-cell lysate showing depletion of endogenous CHMP4B and expression of the eGFP-fusions. g, Micronuclear CHMP4B foci correspond to convoluted envelope regions. Widefield image of live micronucleated HeLaK CHMP4B-eGFP, mCherry-KDEL cells stained with SiR-Hoechst. All microscopy images are representative of 3 independent experiments. Scale bar, 5 µm. Unprocessed western blots are Unprocessed western blots are shown in Source Data Extended Data Fig. 5.

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Extended Data Fig. 6 ESCRT-III drives extensive membrane deformations in ruptured micronuclei.

a, Micronuclear CHMP4B foci correspond to areas of extreme membrane deformations. CLEM of a HeLaK CHMP4B-eGFP cell, treated with AZ3146 to induce micronuclei, fixed, imaged with confocal microscopy and then processed for EM. CHMP4B foci (detected by light microscopy) is shown relative to areas of the micronuclei where the nuclear envelope is dramatically deformed (observed on consecutive EM sections). Sequential confocal planes with the respective electron micrographs are shown. Arrowheads indicate confocal planes of CHMP4B-eGFP foci and the corresponding areas in the electron micrographs. Asterisks are references for micronucleus position. b, CHMP4B foci (detected by light microscopy) correlate with micronuclear envelope deformations (arrowheads) in naturally occurring micronuclei (observed on EM sections, arrowheads) of HeLaK CHMP4B-eGFP cells. c, Endogenous CHMP4B foci (detected by light microscopy) correlate with micronuclear envelope deformations (arrowheads) in micronuclei (observed on tomograms from consecutive EM sections) of HeLaK cells. d, ESCRT-III depletion rescues micronuclear envelope trabecular distortions. CLEM of ruptured micronuclei in HeLaK CHMP4B-eGFP, mRuby3-NES cells transfected with indicated siRNAs, incubated with AZ3146, fixed, imaged with light microscopy and processed for EM. All microscopy images are representative of 3 independent experiments.

Extended Data Fig. 7 Membrane ultrastructure of ruptured micronuclei resembles trabecular membrane networks driven by CHMP7 overexpression.

Electron tomograms from CLEM experiments and 3D models of membrane ultrastructure induced by overexpression of CHMP7NLS-FLAG (upper panels) or CHMP7NES*-FLAG (middle panels). Note the similarity to a spontaneously ruptured micronucleus of HeLaK cells with distinct endogenous CHMP4B foci (from Extended Data Fig. 6c (lower panels). CYT, cytoplasm; NUC, nucleoplasm; PN, primary nucleus; MN, micronucleus. All microscopy images are representative of 3 independent experiments.

Extended Data Fig. 8 Ruptured micronuclei are unable to restrict CHMP7–LEMD2 complexes.

a, Ruptured micronuclei can accumulate CHMP4B to levels sufficient to drive rupture at primary nuclei upon induction of CHMP7NES* expression. CHMP4B intensity was measured in live RPE1 cells during nuclear envelope reassembly (anaphase), in ruptured micronuclei (MN) and in CHMP7NES* DOX-induced foci at primary nuclei (PN DOX + ). Bars, mean and SEM from n = 37, 41, 30 nuclei (from left to right) analysed across 3 experiments. ****P < 0.0001, df=76, 65. *P = 0.0280 df=69, two-tailed unpaired t-test with Welch correction. b, VPS4 is recruited to ruptured micronuclei with similar kinetics as CHMP4B. Live-cell imaging of HeLaK CHMP4B-eGFP, VPS4A-SNAP, mCherry-NLS cells. Scale bar, 5 μm. Representative of n = 41, 4 experiments. c, CHMP2A depletion aggravates micronuclear collapse phenotypes. Widefield images of ruptured live CHMP4B-eGFP, mCherry-KDEL HeLa cells treated with AZ3146 and siRNAs as indicated. Scale bars, 5 μm. d, As in c, immunoblot of whole-cell lysate of HeLaK CHMP4B-eGFP cells showing efficient depletion of CHMP2A upon siRNA treatment. e, Immunoblot from whole-cell lysate showing efficient depletion of endogenous LEMD2 and expression of rescue alleles. Related to Fig. 4e. f, related to Fig. 4f, quantification of the fraction of micronuclei enriched for CHMP4B-eGFP, endogenous LEMD2 and CHMP7. Bars, mean and SEM from n = 3 independent experiments; in total 2666 micronuclei were analysed. All microscopy images are representative of 3 independent experiments. Unprocessed western blots and statistical source data are shown in Source Data Extended Data Fig. 8.

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Extended Data Fig. 9 Several parameters contribute to CHMP7–LEMD2 complex spreading along ruptured micronuclei.

a, as in Fig. 5a, but with CHMP7 as a cytosolic protein. The CHMP7-LEMD2 spreading results for membrane-bound CHMP7 better capture the experimental data. b, Phi line plots from computer simulations showing evolution of CHMP7-LEMD2 complex angular density along the INM from the rupture site (φ to rupture = 0) to the opposite pole (φ to rupture = π) for ER-associated CHMP7 using different LEMD2 occupancy cut-offs for generation of new INM LEMD2 molecules. Results are shown for micronuclei (top panel) and primary nuclei (bottom panel) at different time points after rupture as indicated. c, Decreasing CHMP7 concentration causes CHMP7-LEMD2 complex formation to become more concentrated at the site of rupture. As in Fig. 5a, but with varying cellular CHMP7 concentrations. d, as in c, but here the LEMD2 concentration is altered as indicated. e, micronuclei do not retain XPO1 following rupture. Confocal imaging of micronucleated CHMP4B-eGFP, mRuby3-NES HelaK cells, treated with DOX to induce CHMP7NES*, fixed and stained for XPO1 and Hoechst. Asterisk indicates a ruptured primary nucleus. Arrowheads indicates a ruptured micronucleus. Related to Fig. 5b. Scale bar, 5 μm. All microscopy images are representative of 3 independent experiments.

Extended Data Fig. 10 Unrestrained nuclear ESCRT-III negatively affects genome stability.

a, Nuclear CHMP4B foci associate with local sites of DNA damage. RPE1 CHMP4B-mNG-3HA, mRuby3-NES and inducible CHMP7-FLAG, CHMP7NES*-FLAG or CHMP7-ΔNNES*-FLAG (unable to bind to membranes) were treated with DOX, fixed and stained for the DNA damage marker γH2Ax. Scale bars, 5 µm. b, Nuclear CHMP7 accumulation triggers DNA torsional stress. RPE1 CHMP4B-mNG-3HA, mRuby3-NES and inducible CHMP7-FLAG or CHMP7NES*-FLAG cells were treated with DOX to induce the indicated CHMP7 allele, fixed, stained for Top2B and imaged by confocal microscopy. Quantification of Top2B accumulation in Fig. 6e. Scale bars, 5 µm. c, Depletion of CHMP4B, CHMP7 and LEMD2 does not affect cell cycle progression. HeLaK CHMP4B-eGFP, mRuby3-NES, RPA2-SNAP cells were treated with indicated siRNAs. Cell cycle phase distribution for each treatment was measured by flow cytometry. Bars, mean and SEM from n = 3 independent experiments. P (from left to right) = 0.0737, 0.779, 0.262, 0.5455, 0.0714, 0.8135. Additional statistics are included in Supplementary Fig. 1 and source data. d, The ER-associated TREX1 exonuclease enriches at nuclear CHMP4B foci. RPE1 CHMP4B-mNG-3HA, mRuby3-NES and inducible CHMP7-FLAG or CHMP7NES*-FLAG cells were treated with DOX to induce the indicated CHMP7 allele, fixed, stained for TREX1 and imaged by confocal microscopy. Scale bar, 5 µm. e, TREX1 is enriched at CHMP4B foci in collapsed micronuclei. HeLaK CHMP4B-eGFP, mRuby3-NES, RPA2-SNAP cells were treated with AZ3146 to induce micronuclei, fixed and stained for RPA2 and TREX1. Scale bar, 5 µm. f, Quantification of ruptured micronuclei showing TREX1 enrichment. HeLaK mRuby3-NES, CHMP4B-eGFP cells were treated with Control siRNA or a CHMP4B siRNA targeting both endogenous and GFP-tagged allele. Cells were then treated with AZ3146 to induce formation of micronuclei, fixed and stained for TREX1. Bars indicate mean and SEM, dots represent the mean of each independent experiment. n = 171, 238 micronuclei. **P = 0.0025, df=4. All microscopy images are representative of 3 independent experiments. Statistical source data are shown in Source Data Extended Data Fig. 10.

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Supplementary information

Supplementary Information

Supplementary Fig. 1

Reporting Summary

Supplementary Video 1

Live-cell imaging of HeLaK cells monitoring the relocalization of CHMP4B–eGFP following treatment with the XPO1 inhibitor Leptomycin B (LMB added at t = 0 min).

Supplementary Video 2

Live-cell imaging of HeLaK cells monitoring cellular CHMP4B–eGFP levels following doxycycline-induced expression of LEMD2–SNAP (DOX added at t = 0 min).

Supplementary Video 3

Live-cell imaging of RPE1 cells monitoring CHMP4B–mNG–3HA localization and nucleocytoplasmic compartmentalization (as assessed by mRuby3–NES) following doxycycline-induced expression of CHMP7NES*–FLAG (DOX added at t = 0 min).

Supplementary Video 4

Immunogold electron tomogram showing tight association of CHMP4B–mNG–3HA with the distorted nuclear membranes following doxycycline-induced expression of CHMP7NES*–FLAG. Scale bar, 200 nm.

Supplementary Video 5

Live-cell imaging of HeLaK cells monitoring CHMP4B–eGFP localization to micronuclei following rupture (as assessed by mRuby3–NES micronuclear influx). SiR–Hoechst is used as a DNA dye.

Supplementary Video 6

Live-cell imaging of HeLaK cells monitoring CHMP4B–eGFP localization to micronuclei following rupture (as assessed by mCherry–NLS nuclear efflux). SiR–Hoechst is used as a DNA dye.

Supplementary Video 7

Live-cell imaging of HeLaK cells monitoring the effects of micronuclear rupture and CHMP4B–eGFP accumulation on the architecture of micronuclear membranes, as assessed by mCherry–KDEL.

Supplementary Video 8

Electron tomogram of a CHMP4B-positive ruptured micronucleus to analyse the complex trabecular membrane network at sites of CHMP4B accumulation.

Supplementary Video 9

Live-cell imaging of HeLaK cells monitoring recruitment of CHMP4B–eGFP recruitment and the spreading and accumulation of LEMD2–SNAP at ruptured micronuclei.

Supplementary Video 10

Live-cell imaging of HeLaK cells monitoring the effects of CHMP4B or CHMP7 depletion on the spreading and accumulation of LEMD2–SNAP at ruptured micronuclei. The moment of rupture is indicated by a circle.

Supplementary Video 11

Live-cell imaging of HeLaK cells monitoring LEMD2–SNAP localization and nucleocytoplasmic compartmentalization (as assessed by mRuby3–NES) following brief overexpression of wild-type CHMP7 in cells traversing mitosis.

Supplementary Video 12

Live-cell imaging of HeLaK cells monitoring accumulation of SNAP–RPA2 at ruptured micronuclei (as assessed by mRuby3–NES) following depletion of CHMP4B, CHMP7 and LEMD2. SiR–Hoechst is used as a DNA dye. The moment of rupture is indicated by a circle.

Supplementary Tables

Supplementary Table 1. Stable cell lines used in this study. Supplementary Table 2. Plasmids used in this study. Supplementary Table 3. Variables used for the computer simulations.

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Vietri, M., Schultz, S.W., Bellanger, A. et al. Unrestrained ESCRT-III drives micronuclear catastrophe and chromosome fragmentation. Nat Cell Biol 22, 856–867 (2020). https://doi.org/10.1038/s41556-020-0537-5

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