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Spastin and ESCRT-III coordinate mitotic spindle disassembly and nuclear envelope sealing

Nature volume 522, pages 231–235 (2015)Cite this article

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Abstract

At the onset of metazoan cell division the nuclear envelope breaks down to enable capture of chromosomes by the microtubule-containing spindle apparatus1. During anaphase, when chromosomes have separated, the nuclear envelope is reassembled around the forming daughter nuclei1,2. How the nuclear envelope is sealed, and how this is coordinated with spindle disassembly, is largely unknown. Here we show that endosomal sorting complex required for transport (ESCRT)-III, previously found to promote membrane constriction and sealing during receptor sorting, virus budding, cytokinesis and plasma membrane repair3,4,5,6, is transiently recruited to the reassembling nuclear envelope during late anaphase. ESCRT-III and its regulatory AAA (ATPase associated with diverse cellular activities) ATPase VPS4 are specifically recruited by the ESCRT-III-like protein CHMP7 to sites where the reforming nuclear envelope engulfs spindle microtubules. Subsequent association of another ESCRT-III-like protein, IST1, directly recruits the AAA ATPase spastin to sever microtubules. Disrupting spastin function impairs spindle disassembly and results in extended localization of ESCRT-III at the nuclear envelope. Interference with ESCRT-III functions in anaphase is accompanied by delayed microtubule disassembly, compromised nuclear integrity and the appearance of DNA damage foci in subsequent interphase. We propose that ESCRT-III, VPS4 and spastin cooperate to coordinate nuclear envelope sealing and spindle disassembly at nuclear envelope–microtubule intersection sites during mitotic exit to ensure nuclear integrity and genome safeguarding, with a striking mechanistic parallel to cytokinetic abscission7.

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Figure 1: ESCRT-III is transiently recruited around chromatin discs during nuclear envelope reformation.
Figure 2: ESCRT-III and VPS4 cooperate at sites of NE and MT intersection during nuclear envelope reassembly.
Figure 3: ESCRT-III-dependent recruitment of spastin to the reforming NE mediates mitotic spindle disassembly.
Figure 4: ESCRT-III dysfunction compromises nuclear integrity and leads to DNA damage and cell cycle arrest.

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References

  1. Guttinger, S., Laurell, E. & Kutay, U. Orchestrating nuclear envelope disassembly and reassembly during mitosis. Nature Rev. Mol. Cell Biol. 10, 178–191 (2009)

    Article  Google Scholar 

  2. Burke, B. & Ellenberg, J. Remodelling the walls of the nucleus. Nature Rev. Mol. Cell Biol. 3, 487–497 (2002)

    Article  CAS  Google Scholar 

  3. Babst, M., Katzmann, D. J., Estepa-Sabal, E. J., Meerloo, T. & Emr, S. D. Escrt-III: an endosome-associated heterooligomeric protein complex required for mvb sorting. Dev. Cell 3, 271–282 (2002)

    Article  CAS  Google Scholar 

  4. von Schwedler, U. K. et al. The protein network of HIV budding. Cell 114, 701–713 (2003)

    Article  CAS  Google Scholar 

  5. Carlton, J. G. & Martin-Serrano, J. Parallels between cytokinesis and retroviral budding: a role for the ESCRT machinery. Science 316, 1908–1912 (2007)

    Article  ADS  CAS  Google Scholar 

  6. Jimenez, A. J. et al. ESCRT machinery is required for plasma membrane repair. Science 343, 1247136 (2014)

    Article  Google Scholar 

  7. Guizetti, J. et al. Cortical constriction during abscission involves helices of ESCRT-III-dependent filaments. Science 331, 1616–1620 (2011)

    Article  ADS  CAS  Google Scholar 

  8. Morita, E. et al. Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis. EMBO J. 26, 4215–4227 (2007)

    Article  CAS  Google Scholar 

  9. Poser, I. et al. BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nature Methods 5, 409–415 (2008)

    Article  ADS  CAS  Google Scholar 

  10. Hanson, P. I., Roth, R., Lin, Y. & Heuser, J. E. Plasma membrane deformation by circular arrays of ESCRT-III protein filaments. J. Cell Biol. 180, 389–402 (2008)

    Article  CAS  Google Scholar 

  11. Hetzer, M. W. The nuclear envelope. Cold Spring Harb. Perspect. Biol. 2, a000539 (2010)

    Article  Google Scholar 

  12. Wollert, T., Wunder, C., Lippincott-Schwartz, J. & Hurley, J. H. Membrane scission by the ESCRT-III complex. Nature 458, 172–177 (2009)

    Article  ADS  CAS  Google Scholar 

  13. Stauffer, D. R., Howard, T. L., Nyun, T. & Hollenberg, S. M. CHMP1 is a novel nuclear matrix protein affecting chromatin structure and cell-cycle progression. J. Cell Sci. 114, 2383–2393 (2001)

    CAS  Google Scholar 

  14. Buchkovich, N. J., Henne, W. M., Tang, S. & Emr, S. D. Essential N-terminal insertion motif anchors the ESCRT-III filament during MVB vesicle formation. Dev. Cell 27, 201–214 (2013)

    Article  CAS  Google Scholar 

  15. Henne, W. M., Stenmark, H. & Emr, S. D. Molecular mechanisms of the membrane sculpting ESCRT pathway. Cold Spring Harb. Perspect. Biol. 5, http://dx.doi.org/10.1101/cshperspect.a016766 (2013)

  16. Horii, M. et al. CHMP7, a novel ESCRT-III-related protein, associates with CHMP4b and functions in the endosomal sorting pathway. Biochem. J. 400, 23–32 (2006)

    Article  CAS  Google Scholar 

  17. Hill, C. P. & Babst, M. Structure and function of the membrane deformation AAA ATPase Vps4. Biochim. Biophys. Acta 1823, 172–181 (2012)

    Article  CAS  Google Scholar 

  18. Haraguchi, T. et al. Live cell imaging and electron microscopy reveal dynamic processes of BAF-directed nuclear envelope assembly. J. Cell Sci. 121, 2540–2554 (2008)

    Article  CAS  Google Scholar 

  19. Lu, L., Ladinsky, M. S. & Kirchhausen, T. Formation of the postmitotic nuclear envelope from extended ER cisternae precedes nuclear pore assembly. J. Cell Biol. 194, 425–440 (2011)

    Article  CAS  Google Scholar 

  20. Yang, D. et al. Structural basis for midbody targeting of spastin by the ESCRT-III protein CHMP1B. Nature Struct. Mol. Biol. 15, 1278–1286 (2008)

    Article  CAS  Google Scholar 

  21. Lumb, J. H., Connell, J. W., Allison, R. & Reid, E. The AAA ATPase spastin links microtubule severing to membrane modelling. Biochim. Biophys. Acta 1823, 192–197 (2012)

    Article  CAS  Google Scholar 

  22. Agromayor, M. et al. Essential role of hIST1 in cytokinesis. Mol. Biol. Cell 20, 1374–1387 (2009)

    Article  CAS  Google Scholar 

  23. Reid, E. et al. The hereditary spastic paraplegia protein spastin interacts with the ESCRT-III complex-associated endosomal protein CHMP1B. Hum. Mol. Genet. 14, 19–38 (2005)

    Article  CAS  Google Scholar 

  24. Lattanzi, G., Marmiroli, S., Facchini, A. & Maraldi, N. M. Nuclear damages and oxidative stress: new perspectives for laminopathies. Eur. J. Histochem. 56, e45 (2012)

    Article  CAS  Google Scholar 

  25. Liu, B. et al. Genomic instability in laminopathy-based premature aging. Nature Med. 11, 780–785 (2005)

    Article  CAS  Google Scholar 

  26. Abbas, T. & Dutta, A. p21 in cancer: intricate networks and multiple activities. Nature Rev. Cancer 9, 400–414 (2009)

    Article  CAS  Google Scholar 

  27. Hatch, E. & Hetzer, M. Breaching the nuclear envelope in development and disease. J. Cell Biol. 205, 133–141 (2014)

    Article  CAS  Google Scholar 

  28. Steigemann, P. et al. Aurora B-mediated abscission checkpoint protects against tetraploidization. Cell 136, 473–484 (2009)

    Article  Google Scholar 

  29. Carlton, J. G., Caballe, A., Agromayor, M., Kloc, M. & Martin-Serrano, J. ESCRT-III governs the Aurora B-mediated abscission checkpoint through CHMP4C. Science 336, 220–225 (2012)

    Article  ADS  CAS  Google Scholar 

  30. Thoresen, S. B. et al. ANCHR mediates Aurora-B-dependent abscission checkpoint control through retention of VPS4. Nature Cell Biol. 16, 550–560 (2014)

    Article  CAS  Google Scholar 

  31. Bache, K. G. et al. The ESCRT-III subunit hVps24 is required for degradation but not silencing of the epidermal growth factor receptor. Mol. Biol. Cell 17, 2513–2523 (2006)

    Article  CAS  Google Scholar 

  32. Sagona, A. P. et al. PtdIns(3)P controls cytokinesis through KIF13A-mediated recruitment of FYVE-CENT to the midbody. Nature Cell Biol. 12, 362–371 (2010)

    Article  CAS  Google Scholar 

  33. Mu, F. T. et al. EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine “fingers” and contains a calmodulin-binding IQ motif. J. Biol. Chem. 270, 13503–13511 (1995)

    Article  CAS  Google Scholar 

  34. Raiborg, C., Bache, K. G., Mehlum, A., Stang, E. & Stenmark, H. Hrs recruits clathrin to early endosomes. EMBO J. 20, 5008–5021 (2001)

    Article  CAS  Google Scholar 

  35. Schmitz, M. H. et al. Live-cell imaging RNAi screen identifies PP2A-B55alpha and importin-beta1 as key mitotic exit regulators in human cells. Nature Cell Biol. 12, 886–893 (2010)

    Article  CAS  Google Scholar 

  36. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nature Methods 9, 676–682 (2012)

    Article  CAS  Google Scholar 

  37. McKinney, W. Data structures for statistical computing in Python. In Proc. 9th Python in Science Conference 51–56. (2010)

  38. Dultz, E., Huet, S. & Ellenberg, J. Formation of the nuclear envelope permeability barrier studied by sequential photoswitching and flux analysis. Biophys. J. 97, 1891–1897 (2009)

    Article  ADS  CAS  Google Scholar 

  39. Campeau, E. et al. A versatile viral system for expression and depletion of proteins in mammalian cells. PLoS ONE 4, e6529 (2009)

    Article  ADS  Google Scholar 

  40. Dull, T. et al. A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463–8471 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Kremer, J. R. et al. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank T. Høiby, T. Håve and K. W. Tan for assistance with generation of stable cell lines, M. Smestad and L. Hermansen for assistance with EM, A. Engen, A. Al-Kayssi and B. M. Furulund for assistance with cell cultures, E. Rønning and A. Gro Bergersen for technical support, and C. Bassols for IT support. We thank C. González, R. Syljuåsen, V. Nähse-Kumpf and E. Wenzel for discussions. We are grateful to A. A. Hyman for the gift of CHMP4B–eGFP BAC HeLa cells, and E. Reid for providing spastin constructs. We especially thank J. Carlton for sharing results before publication. The Core Facilities for Confocal Microscopy, Super-Resolution Microscopy and Electron Microscopy at Oslo University Hospital are acknowledged for providing access to relevant microscopes. M.V. is a PhD student and S.B.T. a postdoctoral fellow of the South-Eastern Norway Regional Health Authority. C.C. is a postdoctoral fellow and C.R. a senior research fellow of the Norwegian Cancer Society. H.S. is the recipient of an Advanced Grant from the European Research Council. This work was partly supported by the Research Council of Norway through its Centres of Excellence funding scheme, project number 179571.

Author information

Author notes

  1. Kay O. Schink and Coen Campsteijn: These authors contributed equally to this work.

Authors and Affiliations

  1. Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Montebello, N-0379, Oslo, Norway

    Marina Vietri, Kay O. Schink, Coen Campsteijn, Catherine Sem Wegner, Sebastian W. Schultz, Liliane Christ, Sigrid B. Thoresen, Andreas Brech, Camilla Raiborg & Harald Stenmark

  2. Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, N-0379, Oslo, Norway

    Marina Vietri, Kay O. Schink, Coen Campsteijn, Catherine Sem Wegner, Sebastian W. Schultz, Liliane Christ, Sigrid B. Thoresen, Andreas Brech, Camilla Raiborg & Harald Stenmark

  3. Centre of Molecular Inflammation Research, Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, N-7491, Norway

    Harald Stenmark

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Contributions

M.V. generated plasmid constructs and stable cell lines, performed confocal and live-cell imaging, cell transfections, image processing, data analyses, statistical analyses, and prepared figures; K.O.S. generated plasmid contructs and stable cell lines, performed live-cell and SIM imaging, image processing, developed algorithms for automated data analyses, performed data analyses, statistical analyses, and prepared figures; C.C. developed and co-supervised the project, generated plasmid contructs and stable cell lines, and performed live-cell imaging, image processing, data analyses and prepared figures; C.S.W., S.W.S. and A.B. performed EM and preparation of EM figures; L.C. performed confocal and live-cell imaging and cell transfections; S.B.T. performed confocal imaging and cell transfections; C.R. conceived and co-supervised the study, performed confocal microscopy, cell transfections, image processing and data analyses, and prepared figures; H.S. coordinated the study and oversaw experiments; M.V., C.C. and H.S. wrote the paper with input from all co-authors.

Corresponding authors

Correspondence to Coen Campsteijn or Harald Stenmark.

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Extended data figures and tables

Extended Data Figure 1 ESCRT-III is transiently recruited around chromatin discs during nuclear envelope reformation.

a, Confocal image of HeLa cells treated with control siRNA or CHMP4B targeting siRNA and stained as indicated. Scale bars, 10 μm. Image is representative of at least five captures. b, Immunoblot of whole-cell lysates probed for endogenous CHMP4B and β-actin. c, Confocal images of H199v, hTERT RPE1 and U2OS cells showing localization of endogenous CHMP4B around chromatin discs during late anaphase. Cells were co-stained for α-tubulin and DNA (Hoechst). Scale bars, 10 μm. Images are representative of at least three captures for each cell line. d, Confocal image of fixed HeLa cells stably expressing HA-CHMP4B. Anti-HA in red and DNA (Hoechst) labelled in blue. Scale bar, 5 μm. Image is representative of at least ten captures. e, Confocal images of late anaphase HeLa cells stained for endogenous proteins (CHMP4A, CHMP3, CHMP2 in red) or stably expressing tagged proteins (CHMP1B–Flag, CHMP1A-V5, IST1–mCherry in red) as indicated and DNA (Hoechst) shown in blue. Scale bars, 5 μm. Images are representative of at least three captures. f, Confocal image of HeLa cells labelled for CHMP4B–eGFP, the early endosome marker EEA1, the multivesicular endosome marker Hrs and DNA (Hoechst). Image is representative of five captures. g, As f, but stained for the late endosome marker LAMP1 instead of Hrs. Image is representative of five captures. h, Confocal images of fixed HeLa cells in different phases of mitotic exit, stained for lamin A, endogenous CHMP4B, and DNA (Hoechst). Scale bars, 5 μm. Images are representative of at least five captures for each stage. i, Confocal image of HeLa cells at different phases of mitotic exit stained as indicated. Note the different stage in nuclear pore reassembly in late anaphase (arrow) compared with telophase (arrowhead). Scale bar, 5 μm. Image is representative of at least ten captures. j, SIM reconstruction of HeLa cells (labelled as indicated) shows no co-localization between CHMP4B and nuclear pores (Nup153). Image is representative of five captures.

Extended Data Figure 2 ESCRT-III is transiently recruited around chromatin discs during nuclear envelope reformation.

a, Deconvolved wide-field live-cell imaging of HeLa cells stably expressing CHMP4B–eGFP and mCherry–KDEL, showing recruitment of CHMP4B only at sites where the reforming NE has engulfed the chromatin disc (see gallery). Images are representative of at least ten videos. b, HeLa cells stably expressing CHMP4B–eGFP and mCherry–α-tubulin were fixed and treated with increasing concentrations of DNase I. Cells were then labelled for GFP, mCherry and DNA and imaged with a confocal microscope. Scale bar, 5 μm. Images are representative of at least three captures for each condition. c, Immunoblot showing endogenous CHMP4B knockdown in HeLa cells stably expressing WT CHMP4B–eGFP or a membrane binding defective mutant CHMP4B–eGFP 4DE. Asterisk indicates non-specific immunoreactivity. d, Table showing frequency of cells where CHMP4B was recruited around anaphase chromatin under the indicated siRNA transfections. CHMP4B is not recruited upon CHMP7 depletion. This result is representative of knockdown experiments using three independent CHMP7-targeting siRNAs. e, Representative confocal images of HeLa cells transfected with the indicated siRNAs, fixed and immunolabelled for CHMP4B, α-tubulin and DNA. CHMP7 depletion affects CHMP4B recruitment around anaphase chromatin (upper panel), but does not affect CHMP4B recruitment at the midbody (lower panel). Scale bars, 5 μm. Images are representative of 20 captures each for control and CHMP7 siRNAs. f, Immunoblot showing efficient endogenous CHMP7 knockdown in HeLa cells stably expressing CHMP4B–eGFP.

Extended Data Figure 3 ESCRT-III and VPS4 cooperate at foci of NE–MT intersection during nuclear envelope reassembly.

a, Deconvolved wide-field images of live HeLa cells stably expressing HAeGFP–VPS4A followed through mitotic exit, showing transient localization around chromatin discs during late anaphase. Scale bar, 5 μm. Gallery is representative of three videos. b, CHMP4B–eGFP/H2B–mCherry HeLa cells stably expressing untagged WT CHMP2A or untagged RES CHMP2A (CHMP2A allele resistant for CHMP2A siRNA 1) were transfected with the indicated siRNAs. Cells were then imaged live after anaphase onset and residence time (in minutes) of CHMP4B foci around chromatin was scored and plotted as a percentage of population. Immunoblot shows efficient depletion of CHMP2A in the non-resistant lines. n = 21 cells for WT/control siRNA; n = 24 cells for WT/CHMP2A siRNA 1; n = 11 cells WT/CHMP2A siRNA 2; n = 18 cells for RES/control siRNA; n = 17 cells for RES/CHMP2A siRNA 1; n = 12 cells for RES/CHMP2A siRNA 2. ***P = 0.0002 was derived from an unpaired t-test. c, HeLa cells stably expressing CHMP4B–eGFP and Nup58–mCherry were transfected with the indicated siRNAs for 48 h and imaged every 20 s after anaphase onset. Normalized fluorescence intensity of Nup58–mCherry at NE was plotted over time. n = 8 DNA discs for control siRNA; n = 8 DNA discs for CHMP3 siRNA. Bars, mean and 95% confidence intervals. d, Immunoblot showing efficient endogenous CHMP3 knockdown in HeLa cells stably expressing CHMP4B–eGFP and Nup58–mCherry. e, HeLa cells stably expressing IBB–eGFP and H2B–RFP were transfected with the indicated siRNAs. Cells were then imaged every 20 s after anaphase onset. The mean intensity of the nuclear IBB was normalized to the total cellular IBB signal (nucleus + cytoplasm) and plotted over time. n = 37 cells for control siRNA; n = 32 cells for CHMP2A siRNA. Bars, mean and 95% confidence intervals. f, Electron tomography and three-dimensional reconstruction of kinetochore MTs (light blue) intersecting the reforming NE (magenta), obtained from correlative live-cell imaging (inset) and transmission EM. Nuc, nucleus. The electron tomography image is representative of EM analysis of three anaphase cells.

Extended Data Figure 4 ESCRT-III and VPS4 cooperate at foci of the NE–MT intersection during nuclear envelope reassembly.

CLEM of a whole nucleus from HeLa cells expressing CHMP4B–eGFP and mCherry–CENP-A by combination of a confocal stack and a complete serial EM reconstruction of the same structure. CLEM analysis is representative of three chromatin discs. a, Confocal stack; z1–z20. Pink (z11) and orange (z13) highlighted confocal planes correspond to sections shown in Fig. 2c and in Extended Data Fig. 4f, respectively. b, The strongest CHMP4B–eGFP signals were annotated in an Imaris stack as centre of masses (green spheres in b; blue, DNA; pink and orange bars indicate sections shown in Fig. 2c) and then transferred manually onto a three-dimensional reconstruction from serial EM sections (n = 13 CHMP4B–eGFP mCherry–CENP-A double-positive foci). c, Complete EM serial reconstruction. Bar index indicates corresponding EM sections. d, Immunofluorescence image corresponding to slice 11 through the three-dimensional model in b. e, Electron micrograph corresponding to slice 42 through the three-dimensional model in c (nucleus, blue outline; green circles, approximate positions of centre of masses from b). f, A confocal plane (z13) with the respective electron micrographs is shown, including insets with increasing magnifications. CHMP4B and CENP-A foci (detected by light microscopy) correlate with unsealed NE (observed on EM sections).

Extended Data Figure 5 ESCRT-III and VPS4 cooperate at foci of the NE–MT intersection during nuclear envelope reassembly.

a, Section from a SIM z-stack of a formaldehyde-fixed HeLa cell labelled as indicated. The Imaris surface three-dimensional renderings (three examples) illustrate holes in the NE with intersecting spindle MTs and CHMP4B localization within the NE holes. The SIM image is representative of at least ten captures. Imaris reconstruction was performed on four areas of intersection between CHMP4B, MTs and ER. b, Deconvolved wide-field images of fixed CHMP4B–eGFP HeLa cells labelled as indicated. Note CHMP4B foci localization in connection with polar MTs (left, middle and upper right panels) and to kinetochore MTs (lower right panel). Images are representative of at least 20 videos. c, Deconvolved wide-field image from live HeLa cells stably expressing mCherry–CENP-A, CHMP4B–eGFP and LSSmKate1–α-tubulin, showing localization of CHMP4B on kinetochore MTs and in close proximity to kinetochores. Image is representative of five videos. d, HeLa cells stably expressing mCherry–CENP-A and CHMP4B–eGFP were imaged after anaphase onset. Number of CHMP4B foci not co-localizing or co-localizing with CENP-A, and total number of CENP-A foci, were quantified and plotted over time. Time = 0 equals onset of CHMP4B recruitment. n = 6 cells. Bars, mean and s.d.

Extended Data Figure 6 ESCRT-III and VPS4 cooperate at foci of the NE–MT intersection during nuclear envelope reassembly.

a, HeLa cells stably expressing CHMP4B–eGFP and mCherry–α-tubulin were transfected with the indicated siRNAs and imaged every 30 s during mitotic exit. Microtubule bundles contacting CHMP4B foci around chromatin discs were tracked and shown in representative galleries. Time = 0 indicates CHMP4B enrichment on the MT bundle. Scale bars, 5 μm. Images and galleries are representative of 23 events for control siRNA; 26 events for CHMP3 siRNA; 18 events for CHMP2A siRNA; 22 events for VPS4A+B siRNA. b, CHMP4B–eGFP/mCherry–α-tubulin HeLa cells stably expressing WT or siRNA 1 resistant CHMP2A were transfected with CHMP2A siRNA 1 and imaged every 30 s during mitotic exit. Spindle MTs reaching CHMP4B were tracked and their time of disappearance after CHMP4B onset was scored and plotted. n = 27 MTs for WT CHMP2A; n = 25 MTs for RES CHMP2A. Tukey whiskers extend from the largest value within 1.5 IQR (interquartile range) of the upper quartile to the smallest value within 1.5 IQR of the lower quartile. ***P < 0.0001 derived from unpaired t-test. c, Deconvolved wide-field images of CHMP4B–eGFP/mCherry–α-tubulin HeLa cells where MT intensity around CHMP4B foci was measures using line plots. Measured values were plotted and Spearman's correlation analysed. Scale bars, 5 μm.

Extended Data Figure 7 ESCRT-III-dependent recruitment of spastin to the reforming NE mediates mitotic spindle disassembly.

a, Deconvolved wide-field image of live HeLa cells stably co-expressing mCherry–spastin M1 with CHMP4B–eGFP, illustrating co-localization. Image is representative of at least five videos. b, As in a, but instead co-expressing the mCherry–spastin M87 allele. Image is representative of at least five videos. c, A section from a SIM z-stack of a formaldehyde fixed HeLa cell labelled as indicated. The Imaris surface three-dimensional renderings (right) illustrate how CHMP4B and spastin embrace the spindle MT. SIM image is representative of five captures. d, ESCRT-III-dependent localization of spastin shown by confocal imaging of endogenous spastin (red), endogenous CHMP4B (green) and DNA (blue) after siRNA transfections as indicated. Scale bars, 5 μm. Images are representative of 30 cells for control siRNA and 30 cells for CHMP4B siRNA. e, Quantification of endogenous spastin fluorescence intensity around anaphase chromatin in cells treated and immunolabelled as in d. n = 30 cells for control siRNA; n = 30 cells for CHMP4B siRNA. Bars, mean and s.d. ***P < 0.0001 derived from unpaired t-test. f, Immmunoblots show efficient knockdown of endogenous CHMP1B (upper panel) and endogenous IST1 (lower panel). g, Deconvolved wide-field images of live anaphase HeLa cells stably co-expressing mCherry–spastin M87 with CHMP4B–eGFP, transfected with the indicated siRNA. Scale bars, 5 μm. Images are representative of 46 cells for control siRNA; 14 cells for CHMP2A siRNA; 11 cells for CHMP1B siRNA; 31 cells for IST1 siRNA. h, Confocal images of HeLa cells stably co-expressing CHMP1B–Flag with CHMP4B–eGFP, transfected with control or CHMP2A siRNA, fixed and stained for DNA, CHMP4B–eGFP and IST1 (left panel) or CHMP1B–Flag (CHMP1B-FL) (right panel). Scale bars, 5 μm. Images are representative of at least five captures for each condition.

Extended Data Figure 8 ESCRT-III-dependent recruitment of spastin to the reforming NE mediates mitotic spindle disassembly.

a, Immunoblot of whole-cell lysates showing the efficiency of endogenous spastin depletion by the indicated siRNAs. b, Residence time of CHMP4B localization at anaphase chromatin discs is increased in HeLa cells expressing inducible spastin M87E442Q allele in addition to CHMP4B–eGFP. n = 17 cells for control (non-induced); n = 19 cells for induced spastin M87E442Q. Bars, mean with 95% confidence intervals. ***P < 0.0001 derived from unpaired t-test. c, HeLa cells stably expressing CHMP4B–eGFP were imaged every 20 s after anaphase onset. Taxol or DMSO was added in early anaphase. The percentage of cells with CHMP4B localized at chromatin discs was plotted over time. n = 9 cells for DMSO; n = 12 cells for Taxol. P < 0.0001 derived from unpaired t-test.

Extended Data Figure 9 ESCRT-III dysfunction compromises nuclear integrity and leads to DNA damage and cell cycle arrest.

a, hTERT RPE1 cells were transfected with the indicated siRNAs. Cells were then fixed and labelled for γ-H2AX and DNA. Images were collected on a high-content ScanR microscope and the mean fluorescence intensity of γ-H2AX foci was measured and the percentage of the population plotted. n = 3,414 cells for control siRNA; n = 2,320 for CHMP2A siRNA; n = 3,410 for VPS4A+B siRNA. Tukey whiskers extend from the largest value within 1.5 IQR of the upper quartile to the smallest value within 1.5 IQR of the lower quartile. ***P < 0.0001 derived from unpaired t-test. b, Confocal image of HeLa cells depleted for CHMP2A fixed and labelled for nuclear pore complex (anti-pan NPC) and 53BP1. Scale bar, 5 μm. Image is representative of six captures. c, Immunoblot showing efficient knockdown of CHMP2A, VPS4A, VPS4B and CEP55 in hTERT RPE1 cells. Asterisk indicates non-specific immunoreactivity. d, Confocal images of hTERT RPE1 cells that were fixed 1 h or 5 h after exposure to ionizing radiation. Cells are labelled for DNA, γ-H2AX and CHMP4B. Scale bar, 5 μm. Images are representative of five captures for control; seven captures for 2 Gy, 1 h; eight captures for 2 Gy, 5 h. e, Model for coordination of spindle disassembly and nuclear envelope sealing by ESCRT-III, VPS4 and spastin. During anaphase, ESCRT-III is recruited by CHMP7 to sites where the reforming NE engulfs spindle MTs. ESCRT-III recruits spastin M1 that is embedded in the engulfing NE as well as cytosolic spastin M87, which together promote MT disassembly. As such, ESCRT-III, VPS4 and spastin cooperate to coordinate progressive membrane constriction while severing MTs. Only when the surface of the reforming nucleus is cleared from spindle MTs can NE sealing occur.

Supplementary information

Live cell imaging of HeLa cells stably expressing CHMP4B-eGFP show transient localization of CHMP4B around chromatin discs during anaphase.

Live cell imaging of HeLa cells stably expressing CHMP4B-eGFP show transient localization of CHMP4B around chromatin discs during anaphase. (MOV 1160 kb)

Live cell imaging of HeLa cells stably expressing mCherry-KDEL and CHMP4B-eGFP show recruitment of CHMP4B to the reforming NE.

Live cell imaging of HeLa cells stably expressing mCherry-KDEL and CHMP4B-eGFP show recruitment of CHMP4B to the reforming NE. (MOV 581 kb)

Live cell imaging of HeLa cells stably expressing wt CHMP4B-eGFP or 4DE CHMP4B-eGFP show recruitment around anaphase chromatin of the wt allele, but not of the 4DE mutant allele.

Live cell imaging of HeLa cells stably expressing wt CHMP4B-eGFP or 4DE CHMP4B-eGFP show recruitment around anaphase chromatin of the wt allele, but not of the 4DE mutant allele. (MOV 580 kb)

Live cell imaging of HeLa cells stably expressing CHMP4B-eGFP show that CHMP7 knockdown abolishes recruitment of CHMP4B around anaphase chromatin.

Live cell imaging of HeLa cells stably expressing CHMP4B-eGFP show that CHMP7 knockdown abolishes recruitment of CHMP4B around anaphase chromatin. (MOV 712 kb)

Live cell imaging of HeLa cells stably expressing CHMP4B-eGFP show prolonged residency time of CHMP4B around anaphase chromatin upon CHMP3, CHMP2A and VPS4A+B knockdown compared to control.

Live cell imaging of HeLa cells stably expressing CHMP4B-eGFP show prolonged residency time of CHMP4B around anaphase chromatin upon CHMP3, CHMP2A and VPS4A+B knockdown compared to control. (MOV 2586 kb)

Live cell imaging of CHMP2A depleted HeLa cells stably expressing CHMP4B-eGFP and H2B-mCherry show striking persistency of CHMP4B at chromatin.

Live cell imaging of CHMP2A depleted HeLa cells stably expressing CHMP4B-eGFP and H2B-mCherry show striking persistency of CHMP4B at chromatin. (MOV 7166 kb)

Live cell imaging of HeLa cells stably expressing mCherry-CENP-A and CHMP4B-eGFP show dynamic localization of CHMP4B at different regions of the chromatin disc.

Live cell imaging of HeLa cells stably expressing mCherry-CENP-A and CHMP4B-eGFP show dynamic localization of CHMP4B at different regions of the chromatin disc (first at the rims, then at the core where it localized in juxtaposition with kinetochores). (MOV 973 kb)

Live cell imaging of cells stably expressing CHMP4B-eGFP and H2B-mCherry which undergo mitotic slippage in the presence (Taxol) or not (nocodazole) of microtubules.

Live cell imaging of cells stably expressing CHMP4B-eGFP and H2B-mCherry which undergo mitotic slippage in the presence (Taxol) or not (nocodazole) of microtubules. CHMP4B is recruited to chromatin only in the presence of microtubules. (MOV 1288 kb)

Live cell imaging of HeLa cells stably expressing CHMP4B-eGFP fixed for EM at an identical time point after anaphase onset.

Live cell imaging of HeLa cells stably expressing CHMP4B-eGFP fixed for EM at an identical time point after anaphase onset. At the time of fixation, control cells no longer displayed CHMP4B staining whereas CHMP3 knockdown cells did. (MOV 1036 kb)

Live cell imaging of HeLa cells stably expressing CHMP4B-eGFP showing increased residence time of CHMP4B upon microtubule stabilization (Taxol).

Live cell imaging of HeLa cells stably expressing CHMP4B-eGFP showing increased residence time of CHMP4B upon microtubule stabilization (Taxol). (MOV 1018 kb)

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Vietri, M., Schink, K., Campsteijn, C. et al. Spastin and ESCRT-III coordinate mitotic spindle disassembly and nuclear envelope sealing. Nature 522, 231–235 (2015). https://doi.org/10.1038/nature14408

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