Letter | Published:

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

Nature volume 522, pages 231235 (11 June 2015) | Download Citation

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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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

    • Kay O. Schink
    •  & Coen Campsteijn

    These authors contributed equally to this work.

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, N-7491 Trondheim, 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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Coen Campsteijn or Harald Stenmark.

Extended data

Supplementary information

Videos

  1. 1.

    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.

  2. 2.

    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.

  3. 3.

    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.

  4. 4.

    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.

  5. 5.

    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.

  6. 6.

    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.

  7. 7.

    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).

  8. 8.

    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.

  9. 9.

    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.

  10. 10.

    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).

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DOI

https://doi.org/10.1038/nature14408

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