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ANCHR mediates Aurora-B-dependent abscission checkpoint control through retention of VPS4

Nature Cell Biology volume 16, pages 547557 (2014) | Download Citation

Abstract

During the final stage of cell division, cytokinesis, the Aurora-B-dependent abscission checkpoint (NoCut) delays membrane abscission to avoid DNA damage and aneuploidy in cells with chromosome segregation defects. This arrest depends on Aurora-B-mediated phosphorylation of CHMP4C, a component of the endosomal sorting complex required for transport (ESCRT) machinery that mediates abscission, but the mechanism remains unknown. Here we describe ANCHR (Abscission/NoCut Checkpoint Regulator; ZFYVE19) as a key regulator of the abscission checkpoint, functioning through the most downstream component of the ESCRT machinery, the ATPase VPS4. In concert with CHMP4C, ANCHR associates with VPS4 at the midbody ring following DNA segregation defects to control abscission timing and prevent multinucleation in an Aurora-B-dependent manner. This association prevents VPS4 relocalization to the abscission zone and is relieved following inactivation of Aurora B to allow abscission. We propose that the abscission checkpoint is mediated by ANCHR and CHMP4C through retention of VPS4 at the midbody ring.

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Acknowledgements

A. Engen and H.P. Bjønnes are acknowledged for expert handling of cell cultures, and T. Høiby and C. Herrmann for invaluable technical assistance. O. Mjaavatten at the Proteomics Unit at the University of Bergen (PROBE) and G. de Souza at the Proteomics Core Facility Unit at Oslo University Hospital are acknowledged for performing mass spectrometry analyses of GFP–ANCHR and GFP–VPS4 immunoprecipitates. The confocal microscopy core facility of Oslo University Hospital is acknowledged for providing access to microscopes. We also thank D. Gerlich at the Institute of Molecular Biotechnology (IMBA), Vienna, for supplying plasmids for the generation of GFP–α-tubulin+mCherry–H2B stable cell lines, and J. Martin-Serrano, King’s College London, UK, for advice on NoCut activation. S.B.T. and M.V. are PhD students of the South-Eastern Norway Regional Health Authority. C.R. is a senior researcher and C.C. a postdoctoral fellow of the Norwegian Cancer Society. H.S. was supported by 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

Affiliations

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

    • Sigrid B. Thoresen
    • , Coen Campsteijn
    • , Marina Vietri
    • , Kay O. Schink
    • , Knut Liestøl
    • , Camilla Raiborg
    •  & Harald Stenmark
  2. Department of Biochemistry, Institute for Cancer Research, Oslo University Hospital, Montebello, N-0379 Oslo, Norway

    • Sigrid B. Thoresen
    • , Coen Campsteijn
    • , Marina Vietri
    • , Kay O. Schink
    • , Camilla Raiborg
    •  & Harald Stenmark
  3. Department of Informatics, University of Oslo, N-0373 Oslo, Norway

    • Knut Liestøl
  4. Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense, Denmark

    • Jens S. Andersen

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Contributions

S.B.T. generated plasmid constructs, and performed confocal, high-content and live imaging, biochemical work, cell transfections, image processing, data analysis, statistical analyses and preparation of figures. C.C. generated plasmid constructs and stable cell lines, and performed live microscopy, photoconversion experiments, image processing, in vitro kinase assays and data analysis. C.R. carried out and analysed the siRNA screen targeting PtdIns(3)P-binding proteins, and performed cell transfections for epistasis studies. M.V. performed cell transfections, biochemical work and image analysis. K.O.S. generated plasmid constructs and stable cell lines, and performed photoconversion experiments and image processing. K.L. did the statistical analysis of the siRNA screen. J.S.A. performed mass spectrometry analysis of Aurora-B-phosphorylated ANCHR. H.S. coordinated the study and oversaw experiments. S.B.T., C.C. and H.S. wrote the paper. All authors discussed the results and assisted in revising the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Harald Stenmark.

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    Localization of eGFP-Vps4 to the midbody ring before abscission.

    HeLa cells stably expressing near-endogenous levels of eGFP-VPS4 and the midbody marker mCherry-CEP55 were imaged as the cell proceeded through abscission. Note that eGFP-VPS4 colocalizes with mCherry-CEP55 before abscission (t = 12 min).

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https://doi.org/10.1038/ncb2959

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