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Live-cell imaging RNAi screen identifies PP2A–B55α and importin-β1 as key mitotic exit regulators in human cells

Nature Cell Biology volume 12, pages 886893 (2010) | Download Citation

Abstract

When vertebrate cells exit mitosis various cellular structures are re-organized to build functional interphase cells1. This depends on Cdk1 (cyclin dependent kinase 1) inactivation and subsequent dephosphorylation of its substrates2,3,4. Members of the protein phosphatase 1 and 2A (PP1 and PP2A) families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit5,6, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we use a live-cell imaging assay and RNAi knockdown to screen a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identify a trimeric PP2A–B55α complex as a key factor in mitotic spindle breakdown and postmitotic reassembly of the nuclear envelope, Golgi apparatus and decondensed chromatin. Using a chemically induced mitotic exit assay, we find that PP2A–B55α functions downstream of Cdk1 inactivation. PP2A–B55α isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate, histone H1, and was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor importin-β1, and RNAi depletion of importin-β1 delayed mitotic exit synergistically with PP2A–B55α. This demonstrates that PP2A–B55α and importin-β1 cooperate in the regulation of postmitotic assembly mechanisms in human cells.

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Acknowledgements

The authors thank F. Uhlmann and B. Novak for critical comments on the manuscript. We thank S. Maar, the ETHZ Light Microscopy Centre (LMC), the ETHZ RNAi Screening Centre (RISC), M. Augsburg (The Max Planck Institute of Molecular Cell Biology and Genetics; MPI-CBG), M. Leuschner (MPI-CBG) and A. Ssykor (MPI-CBG) for technical assistance. We thank U. Kutay (ETHZ) for anti-importin-β1 and anti-nucleolin antibodies, J. Rohrer (University of Zurich) for providing GalT–eGFP plasmid, J. Ellenberg (EMBL, Heidelberg) for IBB–eGFP plasmid, M.C. Cardoso (Technical University, Darmstadt) for eGFP–PCNA plasmid and Sanofi Aventis and the National Cancer Institute for providing flavopiridol. This work was supported by Swiss National Science Foundation (SNF) research grant 3100A0-114120, SNF ProDoc grant PDFMP3_124904, a European Young Investigator (EURYI) award of the European Science Foundation to D.W.G., a MBL Summer Research Fellowship by the Evelyn and Melvin Spiegel Fund to D.W.G., a Roche Ph.D. fellowship to M.H.A.S. and a Mueller fellowship of the Molecular Life Sciences Ph.D. programme Zurich to M.H. M.H. and M.H.A.S. are fellows of the Zurich Ph.D. programme in Molecular Life Sciences. V.J. and J.G. were supported by grants of the 'Geconcerteerde OnderzoeksActies' of the Flemish government, the 'Interuniversitary Attraction Poles' of the Belgian Science Policy P6/28 and the 'Fonds voor Wetenschappelijk Onderzoek-Vlaanderen'. A.I.L. is a Wellcome Trust Principal Research Fellow. A.A.H. acknowledges funding by the Max Planck Society, MitoCheck (the EU-FP6 integrated project), and a BMBF (Bundesministerium für Bildung and Forschung) grant, DiGtoP (01GS0859). Work in the groups of K.M. and J.M.P. was supported by MitoCheck (the EU-FP6 integrated project), Boehringer Ingelheim, the GEN-AU programme of the Austrian Federal Ministry of Science and Research (Austrian Proteomics Platform III), MeioSys within the Seventh Framework Programme of the European Commission and by Chromosome Dynamics, which is funded by the Austrian Science Foundation (FWF).

Author information

Author notes

    • Michael H. A. Schmitz

    Current address: Systems and Cell Biology of Neurodegeneration, Division of Psychiatry Research, University of Zurich, August Forel-Strasse 1, CH-8008 Zurich, Switzerland.

Affiliations

  1. Institute of Biochemistry, Swiss Federal Institute of Technology Zurich (ETHZ), Schafmattstrasse 18, CH-8093 Zurich, Switzerland.

    • Michael H. A. Schmitz
    • , Michael Held
    •  & Daniel W. Gerlich
  2. Marine Biological Laboratory, Woods Hole, MA 02543, USA.

    • Michael H. A. Schmitz
    • , Michael Held
    •  & Daniel W. Gerlich
  3. Laboratory of Protein Phosphorylation and Proteomics, Department of Molecular Cell Biology, Faculty of Medicine, KU Leuven, Gasthuisberg O&N1, Herestraat 49 Box 901, B-3000 Leuven, Belgium.

    • Veerle Janssens
    • , Elitsa Ivanova
    •  & Jozef Goris
  4. Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030 Vienna, Austria.

    • James R. A. Hutchins
    • , Karl Mechtler
    •  & Jan-Michael Peters
  5. Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Dr. Bohr-Gasse 3, 1030 Vienna, Austria.

    • Otto Hudecz
    •  & Karl Mechtler
  6. Department of Cellular & Molecular Medicine and the Ottawa Institute of Systems Biology, University of Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada.

    • Laura Trinkle-Mulcahy
  7. Wellcome Trust Centre for Gene Regulation & Expression, MSI/WTB/JBC Complex, University of Dundee, Dundee, DD1 5EH, UK.

    • Angus I. Lamond
  8. Max-Planck-Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany.

    • Ina Poser
    •  & Anthony A. Hyman

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Contributions

M.H.A.S. performed all experiments, except the mass spectrometry and in vitro phosphatase assays, and wrote part of the paper. M.H. implemented software for automated imaging and data analysis. V.J., E.I. and J.G. performed in vitro phosphatase assays and B55α phospho-mutant analysis. J.H. and J.M.P. designed and performed PP2A purification. K.M. and O.H. performed mass spectrometry. L.T.M. and A.I.L. compiled the phosphatase screening library. I.P. and A.A.H. generated the cell lines stably expressing LAP-tagged PP2A subunits. D.W.G. conceived the project, designed the screening strategy and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Daniel W. Gerlich.

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DOI

https://doi.org/10.1038/ncb2092

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