Live-cell imaging RNAi screen identifies PP2A-B55 alpha and importin-beta 1 as key mitotic exit regulators in human cells

Michael H. A. Schmitz, Michael Held, Veerle Janssens, James R. A. Hutchins, Otto Hudecz, Elitsa Ivanova, Jozef Goris, Laura Trinkle-Mulcahy, Angus I. Lamond, Ina Poser, Anthony A. Hyman, Karl Mechtler, Jan-Michael Peters, Daniel W. Gerlich

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    218 Citations (Scopus)

    Abstract

    When vertebrate cells exit mitosis various cellular structures are re-organized to build functional interphase cells(1). This depends on Cdk1 (cyclin dependent kinase 1) inactivation and subsequent dephosphorylation of its substrates(2-4). Members of the protein phosphatase 1 and 2A (PP1 and PP2A) families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit(5,6), 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-B55 alpha 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-B55 alpha functions downstream of Cdk1 inactivation. PP2A-B55 alpha 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-beta 1, and RNAi depletion of importin-beta 1 delayed mitotic exit synergistically with PP2A-B55 alpha. This demonstrates that PP2A-B55 alpha and importin-beta 1 cooperate in the regulation of postmitotic assembly mechanisms in human cells.

    Original languageEnglish
    Pages (from-to)886-893
    Number of pages8
    JournalNature Cell Biology
    Volume12
    Issue number9
    DOIs
    Publication statusPublished - Sep 2010

    Keywords

    • XENOPUS EGG EXTRACTS
    • CHROMOSOME SEGREGATION
    • PROTEIN PHOSPHATASE-2A
    • VERTEBRATE CELLS
    • LIVING CELLS
    • MITOSIS
    • DROSOPHILA
    • SUBUNIT
    • DEPHOSPHORYLATION
    • CALCINEURIN

    Cite this

    Schmitz, M. H. A., Held, M., Janssens, V., Hutchins, J. R. A., Hudecz, O., Ivanova, E., Goris, J., Trinkle-Mulcahy, L., Lamond, A. I., Poser, I., Hyman, A. A., Mechtler, K., Peters, J-M., & Gerlich, D. W. (2010). Live-cell imaging RNAi screen identifies PP2A-B55 alpha and importin-beta 1 as key mitotic exit regulators in human cells. Nature Cell Biology, 12(9), 886-893. https://doi.org/10.1038/ncb2092