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

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Schmitz, Michael H. A.
Held, Michael
Janssens, Veerle
Hutchins, James R. A.
Hudecz, Otto
Ivanova, Elitsa
Goris, Jozef
Trinkle-Mulcahy, Laura
Lamond, Angus I.
Poser, Ina
Hyman, Anthony A.
Mechtler, Karl
Peters, Jan-Michael
Gerlich, Daniel W.
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When vertebrate cells exit mitosis, they reorganize various cellular structures to build functional interphase cells1. This depends on Cdk1 inactivation and subsequent dephosphorylation of its substrates2-4. Members of PP1 and PP2A phosphatase 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 used a live imaging assay to screen by RNAi a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identified a trimeric PP2A-B55α complex as a key factor for postmitotic reassembly of the nuclear envelope, the Golgi apparatus, and decondensed chromatin, as well as for mitotic spindle breakdown. Using a chemically-induced mitotic exit assay, we found 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 it 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.
Author Posting. © The Authors, 2010. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in Nature Cell Biology 12 (2010): 886-893, doi:10.1038/ncb2092.
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