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

Institute of Biochemistry, Swiss Federal Institute of Technology Zurich (ETHZ), Schafmattstrasse 18, CH-8093 Zurich, Switzerland.
Nature Cell Biology (Impact Factor: 19.68). 09/2010; 12(9):886-93. DOI: 10.1038/ncb2092
Source: PubMed


When vertebrate cells exit mitosis various cellular structures are re-organized to build functional interphase cells. This depends on Cdk1 (cyclin dependent kinase 1) inactivation and subsequent dephosphorylation of its substrates. Members of the protein phosphatase 1 and 2A (PP1 and PP2A) families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit, 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-B55alpha 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-B55alpha functions downstream of Cdk1 inactivation. PP2A-B55alpha 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-beta1, and RNAi depletion of importin-beta1 delayed mitotic exit synergistically with PP2A-B55alpha. This demonstrates that PP2A-B55alpha and importin-beta1 cooperate in the regulation of postmitotic assembly mechanisms in human cells.

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    • "Interestingly, in yeast, hypertonic stress can promote activation of Cdc14 phosphatase (109), which then dephosphorylates Cdk substrates driving cells out of mitosis, suggesting that phosphatases can drive slippage. However, in humans the role of Cdc14 is not conserved (110), and PP2A appears to be the primary phosphatase responsible for removing mitotic Cdk1 phosphorylations (111, 112). If PP2A is directly activated in response to hypertonic stress it could promote mitotic slippage in human cells, providing a rational for future research focusing on the effectiveness of PP2A inhibitors in combination with mitotic chemotherapies. "
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    • "Machine learning in cell biology – conclusions and outlook Machine learning has tremendous power in the analysis of largescale microscopic image data. Some representative examples for typical machine-learning applications are screens for mitotic regulators (Kittler et al., 2004; Neumann et al., 2010; Schmitz et al., 2010; Wurzenberger et al., 2012), control of cellular stress responses (Wippich et al., 2013), factors involved in ribosome biogenesis (Wild et al., 2010) and cellular host factors involved in virus infection (Mercer et al., 2012). Unsupervised machine learning has been used, for example, to study the heterogeneity of cell responses to diverse drugs (Loo et al., 2009; Singh et al., 2010), to construct genetic interaction profiles (Horn et al., 2011) and for automatic staging of mitotic progression (Zhong et al., 2012). "
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    • "However, this hypothesis and its functional relevance for cell cycle progression have not yet been studied. Additionally, the scaffold subunit of PP2A physically and functionally interacts with the nuclear import factor importin β1 during mitosis, which could also be part of another unidentified PP2A regulatory mechanism (42). "
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    ABSTRACT: To maintain cellular homeostasis against the demands of the extracellular environment, a precise regulation of kinases and phosphatases is essential. In cell cycle regulation mechanisms, activation of the cyclin-dependent kinase (CDK1) and cyclin B complex (CDK1:cyclin B) causes a remarkable change in protein phosphorylation. Activation of CDK1:cyclin B is regulated by two auto-amplification loops-CDK1:cyclin B activates Cdc25, its own activating phosphatase, and inhibits Wee1, its own inhibiting kinase. Recent biological evidence has revealed that the inhibition of its counteracting phosphatase activity also occurs, and it is parallel to CDK1:cyclin B activation during mitosis. Phosphatase regulation of mitotic kinases and their substrates is essential to ensure that the progression of the cell cycle is ordered. Outlining how the mutual control of kinases and phosphatases governs the localization and timing of cell division will give us a new understanding about cell cycle regulation. [BMB Reports 2013; 46(6): 289-294].
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