Assessing kinetics from fixed cells reveals activation of the mitotic entry network at the S/G2 transition

Karen Akopyan, Helena SilvaCascales, Elvira Hukasova, Adrian T. Saurin, Erik Müllers, Himjyot Jaiswal, Danielle A A Hollman, Geert J P L Kops, RenéH Medema, Arne Lindqvist

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    Abstract

    During the cell cycle, DNA duplication in S phase must occur before a cell divides in mitosis. In the intervening G2 phase, mitotic inducers accumulate, which eventually leads to a switch-like rise in mitotic kinase activity that triggers mitotic entry. However, when and how activation of the signaling network that promotes the transition to mitosis occurs remains unclear. We have developed a system to reduce cell-cell variation and increase accuracy offluorescence quantification in single cells. This allows us to use immunofluorescence of endogenous marker proteins to assess kinetics from fixed cells. We find that mitotic phosphorylations initially occur at the completion of S phase, showing that activation of the mitotic entry network does not depend on protein accumulation through G2. Our data show insights into how mitotic entry is linked to the completion of S phase and forms a quantitative resource for mathematical models of the human cell cycle.

    Original languageEnglish
    Pages (from-to)843-853
    Number of pages11
    JournalMolecular Cell
    Volume53
    Issue number5
    Early online date27 Feb 2014
    DOIs
    Publication statusPublished - 6 Mar 2014

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    Akopyan, K., SilvaCascales, H., Hukasova, E., Saurin, A. T., Müllers, E., Jaiswal, H., Hollman, D. A. A., Kops, G. J. P. L., Medema, R., & Lindqvist, A. (2014). Assessing kinetics from fixed cells reveals activation of the mitotic entry network at the S/G2 transition. Molecular Cell, 53(5), 843-853. https://doi.org/10.1016/j.molcel.2014.01.031