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Mol Cell. 2014 Mar 6;53(5):843-53. doi: 10.1016/j.molcel.2014.01.031. Epub 2014 Feb 27.

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

Author information

1
Department of Cell and Molecular Biology, Karolinska Institutet, von Eulers väg 3, 171 77 Stockholm, Sweden.
2
Department of Medical Oncology, Department of Molecular Cancer Research, and Cancer Genomics Centre, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, the Netherlands; Division of Cancer Research, Medical Research Institute, University of Dundee, James Arrot Drive, Dundee DD1 9NT, UK.
3
Department of Medical Oncology and Cancer Genomics Center, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, the Netherlands.
4
Department of Medical Oncology, Department of Molecular Cancer Research, and Cancer Genomics Centre, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, the Netherlands.
5
Department of Medical Oncology and Cancer Genomics Center, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, the Netherlands; Division of Cell Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands.
6
Department of Cell and Molecular Biology, Karolinska Institutet, von Eulers väg 3, 171 77 Stockholm, Sweden; Department of Medical Oncology and Cancer Genomics Center, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, the Netherlands. Electronic address: arne.lindqvist@ki.se.

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 of fluorescence 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.

PMID:
24582498
DOI:
10.1016/j.molcel.2014.01.031
[Indexed for MEDLINE]
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