Abstract
To complete cell division and to exit from mitosis into the next G1 phase, eukaryotic cells need to inactivate the cyclin-dependent kinase (Cdk) and reverse Cdk-phosphorylation events. In budding yeast mitotic exit depends on the phosphatase Cdc14. During the majority of the cell cycle Cdc14 is sequestered and kept inactive in the nucleolus. Activation of Cdc14 at anaphase onset coincides with its release from the nucleolus into the nucleus and subsequently into the cytoplasm. Here we describe a microscopy method, originally developed in the laboratory of Frederick Cross (Lu and Cross, Cell 141:268–279, 2010), that allows quantifying Cdc14 release in live cells using the open source software FIJI. We adapted this method and show that it is able to distinguish between Cdc14 activation defects caused by mutations in the “cdcFourteen Early Anaphase Release”-(FEAR) and the mitotic exit network (MEN) using slk19∆ and cdc15-1 mutant strains.
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Acknowledgments
We would like to thank Angelika Amon and Jill Falk for helpful comments, and the CRG Advanced Light Microscopy Unit. Research in the Mendoza laboratory is supported by grants from the European Research Council (ERC Starting Grant 260965) and the Spanish Ministry of Science (BFU09-08213). GN was supported by a Fellowship from La Caixa and SNSF.
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Neurohr, G., Mendoza, M. (2017). Cdc14 Localization as a Marker for Mitotic Exit: In Vivo Quantitative Analysis of Cdc14 Release. In: Monje-Casas, F., Queralt, E. (eds) The Mitotic Exit Network. Methods in Molecular Biology, vol 1505. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6502-1_5
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DOI: https://doi.org/10.1007/978-1-4939-6502-1_5
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