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Synchronization and Desynchronization of Cells by Interventions on the Spindle Assembly Checkpoint

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Book cover Cell Cycle Synchronization

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1524))

Abstract

Cell cycle checkpoints are surveillance mechanisms that sequentially and continuously monitor cell cycle progression thereby contributing to the preservation of genetic stability. Among them, the spindle assembly checkpoint (SAC) prevents the occurrence of abnormal divisions by halting the metaphase to anaphase transition following the detection of erroneous microtubules-kinetochore attachment(s). Most synchronization strategies are based on the activation of cell cycle checkpoints to enrich the population of cells in a specific phase of the cell cycle. Here, we develop a two-step protocol of sequential cell synchronization and desynchronization employing antimitotic SAC-inducing agents (i.e., nocodazole or paclitaxel) in combination with the depletion of the SAC kinase MPS1. We describe cytofluorometric and time-lapse videomicroscopy methods to detect cell cycle progression, including the assessment of cell cycle distribution, quantification of mitotic cell fraction, and analysis of single cell fate profile of living cells. We applied these methods to validate the synchronization-desynchronization protocol and to qualitatively and quantitatively determine the impact of SAC inactivation on the activity of antimitotic agents.

Both authors equally contributed to this chapter.

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Acknowledgements

GM was funded by Associazione Italiana per la Ricerca sul Cancro (AIRC: Triennal Fellowship “Antonietta Latronico”, 2014). IV was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC: MFAG 2013 cod. 14641), Ministero Italiano della Salute (RF_GR-2011-02351355), and the Programma per i Giovani Ricercatori “Rita Levi Montalcini” 2010.

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    Authors and Affiliations

    1. Department of Cardiology, Vascular Medicine and Physiology, University of Tuebingen, Tuebingen, Germany

      Mohamed Jemaà

    2. Regina Elena National Cancer Institute, Rome, Italy

      Gwenola Manic & Ilio Vitale

    3. Department of Biology, University of Rome “Tor Vergata”, Rome, Italy

      Ilio Vitale

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    1. Mohamed Jemaà
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    2. Gwenola Manic
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    3. Ilio Vitale
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    Correspondence to Ilio Vitale .

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    Editors and Affiliations

    1. Department of Biotechnology and Microbiology, University of Debrecen, Debrecen, Hungary

      Gaspar Banfalvi

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    Jemaà, M., Manic, G., Vitale, I. (2017). Synchronization and Desynchronization of Cells by Interventions on the Spindle Assembly Checkpoint. In: Banfalvi, G. (eds) Cell Cycle Synchronization. Methods in Molecular Biology, vol 1524. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6603-5_5

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    • DOI: https://doi.org/10.1007/978-1-4939-6603-5_5

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    • Publisher Name: Humana Press, New York, NY

    • Print ISBN: 978-1-4939-6602-8

    • Online ISBN: 978-1-4939-6603-5

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