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Elucidating Human Mitosis Using an Anaphase-Like Cell-Free System

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Cell Cycle Oscillators

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2329))

Abstract

A balanced progression through mitosis and cell division is largely dependent on orderly phosphorylation and ubiquitin-mediated proteolysis of regulatory and structural proteins. These series of events ultimately secure genome stability and time-invariant cellular properties during cell proliferation. Two of the core enzymes regulating mitotic milestones in all eukaryotes are cyclin dependent kinase 1 (CDK1) with its coactivator cyclin B, and the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Discovering mechanisms and substrates for these enzymes is vital to understanding how cells move through mitosis and segregate chromosomes with high fidelity. However, the study of these enzymes has significant challenges. Purely in vitro studies discount the contributions of yet to be described regulators and misses the physiological context of cellular environment. In vivo studies are complicated by the fact that each of these enzymes, as well as many of their regulators and downstream targets, are essential. Moreover, long-term in vivo manipulations can result in cascading, indirect effects that can distort data analysis and interpretation. Many of these challenges can be circumvented using cell-free systems, which have historically played a critical role in identifying these enzymes and their contributions under quasicellular environments. Here, we describe the preparation of a newly developed human cell-free system that recapitulates an anaphase-like state of human cells. This new toolkit complements traditional cell-free systems from human cells and frog eggs and can be easily implemented in cell biology labs for direct and quantitative studies of mitotic signaling regulated by phosphorylation, APC/C-mediated proteolysis, and beyond.

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Acknowledgments

This study was by the Israel Cancer Research Fund (ICRF), Grant no. RCDA00102, and the Israel Science Foundation (ISF) Grant no. 659/16 and 2038/19. The Emanuele lab is supported by funds from the UNC University Cancer Research Fund, National Institutes of Health (R01GM120309, R01GM134231), American Cancer Society (RSG-18-220-01-TBG) and donations from the Brookside Foundation.

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Authors and Affiliations

  1. Faculty of Life Sciences and Institute of Nanotechnology and Advanced Materials, Bar-llan University, Ramat-Gan, Israel

    Danit Wasserman, Sapir Nachum, Meirav Noach-Hirsh, Naomi Auerbach, Evelin Sheinberger-Chorni, Roxane Lahmi & Amit Tzur

  2. Lineberger Comprehensive Cancer Center, Department of Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Taylor P. Enrico & Michael J. Emanuele

Authors
  1. Danit Wasserman
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  2. Sapir Nachum
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  3. Meirav Noach-Hirsh
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  4. Naomi Auerbach
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  5. Evelin Sheinberger-Chorni
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  6. Taylor P. Enrico
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  7. Roxane Lahmi
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  8. Michael J. Emanuele
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  9. Amit Tzur
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Corresponding author

Correspondence to Amit Tzur .

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Editors and Affiliations

  1. School of Science and Technology, Nottingham Trent University, Nottingham, UK

    Amanda S. Coutts

  2. Scientific Writer, Newcastle upon Tyne, UK

    Louise Weston

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Wasserman, D. et al. (2021). Elucidating Human Mitosis Using an Anaphase-Like Cell-Free System. In: Coutts, A.S., Weston, L. (eds) Cell Cycle Oscillators . Methods in Molecular Biology, vol 2329. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1538-6_11

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  • DOI: https://doi.org/10.1007/978-1-0716-1538-6_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1537-9

  • Online ISBN: 978-1-0716-1538-6

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