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Assays to Study Mitotic Centrosome and Spindle Pole Assembly and Regulation

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The Mitotic Spindle

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1413))

Abstract

Faithful chromosome segregation during cell division requires proper bipolar spindle assembly and critically depends on spindle pole integrity. In most animal cells, spindle poles form as the result of the concerted action of various factors operating in two independent pathways of microtubule assembly mediated by chromatin/RanGTP and by centrosomes. Mutation or deregulation of a number of spindle pole-organizing proteins has been linked to human diseases, including cancer and microcephaly. Our knowledge on how the spindle pole-organizing factors function at the molecular level and cooperate with one another is still quite limited. As the list of these factors expands, so does the need for the development of experimental approaches to study their function. Cell-free extracts from Xenopus laevis eggs have played an instrumental role in the dissection of the mechanisms of bipolar spindle assembly and have recently allowed the reconstitution of the key steps of the centrosome-driven microtubule nucleation pathway (Joukov et al., Mol Cell 55:578–591, 2014). Here we describe assays to study both centrosome-dependent and centrosome-independent spindle pole formation in Xenopus egg extracts. We also provide experimental procedures for the use of artificial centrosomes, such as microbeads coated with an anti-Aurora A antibody or a recombinant fragment of the Cep192 protein, to model and study centrosome maturation in egg extract. In addition, we detail the protocol for a microtubule regrowth assay that allows assessment of the centrosome-driven spindle microtubule assembly in mammalian cells.

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Author information

Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Room C1-226A, 240 Longwood Ave., Boston, MA, 02115, USA

    Vladimir Joukov

  2. Department of Biological Chemistry and Molecular Pharmacology, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA

    Johannes C. Walter

  3. Department of Medicine, Harvard Medical School, Boston, MA, USA

    Arcangela De Nicolo

  4. Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA

    Arcangela De Nicolo

Authors
  1. Vladimir Joukov
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  2. Johannes C. Walter
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  3. Arcangela De Nicolo
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Corresponding author

Correspondence to Vladimir Joukov .

Editor information

Editors and Affiliations

  1. Massachusetts Institute of Technolo, Cambridge, Massachusetts, USA

    Paul Chang

  2. Dept of Cell & Developmental Biolog, Vanderbilt University, Nashville, Tennessee, USA

    Ryoma Ohi

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© 2016 Springer Science+Business Media New York

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Joukov, V., Walter, J.C., De Nicolo, A. (2016). Assays to Study Mitotic Centrosome and Spindle Pole Assembly and Regulation. In: Chang, P., Ohi, R. (eds) The Mitotic Spindle. Methods in Molecular Biology, vol 1413. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3542-0_14

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  • DOI: https://doi.org/10.1007/978-1-4939-3542-0_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3540-6

  • Online ISBN: 978-1-4939-3542-0

  • eBook Packages: Springer Protocols

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