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Analysis of Mitotic Protein Dynamics and Function in Drosophila Embryos by Live Cell Imaging and Quantitative Modeling

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Mitosis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1136))

Abstract

Mitosis depends upon the mitotic spindle, a dynamic protein machine that uses ensembles of dynamic microtubules (MTs) and MT-based motor proteins to assemble itself, control its own length (pole–pole spacing), and segregate chromosomes during anaphase A (chromosome-to-pole motility) and anaphase B (spindle elongation). In this review, we describe how the molecular and biophysical mechanisms of these processes can be analyzed in the syncytial Drosophila embryo by combining (1) time-lapse imaging and other fluorescence light microscopy techniques to study the dynamics of mitotic proteins such as tubulins, mitotic motors, and chromosome or centrosome proteins; (2) the perturbation of specific mitotic protein function using microinjected inhibitors (e.g., antibodies) or mutants to infer protein function; and (3) mathematical modeling of the qualitative models derived from these experiments, which can then be used to make predictions which are in turn tested experimentally. We provide details of the methods we use for embryo preparation, fluorescence imaging, and mathematical modeling.

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Acknowledgements

This protocol is currently used in our laboratory and has been refined over the years by many people including Drs. David Sharp, Mijung Kwon, Patrizia Sommi, and Dhanya Cheerambathur. We thank Dr. Bill Sullivan (UCSC), who provided us with excellent advice on the manipulation and microinjection of early Drosophila embryos when our work in this system was being initiated. We thank all members of the Scholey laboratory. Our work on mitosis in Drosophila is supported by NIH grant GM55507.

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Authors and Affiliations

  1. Department of Molecular and Cellular Biology, University of California, Davis, One Shields Avenue, Davis, CA, 95616, USA

    Ingrid Brust-Mascher, Gul Civelekoglu-Scholey & Jonathan M. Scholey

Authors
  1. Ingrid Brust-Mascher
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  2. Gul Civelekoglu-Scholey
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  3. Jonathan M. Scholey
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Corresponding author

Correspondence to Jonathan M. Scholey .

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Editors and Affiliations

  1. Dept Physiology Biophysics, Albert Einstein College of Medicine, Bronx, New York, USA

    David J. Sharp

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Brust-Mascher, I., Civelekoglu-Scholey, G., Scholey, J.M. (2014). Analysis of Mitotic Protein Dynamics and Function in Drosophila Embryos by Live Cell Imaging and Quantitative Modeling. In: Sharp, D. (eds) Mitosis. Methods in Molecular Biology, vol 1136. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0329-0_1

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  • DOI: https://doi.org/10.1007/978-1-4939-0329-0_1

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0328-3

  • Online ISBN: 978-1-4939-0329-0

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