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In Vitro Reconstitution of Microtubule Dynamics and Severing Imaged by Label-Free Interference-Reflection Microscopy

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Microtubules

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2430))

Abstract

The dynamic architecture of the microtubule cytoskeleton is crucial for cell division, motility and morphogenesis. The dynamic properties of microtubules—growth, shrinkage, nucleation, and severing—are regulated by an arsenal of microtubule-associated proteins (MAPs). The activities of many of these MAPs have been reconstituted in vitro using microscope assays. As an alternative to fluorescence microscopy, interference-reflection microscopy (IRM) has been introduced as an easy-to-use, wide-field imaging technique that allows label-free visualization of microtubules with high contrast and speed. IRM circumvents several problems associated with fluorescence microscopy including the high concentrations of tubulin required for fluorescent labeling, the potential perturbation of function caused by the fluorophores, and the risks of photodamage. IRM can be implemented on a standard epifluorescence microscope at low cost and can be combined with fluorescence techniques like total-internal-reflection-fluorescence (TIRF) microscopy. Here we describe the experimental procedure to image microtubule dynamics and severing using IRM , providing practical tips and guidelines to resolve possible experimental hurdles.

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Acknowledgments

We thank Dr. Mohammed Mahamdeh and Dr. Anna Luchniak for comments and discussions on the manuscript. This work was supported by NIH grants R01 GM139337, DP1 MH110065, and R01 NS118884 (to J.H.) and a fellowship from the Ministry of Education in Taiwan (to Y-W.K.).

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Authors and Affiliations

  1. Department of Chemistry, Yale University, New Haven, CT, USA

    Yin-Wei Kuo

  2. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

    Yin-Wei Kuo & Jonathon Howard

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  1. Yin-Wei Kuo
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  2. Jonathon Howard
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Correspondence to Jonathon Howard .

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Editors and Affiliations

  1. Dept Chemistry and Biotechnology, Tottori University, Tottori, Japan

    Hiroshi Inaba

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Kuo, YW., Howard, J. (2022). In Vitro Reconstitution of Microtubule Dynamics and Severing Imaged by Label-Free Interference-Reflection Microscopy. In: Inaba, H. (eds) Microtubules. Methods in Molecular Biology, vol 2430. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1983-4_5

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  • DOI: https://doi.org/10.1007/978-1-0716-1983-4_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1982-7

  • Online ISBN: 978-1-0716-1983-4

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