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In Vitro Reconstitution Assays of Microtubule Amplification and Lattice Repair by the Microtubule-Severing Enzymes Katanin and Spastin

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Cytoskeleton Dynamics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2101))

Abstract

Microtubules are non-covalent dynamic polymers essential for the life of all eukaryotic cells. Their dynamic behavior is regulated by a large array of cellular effectors. In vitro microtubule assays have been instrumental in dissecting the mechanism of microtubule-associated proteins. In this chapter, we focus on microtubule-severing enzymes katanin and spastin. They are AAA ATPases that generate internal breaks in microtubules by extracting tubulin dimers out of the microtubule lattice. We present protocols for TIRF microscopy-based assays that were instrumental in proving that these enzymes not only sever microtubules but also remodel the microtubule lattice by promoting the exchange of lattice GDP-tubulin with GTP-tubulin from the soluble pool. This activity can modulate microtubule dynamics and support microtubule-dependent microtubule amplification in the absence of a nucleating factor.

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Acknowledgment

A.R.M. is supported by the intramural programs of the National Institute of Neurological Disorders and Stroke (NINDS) and the National, Heart, Lung, and Blood Institute (NHLBI).

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Authors and Affiliations

  1. Cell Biology and Biophysics Unit, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, Bethesda, MD, USA

    Annapurna Vemu, Ewa Szczesna & Antonina Roll-Mecak

  2. Biochemistry and Biophysics Center, National Heart, Lung and Blood Institute, Bethesda, MD, USA

    Antonina Roll-Mecak

Authors
  1. Annapurna Vemu
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  2. Ewa Szczesna
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  3. Antonina Roll-Mecak
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Corresponding author

Correspondence to Antonina Roll-Mecak .

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Editors and Affiliations

  1. i3S University of Porto, Porto, Portugal

    Helder Maiato

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Vemu, A., Szczesna, E., Roll-Mecak, A. (2020). In Vitro Reconstitution Assays of Microtubule Amplification and Lattice Repair by the Microtubule-Severing Enzymes Katanin and Spastin. In: Maiato, H. (eds) Cytoskeleton Dynamics. Methods in Molecular Biology, vol 2101. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0219-5_3

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  • DOI: https://doi.org/10.1007/978-1-0716-0219-5_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0218-8

  • Online ISBN: 978-1-0716-0219-5

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