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In vitro reconstitution of branching microtubule nucleation

Eukaryotic cell division requires the mitotic spindle, a microtubule (MT)-based structure which accurately aligns and segregates duplicated chromosomes. The dynamics of spindle formation are determined primarily by correctly localising the MT nucleator, γ-Tubulin Ring Complex (γ-TuRC), within the ce...

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Autores principales: Tariq, Ammarah, Green, Lucy, Jeynes, J Charles G, Soeller, Christian, Wakefield, James G
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959987/
https://www.ncbi.nlm.nih.gov/pubmed/31933481
http://dx.doi.org/10.7554/eLife.49769
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author Tariq, Ammarah
Green, Lucy
Jeynes, J Charles G
Soeller, Christian
Wakefield, James G
author_facet Tariq, Ammarah
Green, Lucy
Jeynes, J Charles G
Soeller, Christian
Wakefield, James G
author_sort Tariq, Ammarah
collection PubMed
description Eukaryotic cell division requires the mitotic spindle, a microtubule (MT)-based structure which accurately aligns and segregates duplicated chromosomes. The dynamics of spindle formation are determined primarily by correctly localising the MT nucleator, γ-Tubulin Ring Complex (γ-TuRC), within the cell. A conserved MT-associated protein complex, Augmin, recruits γ-TuRC to pre-existing spindle MTs, amplifying their number, in an essential cellular phenomenon termed ‘branching’ MT nucleation. Here, we purify endogenous, GFP-tagged Augmin and γ-TuRC from Drosophila embryos to near homogeneity using a novel one-step affinity technique. We demonstrate that, in vitro, while Augmin alone does not affect Tubulin polymerisation dynamics, it stimulates γ-TuRC-dependent MT nucleation in a cell cycle-dependent manner. We also assemble and visualise the MT-Augmin-γ-TuRC-MT junction using light microscopy. Our work therefore conclusively reconstitutes branching MT nucleation. It also provides a powerful synthetic approach with which to investigate the emergence of cellular phenomena, such as mitotic spindle formation, from component parts.
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spelling pubmed-69599872020-01-16 In vitro reconstitution of branching microtubule nucleation Tariq, Ammarah Green, Lucy Jeynes, J Charles G Soeller, Christian Wakefield, James G eLife Cell Biology Eukaryotic cell division requires the mitotic spindle, a microtubule (MT)-based structure which accurately aligns and segregates duplicated chromosomes. The dynamics of spindle formation are determined primarily by correctly localising the MT nucleator, γ-Tubulin Ring Complex (γ-TuRC), within the cell. A conserved MT-associated protein complex, Augmin, recruits γ-TuRC to pre-existing spindle MTs, amplifying their number, in an essential cellular phenomenon termed ‘branching’ MT nucleation. Here, we purify endogenous, GFP-tagged Augmin and γ-TuRC from Drosophila embryos to near homogeneity using a novel one-step affinity technique. We demonstrate that, in vitro, while Augmin alone does not affect Tubulin polymerisation dynamics, it stimulates γ-TuRC-dependent MT nucleation in a cell cycle-dependent manner. We also assemble and visualise the MT-Augmin-γ-TuRC-MT junction using light microscopy. Our work therefore conclusively reconstitutes branching MT nucleation. It also provides a powerful synthetic approach with which to investigate the emergence of cellular phenomena, such as mitotic spindle formation, from component parts. eLife Sciences Publications, Ltd 2020-01-14 /pmc/articles/PMC6959987/ /pubmed/31933481 http://dx.doi.org/10.7554/eLife.49769 Text en © 2020, Tariq et al http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Cell Biology
Tariq, Ammarah
Green, Lucy
Jeynes, J Charles G
Soeller, Christian
Wakefield, James G
In vitro reconstitution of branching microtubule nucleation
title In vitro reconstitution of branching microtubule nucleation
title_full In vitro reconstitution of branching microtubule nucleation
title_fullStr In vitro reconstitution of branching microtubule nucleation
title_full_unstemmed In vitro reconstitution of branching microtubule nucleation
title_short In vitro reconstitution of branching microtubule nucleation
title_sort in vitro reconstitution of branching microtubule nucleation
topic Cell Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959987/
https://www.ncbi.nlm.nih.gov/pubmed/31933481
http://dx.doi.org/10.7554/eLife.49769
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