Abstract
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.
Keywords:
D. melanogaster; cell biology; microtubule; mitosis; mitotic spindle.
© 2020, Tariq et al.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Cell Cycle
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Cell Cycle Proteins / metabolism
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Chromosomes / metabolism
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Drosophila Proteins / metabolism
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Drosophila melanogaster / embryology*
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Gene Expression Regulation, Developmental
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Green Fluorescent Proteins / metabolism
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In Vitro Techniques
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Microtubule Proteins / metabolism
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Microtubule-Associated Proteins / metabolism
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Microtubule-Organizing Center / metabolism
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Microtubules / metabolism*
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Mitosis
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Multiprotein Complexes / metabolism
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Protein Binding
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Spindle Apparatus / metabolism
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Tubulin / metabolism*
Substances
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Cell Cycle Proteins
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Drosophila Proteins
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Microtubule Proteins
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Microtubule-Associated Proteins
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Multiprotein Complexes
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Tubulin
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augmin protein, Drosophila
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Green Fluorescent Proteins
Grants and funding
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.