In vitro reconstitution of branching microtubule nucleation

• Published in: eLife Vol. 9
• Main Authors: Tariq, Ammarah, Green, Lucy, Jeynes, J Charles G, Soeller, Christian, Wakefield, James G
• Format: Journal Article
• Language: English
• Published: England eLife Science Publications, Ltd 14.01.2020; eLife Sciences Publications Ltd; eLife Sciences Publications, Ltd
• Subjects: Animals; Cell Biology; Cell Cycle; Cell Cycle Proteins - metabolism; Cell division; Chromosomes; Chromosomes - metabolism; Drosophila; Drosophila melanogaster - embryology; Drosophila Proteins - metabolism; Embryos; Gene Expression Regulation, Developmental; Glycerol; Green Fluorescent Proteins - metabolism; In Vitro Techniques; Insects; Light microscopy; Microscopy; microtubule; Microtubule-Associated Proteins - metabolism; Microtubule-Organizing Center - metabolism; Microtubules - metabolism; Mitosis; mitotic spindle; Novels; Nucleation; Polymerization; Protein Binding; Proteins; Sedimentation & deposition; Sediments; Short Report; Spindle Apparatus - metabolism; Tubulin; Tubulin - metabolism; United Kingdom; microtubule; cell biology; D. melanogaster; mitosis; mitotic spindle
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/eLife.49769

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.