In vitro reconstitution of branched microtubule nucleation
Tariq, A.; Green, L.; Jeynes, J. C. G.; Soeller, C.; Wakefield, J.
Show 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,{gamma} -Tubulin Ring Complex ({gamma}-TuRC)1-4, within the cell. A conserved MT-associated protein complex, Augmin, recruits{gamma} -TuRC to pre-existing spindle MTs, amplifying their number, in an essential cellular phenomenon termed \"branched\" MT nucleation5-9. Here, we purify endogenous, GFP-tagged Augmin and{gamma} -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{gamma} -TuRC-dependent MT nucleation in a cell cycle-dependent manner. We also assemble and visualise the MT-Augmin-{gamma}-TuRC-MT junction using light microscopy. Our work therefore conclusively reconstitutes branched 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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