In vitro reconstitution of branched 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 - Tu bulin R ing C omplex ( γ -TuRC) 1-4 , 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 “branched” MT nucleation 5-9 . 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 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.