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, g-Tubulin Ring
Complex (g-TuRC), within the cell. A conserved MT-associated ...
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, g-Tubulin Ring
Complex (g-TuRC), within the cell. A conserved MT-associated protein complex, Augmin, recruits g-
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 g-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 g-TuRC-dependent MT nucleation in a cell cycle-dependent manner. We also assemble
and visualise the MT-Augmin-g-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.