In vitro reconstitution of branched microtubule nucleation

Abstract

Eukaryotic cell division requires the mitotic spindle, a microtubule (MT)-based 1 structure which accurately aligns and segregates duplicated chromosomes. The 2 dynamics of spindle formation are determined primarily by correctly localising the MT 3 nucleator, g-Tubulin Ring Complex (g-TuRC) 1-4 , within the cell. A conserved MT-4 associated protein complex, Augmin, recruits g-TuRC to pre-existing spindle MTs, 5 amplifying their number, in an essential cellular phenomenon termed "branched" MT 6 nucleation 5-9 . Here, we purify endogenous, GFP-tagged Augmin and g-TuRC from 7Drosophila embryos to near homogeneity using a novel one-step affinity technique. 8We demonstrate that, in vitro, while Augmin alone does not affect Tubulin 9 polymerisation dynamics, it stimulates g-TuRC-dependent MT nucleation in a cell 10 cycle-dependent manner. We also assemble and visualise the MT-Augmin-g-TuRC-11MT junction using light microscopy. Our work therefore conclusively reconstitutes 12 branched MT nucleation. It also provides a powerful synthetic approach with which to 13 investigate the emergence of cellular phenomena, such as mitotic spindle formation, 14 from component parts. 15 16 17 18 19Branched MT nucleation, and its dependence on Augmin and g-TuRC, generates the 20 bulk of MTs required for both meiotic and mitotic spindle formation 10,11 and has been 21 visualised in vivo in Drosophila, Xenopus, plants, and humans 5,6,[9][10][11][12][13][14] 22 understanding, and in vitro reconstitution of, this phenomenon has been hampered