| dc.description |
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, I purify endogenous, GFP-tagged Augmin and γ-TuRC from Drosophila embryos to near homogeneity using a novel one-step affinity technique I term cl-AP (cleavable Affinity Purification) and demonstrate their direct functional relationship by the reconstitution of branched MT nucleation in vitro. I show that branched MT nucleation requires only γ-TuRC, Augmin and Tubulin and that it is cell cycle dependent. I also reconstitute the MT-Augmin- γ-TuRC-MT junction. I have identified mitosis-specific phosphorylation sites on two Augmin subunits, Msd5 and Dgt6, and demonstrated that mutant Augmin, purified from embryos expressing the phospho-deficient mutant of Dgt6, suppresses γ-TuRC dependent MT nucleation and branching. Finally, I have shown that expression of the phospho-mutant Dgt6/Augmin in the syncytial Drosophila embryo results in mitotic defects such as chromosomal aneuploidy and abnormal spindle formation. Together, my work demonstrates the synergistic and spatial action of Augmin on γ-TuRC-dependent MT generation, highlighting the importance of Augmin in faithful chromosome segregation during mitosis. More broadly, by developing and optimising cl-AP to isolate intact, functional protein complexes from their endogenous source, for in vitro analysis I anticipate this approach will provide a powerful new tool with which to understand biological process. |
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