Drosophila Dgt6 Interacts with Ndc80, Msps/XMAP215, and γ-Tubulin to Promote Kinetochore-Driven MT Formation

SummaryIn centrosome-containing cells, spindle assembly relies on microtubules (MTs) nucleated from both centrosomes and chromosomes 1 and 2. Recent work has suggested that additional spindle MTs can be nucleated by γ-tubulin ring complexes (γ-TuRCs) that associate laterally with preexisting spindle MTs, leading to spindle amplification. It has been proposed that in Drosophila S2 cells, γ-TuRCs are anchored to the spindle MTs by augmin, a multiprotein complex that contains at least eight subunits 3, 4 and 5. Here we show that the Dgt6 component of augmin is primarily required for kinetochore fiber (k-fiber) formation. An analysis of MT regrowth after cold exposure showed that formation of kinetochore-driven k-fibers is severely impaired in Dgt6-depleted cells. In control cells, these fibers are enriched in Dgt6, γ-tubulin, and Msps/XMAP215. Consistent with these results, Dgt6 coprecipitates with Msps, D-TACC, γ-tubulin, Ndc80, and Nuf2. However, RNA interference (RNAi)-mediated inhibition of γ-tubulin, Msps/XMAP215, or Ndc80/Hec1 reduced but did not abolish k-fiber regrowth. These results indicate that Dgt6 plays a pivotal role in kinetochore-driven k-fiber formation, mediating nucleation and/or initial stabilization of chromosome-induced MTs. We propose that Dgt6 binds and stabilizes nascent chromatin-induced MTs, facilitating their interaction with the Ndc80-Nuf2 complex. Dgt6 may also promote elongation of kinetochore-driven k-fibers through its interaction with γ-tubulin and Msps.