Drosophila Dgt6 interacts with Ndc80, Msps/XMAP215, and gamma-tubulin to promote kinetochore-driven MT formation

Curr Biol. 2009 Nov 17;19(21):1839-45. doi: 10.1016/j.cub.2009.09.043. Epub 2009 Oct 15.

Abstract

In centrosome-containing cells, spindle assembly relies on microtubules (MTs) nucleated from both centrosomes and chromosomes. Recent work has suggested that additional spindle MTs can be nucleated by gamma-tubulin ring complexes (gamma-TuRCs) that associate laterally with preexisting spindle MTs, leading to spindle amplification. It has been proposed that in Drosophila S2 cells, gamma-TuRCs are anchored to the spindle MTs by augmin, a multiprotein complex that contains at least eight subunits. 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, gamma-tubulin, and Msps/XMAP215. Consistent with these results, Dgt6 coprecipitates with Msps, D-TACC, gamma-tubulin, Ndc80, and Nuf2. However, RNA interference (RNAi)-mediated inhibition of gamma-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 gamma-tubulin and Msps.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromosome Segregation / genetics
  • Chromosomes / metabolism
  • Drosophila / genetics
  • Drosophila / metabolism*
  • Drosophila / ultrastructure
  • Drosophila Proteins / analysis
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism*
  • Drosophila Proteins / physiology*
  • Kinetochores / metabolism*
  • Kinetochores / physiology
  • Microtubule-Associated Proteins / analysis
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism*
  • Microtubule-Associated Proteins / physiology*
  • Microtubules / metabolism*
  • Spindle Apparatus / metabolism
  • Tubulin / metabolism*
  • Tubulin / physiology

Substances

  • Dgt6 protein, Drosophila
  • Drosophila Proteins
  • Microtubule-Associated Proteins
  • Ndc80 protein, Drosophila
  • Tubulin
  • cmet protein, Drosophila
  • msps protein, Drosophila