Drosophila Dgt6 interacts with Ndc80, Msps/XMAP215, and gamma-tubulin to promote kinetochore-driven MT formation.
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.
Bucciarelli
Elisabetta
E
Istituto Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del Consiglio Nazionale delle Ricerche (CNR), Dipartimento di Genetica e Biologia Molecolare, Università di Roma "La Sapienza," P.A. Moro 5, 00185 Roma, Italy.
Pellacani
Claudia
C
Naim
Valeria
V
Palena
Antonella
A
Gatti
Maurizio
M
Somma
Maria Patrizia
MP
D000818:Animals; D020090:Chromosome Segregation / Q000235:genetics; D002875:Chromosomes / Q000378:metabolism; D004330:Drosophila / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029721:Drosophila Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D018386:Kinetochores / Q000378:metabolism* / Q000502:physiology; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D008870:Microtubules / Q000378:metabolism*; D008941:Spindle Apparatus / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism* / Q000502:physiology
C547531:Dgt6 protein, Drosophila; D029721:Drosophila Proteins; D008869:Microtubule-Associated Proteins; C547532:Ndc80 protein, Drosophila; D014404:Tubulin; C411730:cmet protein, Drosophila; C121511:msps protein, Drosophila