The highly conserved Ndc80 complex is required for kinetochore assembly, chromosome congression, and spindle checkpoint activity

Abstract

We show that the Xenopus homologs of Ndc80/Tid3/HEC1 (xNdc80) and Nuf2/MPP1/Him-10 (xNuf2) proteins physically interact in a 190-kD complex that associates with the outer kinetochore from prometaphase through anaphase. Injecting function-blocking antibodies to either xNdc80 or xNuf2 into XTC cells caused premature exit from mitosis without detectable chromosome congression or anaphase movements. Injected cells did not arrest in response to microtubule drugs, showing that the complex is required for the spindle checkpoint. Kinetochores assembled in Xenopus extracts after immunodepletion of the complex did not contain xRod, xZw10, xP150 glued (Dynactin), xMad1, xMad2, xBub1, and xBub3, demonstrating that the xNdc80 complex is required for functional kinetochore assembly. In contrast, function-blocking antibodies did not affect the localization of other kinetochore proteins when added to extracts containing previously assembled kinetochores. These extracts with intact kinetochores were deficient in checkpoint signaling, suggesting that the Ndc80 complex participates in the spindle checkpoint. We also demonstrate that the spindle checkpoint can arrest budding yeast cells lacking Ndc80 or Nuf2, whereas yeast lacking both proteins fail to arrest in mitosis. Systematic deletion of yeast kinetochore genes suggests that the Ndc80 complex has a unique role in spindle checkpoint signaling. We propose that the Ndc80 complex has conserved roles in kinetochore assembly, chromosome congression, and spindle checkpoint signaling.

Silencing of NUF2 Inhibits Tumor Growth and Induces Apoptosis in Human Hepatocellular Carcinomas

“…The microtubule-binding interface of the kinetochore, namely the outer kinetochore, is critical for accurate chromosome segregation. As a core component of the outer kinetochore, the NDC80 complex is evolutionarily conserved and essential for stabilization of the kinetochore-microtubule anchoring and supporting the centromeric tension implicated in the establishment of correct chromosome congression (McCleland et al, 2003). The NDC80 complex has four components: NDC80 (also called HEC1 or KNTC2), NUF2 (also called CDCA1), SPC24 and SPC25, which together form a dumbbell-like heterotramer (Cheeseman et al, 2006).…”

SAC‐ing mitotic errors: How the spindle assembly checkpoint (SAC) plays defense against chromosome mis‐segregation

“…Ipl1p/Aurora is a kinetochore-associated protein kinase required for the biorientation of sister chromatids [Pinsky et al, 2003] and for the SAC response to a lack of tension across sister kinetochores [Biggins and Murray, 2001]. The Ndc10 kinetochore protein and the Ndc80p kinetochore complex are also required for SAC function in budding yeast [Janke et al, 2001;McCleland et al, 2003;Poddar et al, 2004;Tavormina and Burke 1998]. …”

“…It remains unclear whether the SAC component, Mps1p, also plays a role in centrosome duplication in higher eukaryotes [Fischer et al, 2004;Fisk et al, 2003;Liu et al, 2003;Stucke et al, 2004;Stucke et al, 2002] as it does in budding [Winey et al, 1991] but not in fission yeast [He et al, 1998]. The mammalian Ndc80p complex [McCleland et al, 2003], the ROD and ZW10 kinetochore proteins [Basto et al, 2000;Chan et al, 2000], the outer kinetochore protein Zwint-1 [Obuse et al, 2004;Wang et al, 2004a], and the kinetochore-associated NEK2A protein [Lou et al, 2004] are also required for the spindle checkpoint, indicating that the kinetochore's role in SAC signaling is evolutionarily conserved, although no ROD, ZW10, or Zwint-1 homologues have been identified in yeast. The mammalian survivin/Aurora B complex regulates BubR1p and Mad2p localization to kinetochores [Lens et al, 2003] and, like the budding yeast Ipl1p/Aurora, is required for SAC function when insufficient tension is applied across sister kinetochores [Carvalho et al, 2003;Hauf et al, 2003;Lens et al, 2003].…”

PIASy mediates SUMO-2 conjugation of Topoisomerase-II on mitotic chromosomes

“…The rabbit antisera were subjected to affinity purification on NHS Sepharose columns with their covalently bound antigens. Affinity-purified chicken antiXenopus CENP-A antibodies were as described (kind gift of PT Stukenberg, University of Virginia Medical School; McCleland et al, 2003). Rabbit anti-Topoisomerase-II was the kind gift of PL Jones (University of Illinois at Urbana-Champaign, Urbana, IL; Luke and Bogenhagen, 1989).…”