A quantitative description of Ndc80 complex linkage to human kinetochores.

Aussie Suzuki,Edward D Salmon,Benjamin L Badger

doi:10.1038/ncomms9161

Aussie Suzuki, Edward D Salmon + Show 1 more

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https://doi.org/10.1038/ncomms9161

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Abstract

The Ndc80 complex, which mediates end-on attachment of spindle microtubules, is linked to centromeric chromatin in human cells by two inner kinetochore proteins, CENP-T and CENP-C. Here to quantify their relative contributions to Ndc80 recruitment, we combine measurements of kinetochore protein copy number with selective protein depletion assays. This approach reveals about 244 Ndc80 complexes per human kinetochore (∼14 per kinetochore microtubule), 215 CENP-C, 72 CENP-T and only 151 Ndc80s as part of the KMN protein network (1:1:1 Knl1, Mis12 and Ndc80 complexes). Each CENP-T molecule recruits ∼2 Ndc80 complexes; one as part of a KMN network. In contrast, ∼40% of CENP-C recruits only a KMN network. Replacing the CENP-C domain that binds KMN with the CENP-T domain that recruits both an Ndc80 complex and KMN network yielded functional kinetochores. These results provide a quantitative picture of the linkages between centromeric chromatin and the microtubule-binding Ndc80 complex at the human kinetochore.

Highlights

The Ndc[80] complex, which mediates end-on attachment of spindle microtubules, is linked to centromeric chromatin in human cells by two inner kinetochore proteins, CENP-T and CENP-C
These artificial kinetochores lack CENP-A and the other centromere-associated protein network (CCAN) proteins but contain all members of the KMN network and other outer kinetochore proteins like those of the spindle assembly checkpoint. These findings indicate that CENP-T and CENP-C can both function independently as major recruiters of Ndc[80] protein complex (Ndc80c) as well as the KMN network
Each enhanced green fluorescent protein (EGFP) fusion was expressed near the level of the endogenous protein following removal of the endogenous protein by RNA interference (RNAi) (Fig. 1c; Table 1; Supplementary Fig. 1); the RNAi penetrance was sufficient to reduce the target protein levels to below the detection limit by both western blot (Fig. 1c) and immunofluorescence (Supplementary Fig. 2a)

Summary

Introduction

The Ndc[80] complex, which mediates end-on attachment of spindle microtubules, is linked to centromeric chromatin in human cells by two inner kinetochore proteins, CENP-T and CENP-C. To quantify their relative contributions to Ndc[80] recruitment, we combine measurements of kinetochore protein copy number with selective protein depletion assays. Recent studies have shown that the N-terminal half of either CENP-T or CENP-C alone tethered to chromatin in high numbers by LacO–LacI (B256) can establish a functional artificial kinetochore[18] These artificial kinetochores lack CENP-A and the other CCAN proteins but contain all members of the KMN network and other outer kinetochore proteins like those of the spindle assembly checkpoint.

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