A quantitative description of Ndc80 complex linkage to human kinetochores.
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.
Department of Biology, University of North Carolina at Chapel Hill, Chapel hill, North Carolina 27599, USA.
Aussie
A
Suzuki
Department of Biology, University of North Carolina at Chapel Hill, Chapel hill, North Carolina 27599, USA.
Benjamin L
BL
Badger
Department of Biology, University of North Carolina at Chapel Hill, Chapel hill, North Carolina 27599, USA.
Edward D
ED
Salmon
D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D018386:Kinetochores / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D061848:Optical Imaging
C534001:CENPT protein, human; D002868:Chromosomal Proteins, Non-Histone; D003598:Cytoskeletal Proteins; C472643:Knl1 protein, human; C492664:MIS12 protein, human; D008869:Microtubule-Associated Proteins; C120529:NDC80 protein, human; D009687:Nuclear Proteins; C060905:centromere protein C; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins