The Architecture of CCAN Proteins Creates a Structural Integrity to Resist Spindle Forces and Achieve Proper Intrakinetochore Stretch

• Published in: Developmental cell 2014-09, Vol.30 (6), p.717-730
• Main Authors: Suzuki, Aussie, Badger, Benjamin L., Wan, Xiaohu, DeLuca, Jennifer G., Salmon, Edward D.
• Format: Article
• Language: English
• Subjects: Aurora Kinase B - metabolism; Autoantigens - metabolism; Centromere Protein A; Chromosomal Proteins, Non-Histone - metabolism; HeLa Cells; Humans; Kinetochores - metabolism; Nuclear Proteins - metabolism; Phosphorylation; Protein Binding; Spindle Apparatus - metabolism
• ISSN: 1534-5807; 1878-1551
• DOI: 10.1016/j.devcel.2014.08.003

Constitutive centromere-associated network (CCAN) proteins, particularly CENP-C, CENP-T, and the CENP-H/-I complex, mechanically link CENP-A-containing centromeric chromatin within the inner kinetochore to outer kinetochore proteins, such as the Ndc80 complex, that bind kinetochore microtubules. Accuracy of chromosome segregation depends critically upon Aurora B phosphorylation of Ndc80/Hec1. To determine how CCAN protein architecture mechanically constrains intrakinetochore stretch between CENP-A and Ndc80/Hec1 for proper Ndc80/Hec1 phosphorylation, we used super-resolution fluorescence microscopy and selective protein depletion. We found that at bi-oriented chromosomes in late prometaphase cells, CENP-T is stretched ∼16 nm to the inner end of Ndc80/Hec1, much less than expected for full-length CENP-T. Depletion of various CCAN linker proteins induced hyper-intrakinetochore stretch (an additional 20–60 nm) with corresponding significant decreases in Aurora B phosphorylation of Ndc80/Hec1. Thus, proper intrakinetochore stretch is required for normal kinetochore function and depends critically on all the CCAN mechanical linkers to the Ndc80 complex. •High-resolution microscopy reveals CCAN protein architecture in human kinetochores•Proper intrakinetochore stretch depends on the CCAN linkers to the Ndc80 complex•Ndc80/Hec1 phosphorylation is inhibited by hyper-intrakinetochore stretch•CCAN DNA binding proteins play a minor role in the compaction of CENP-A chromatin Using super-resolution microscopy, Suzuki et al. find that constitutive centromere-associated network proteins mechanically constrain the distance between inner and outer kinetochore components under tension. This constraint on intrakinetochore stretch is important to ensure Aurora B access to its substrates and thus to correct errors in kinetochore-microtubule attachment.