CENP-T provides a structural platform for outer kinetochore assembly

Abstract

The kinetochore forms a dynamic interface with microtubules from the mitotic spindle during mitosis. The Ndc80 complex acts as the key microtubule-binding complex at kinetochores. However, it is unclear how the Ndc80 complex associates with the inner kinetochore proteins that assemble upon centromeric chromatin. Here, based on a highresolution structural analysis, we demonstrate that the N-terminal region of vertebrate CENP-T interacts with the 'RWD' domain in the Spc24/25 portion of the Ndc80 complex. Phosphorylation of CENP-T strengthens a cryptic hydrophobic interaction between CENP-T and Spc25 resulting in a phospho-regulated interaction that occurs without direct recognition of the phosphorylated residue. The Ndc80 complex interacts with both CENP-T and the Mis12 complex, but we find that these interactions are mutually exclusive, supporting a model in which two distinct pathways target the Ndc80 complex to kinetochores. Our results provide a model for how the multiple protein complexes at kinetochores associate in a phospho-regulated manner.

Regulation of outer kinetochore Ndc80 complex-based microtubule attachments by the central kinetochore Mis12/MIND complex

“…By reconstituting the yeast MIND/Ndc80 co-complex and using cross-linking analysis, we have identified an intricate set of interactions involving five of the eight proteins within the two complexes. In addition to the previously identified Spc24-Spc25 interface shared by both MIND and Cnn1 (15,22), we found a unique connection between Nsl1 and a hydrophobic Spc24/Spc25 cleft. This identification of a second unique interface suggests that the Ndc80 complex may differentially interact with MIND and Cnn1, raising the possibility that each receptor might distinctly regulate Ndc80c function.…”

“…It could also explain why Ndc80 is detected only at the kinetochore whereas other microtubule binding components of the kinetochore are also detected all along the spindle microtubules (36). Cnn1 provides a distinct Ndc80 receptor during anaphase (13,15,16). It will therefore be interesting to learn whether and how Cnn1 affects Ndc80's microtubule affinity.…”

“…However, it is unknown whether these structural changes correlate with changes in microtubule affinity. The Ndc80 complex also interacts with a second kinetochore receptor, CENP-T/Cnn1 (13)(14)(15)(16). It is unclear how central kinetochore components influence the activity of the Ndc80 complex throughout mitosis.…”

The Centromere: Epigenetic Control of Chromosome Segregation during Mitosis

“…For example, the role of a CENP-T-containing complex in recruiting the Ndc80 complex and the function of CENP-C in recruiting of the Mis12 complex, suggests different functions of the kinetochore are brought to centromeres through different activities of core centromere proteins. However, despite this modularity, there is significant cross talk between centromere components to stably form a functional centromere and kinetochore; Mis12 and CENP-T compete for Ndc80 binding (Bock et al 2012;Schleiffer et al 2012;Nishino et al 2013) and a CENP-N/L heterodimer binds CENP-C (Carroll et al 2009;Hinshaw and Harrison 2013), presumably as part of a CENP-A nucleosome-containing complex. Pinpointing how and when different centromere modules interact remains an exciting challenge.…”

“…A second essential step in KMN protein assembly at centromeres is binding of the Spc24/25 components of the Ndc80 complex by the phosphorylated aminoterminal tail of CENP-T. This conserved interaction forms stable, load-bearing attachments to microtubules via the Ndc80 complex (Bock et al 2012;Schleiffer et al 2012;Malvezzi et al 2013;Nishino et al 2013). A nonphosphorylatable CENP-T amino-terminal tail disrupts kinetochore function as it cannot fulfill these functions (Gascoigne et al 2011).…”