Direct Binding of Cenp-C to the Mis12 Complex Joins the Inner and Outer Kinetochore

Abstract

Summary Kinetochores are proteinaceous scaffolds implicated in the formation of load-bearing attachments of chromosomes to microtubules during mitosis. Kinetochores contain distinct chromatin- and microtubule-binding interfaces, generally defined as inner and outer kinetochore, respectively [reviewed in 1]. The constitutive centromere-associated network (CCAN) and the Knl1-Mis12-Ndc80 complexes (KMN) network are the main multi-subunit protein assemblies in the inner and outer kinetochore, respectively. The point of contact between the CCAN and the KMN network is unknown. Cenp-C is a conserved CCAN component whose central and C-terminal regions have been implicated in chromatin binding and dimerization [2–10]. Here, we show that a conserved motif in the N-terminal region of Cenp-C binds directly and with high affinity to the Mis12 complex. Expression in HeLa cells of the isolated N-terminal motif of Cenp-C prevents outer kinetochore assembly, causing chromosome mis-segregation. The KMN network is also responsible for kinetochore recruitment of the components of the spindle assembly checkpoint, and we observe checkpoint impairment in cells expressing the Cenp-C N-terminal segment. Our studies unveil a crucial and likely universal link between the inner and outer kinetochore.

Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function

“…4A). This functional analysis was supported by staining for CENP-C -a crucial kinetochore protein, which links the inner and outer kinetochore components (Saitoh et al, 1992;Tomkiel et al, 1994;Gascoigne et al, 2011;Przewloka et al, 2011;Screpanti et al, 2011). CENP-C was severely depleted from the HAC kinetochore following the tethering of tetR-EYFP-VP16, but could be detected at HAC kinetochores in the presence of tetR-EYFP-p65 and the control, tetR-EYFP (Fig.…”

Synthetic physical interactions map kinetochore regulators and regions sensitive to constitutive Cdc14 localization

“…Mtw1 is a member of the essential Mtw1/Mis12/MIND complex (Mtw1, Dsn1, Nnf1, and Nsl1) (26,27). The MIND complex connects the inner constitutive centromere-associated network kinetochore proteins, which are adjacent to the centromeric DNA, with the outer, microtubuleassociated proteins in conjunction with Mif2 (28)(29)(30)(31). We linked the MTW1 kinetochore gene with that of the GBP (MTW1-GBP).…”

Human centromere genomics: now it's personal

“…From the perspective of the underlying DNA, these interactions introduce a combination of multi-protein interactions assuming sequence flexibility to tolerate the induced supercoiling, spacing, and bending necessary for nucleosome wrapping to accommodate centromere-specific chromatin (Furuyama and Henikoff 2009;Hori et al 2008;Nishino et al 2012). In addition to direct sequence-based interactions with inner kinetochore proteins, centromeric sequences also accommodate a hierarchy of constitutive and dynamic protein-based interactions that bridge the inner kinetochore and the outer kinetochore in a protein-mediated transfer of tension from spindle forces to those proteins that directly bind DNA (Hori et al 2008;Screpanti et al 2011). Moreover, proper assembly of pericentromeric heterochromatin, typically correlated with functional kinetochores, may be influenced by sequence-based factors, such as transcriptional regulation and specialized chromatin compaction (Folco et al 2008).…”