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

Department of Experimental Oncology, European Institute of Oncology, Milan, Italy.
Current biology: CB (Impact Factor: 9.57). 02/2011; 21(5):391-8. DOI: 10.1016/j.cub.2010.12.039
Source: PubMed


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 the inner and outer kinetochore, respectively (reviewed in). The constitutive centromere-associated network (CCAN) and the Knl1-Mis12-Ndc80 complexes (KMN) network are the main multisubunit 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. 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 missegregation. 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.

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    • "Tethering of domain II also recruited Halo-M18BP1 in 75% of cells, probably via CENP-I. We also observed the interactions between CENP-C domain I (1-71 aa) and NSL1 (data not shown), which is consistent with previous in vitro analyses (Screpanti et al., 2011). "
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    ABSTRACT: Although it is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity, the pathways leading to the formation and maintenance of centromere chromatin remain unclear. We previously generated human artificial chromosomes (HACs) whose centromeres contain a synthetic alpha-satellite (alphoid) DNA array containing tetracycline operator (alphoid(tetO)). We also obtained cell lines bearing the alphoid(tetO) array at ectopic integration sites on chromosomal arms. Here, we have examined the regulation of CENP-A assembly at centromeres as well as de novo assembly on the ectopic arrays by tethering tetracycline repressor (tetR) fusions of substantial centromeric factors and chromatin modifiers. This analysis revealed four classes of factors that influence CENP-A assembly. Interestingly, many kinetochore structural components induced de novo CENP-A assembly at the ectopic site. We showed that these components work by recruiting CENP-C, subsequently recruiting M18BP1. Furthermore, we found that CENP-I also can recruit M18BP1 and, in consequence, enhances M18BP1 assembly on centromeres in the downstream of CENP-C. Thus we suggest that CENP-C and CENP-I are key factors connecting kinetochore to CENP-A assembly.
    Full-text · Article · Nov 2015 · Journal of Cell Science
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    • "Second, the CCAN recruits the KMN complex (KNL- 1, Mis12, and Ndc80 complex) (Gascoigne et al. 2011; Przewloka et al. 2011; Screpanti et al. 2011), which is a central mediator of both SAC signaling and microtubule binding. KMN is recruited by two interactions through the CCAN; CENP-T binds the Ndc80 complex and CENP-C binds to the Mis12 complex, which in turn binds both KNL-1 protein and the Ndc80 complex (Petrovic et al. 2010; Schleiffer et al. 2012; Screpanti et al. 2011). The Ndc80 complex directly binds microtubules and is pivotal for both alignments to the metaphase plate and anaphase chromosome movements where it couples chromosome movement to microtubule depolymerization (Cheeseman et al. 2006; Ciferri et al. 2008; Miller et al. 2008; Tooley et al. 2011). "
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    ABSTRACT: Kinetochores generate a signal that inhibits anaphase progression until every kinetochore makes proper attachments to spindle microtubules. This spindle assembly checkpoint (SAC) increases the fidelity of chromosome segregation. We will review the molecular mechanisms by which kinetochores generate the SAC and extinguish the signal after making proper attachments, with the goal of identifying unanswered questions and new research directions. We will emphasize recent breakthroughs in how phosphorylation changes drive the activation and inhibition of the signal. We will also emphasize the dramatic changes in kinetochore structure that occur after attaching to microtubules and how these coordinate SAC function with microtubule attachment status. Finally, we will review the emerging cross talk between the DNA damage response and the SAC.
    Full-text · Article · Apr 2015 · Chromosoma
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    • "A second artificial kinetochore was also constructed by similar tethering of CENP-C to a Lac operator array (Hori et al., 2013). CENP-C recruits the Mis12 complex, which binds the Ndc80 complex (Gascoigne et al., 2011; Przewloka et al., 2011; Screpanti et al., 2011; Figure 7). Surprisingly, many centromere proteins, including CENP-A, were not detected in either artificial kinetochore (Hori et al., 2013). "
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    ABSTRACT: Since discovery of the centromere-specific histone H3 variant CENP-A, centromeres have come to be defined as chromatin structures that establish the assembly site for the complex kinetochore machinery. In most organisms, centromere activity is defined epigenetically, rather than by specific DNA sequences. In this review, we describe selected classic work and recent progress in studies of centromeric chromatin with a focus on vertebrates. We consider possible roles for repetitive DNA sequences found at most centromeres, chromatin factors and modifications that assemble and activate CENP-A chromatin for kinetochore assembly, plus the use of artificial chromosomes and kinetochores to study centromere function.
    Full-text · Article · Sep 2014 · Developmental Cell
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