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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    • "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.
    Developmental Cell 09/2014; 30(5):496-508. DOI:10.1016/j.devcel.2014.08.016 · 9.71 Impact Factor
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    • "CENP-E only localizes to active centromeres and whose knockout is embryonically lethal with disorganized/disordered chromosome segregation [26]. Recent research has shown that CENP-C and CENP-T/W, not CENP-A and CENP-B, act as a type of mediator between the centromeric chromatin platform (constitutive centromere-associated network) and the Knl1-Mis12-Ndc80 complex network, which is beneficial in constructing a bridge between the inner and outer kinetochore [27]–[29]. Up to date, there is limited evidence of direct interaction between CENP-A and any of other CENPs [30], and however CENP-B can interact with CENP-C. These data imply that CENP-B most likely participates in more subtle activities than simple kinetochore assembly. "
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    ABSTRACT: CENP-B is a highly conserved protein that facilitates the assembly of specific centromere structures both in interphase nuclei and on mitotic chromosomes. INMAP is a conserved protein that localizes at nucleus in interphase cells and at mitotic apparatus in mitotic cells. Our previous results showed that INMAP over-expression leads to spindle defects, mitotic arrest and formation of polycentrosomal and multinuclear cells, indicating that INMAP may modulate the function of (a) key protein(s) in mitotic apparatus. In this study, we demonstrate that INMAP interacts with CENP-B and promotes cleavage of the N-terminal DNA binding domain from CENP-B. The cleaved CENP-B cannot associate with centromeres and thus lose its centromere-related functions. Consistent with these results, CENP-B in INMAP knockdown cells becomes more diffused around kinetochores. Although INMAP knockdown cells do not exhibit gross defects in mitotic spindle formation, these cells go through mitosis, especially prophase and metaphase, with different relative timing, indicating subtle abnormality. These results identify INMAP as a model regulator of CENP-B and support the notion that INMAP regulates mitosis through modulating CENP-B-mediated centromere organization.
    PLoS ONE 03/2014; 9(3):e91937. DOI:10.1371/journal.pone.0091937 · 3.23 Impact Factor
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    • "Previous biochemical studies revealed that the Ndc80 complex associates with the Mis12 complex, which in turn interacts with the inner kinetochore protein CENP-C (Cheeseman et al, 2004; Obuse et al, 2004; Gascoigne et al, 2011; Przewloka et al, 2011; Screpanti et al, 2011). Petrovic et al (2010) demonstrated that the Nsl1 subunit of the Mis12 complex binds to tightly to Spc24/25. "
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    ABSTRACT: The kinetochore forms a dynamic interface with micro-tubules from the mitotic spindle during mitosis. The Ndc80 complex acts as the key microtubule-binding com-plex 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 high-resolution 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 kineto-chores. Our results provide a model for how the multiple protein complexes at kinetochores associate in a phospho-regulated manner. The EMBO Journal advance online publication, 18 January 2013; doi:10.1038/emboj.2012.348
    The EMBO Journal 01/2013; 32(3). DOI:10.1038/emboj.2012.348 · 10.43 Impact Factor
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