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.

46 Reads
    • "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). "
    [Show abstract] [Hide abstract]
    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.
    Journal of Cell Science 11/2015; DOI:10.1242/jcs.180786 · 5.43 Impact Factor
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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
Show more