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.
The NSL1 subunit structures interactions between the MIS12, NDC80, and KNL1 kinetochore complexes (see also a related paper by Maskell et al. in this issue).
SummaryKinetochores, multisubunit protein assemblies, connect chromosomes to spindle microtubules to promote chromosome segregation. The 10-subunit KMN assembly (comprising KNL1, MIS12, and NDC80 complexes, designated KNL1C, MIS12C, and NDC80C) binds microtubules and regulates mitotic checkpoint function through NDC80C and KNL1C, respectively. MIS12C, on the other hand, connects the KMN to the chromosome-proximal domain of the kinetochore through a direct interaction with CENP-C. The structural basis for this crucial bridging function of MIS12C is unknown. Here, we report crystal structures of human MIS12C associated with a fragment of CENP-C and unveil the role of Aurora B kinase in the regulation of this interaction. The structure of MIS12:CENP-C complements previously determined high-resolution structures of functional regions of NDC80C and KNL1C and allows us to build a near-complete structural model of the KMN assembly. Our work illuminates the structural organization of essential chromosome segregation machinery that is conserved in most eukaryotes.
Chromosomes are carriers of genetic material and their accurate transfer from a mother cell to its two daughters during cell division is of paramount importance for life. Kinetochores are crucial for this process, as they connect chromosomes with microtubules in the mitotic spindle. Kinetochores are multi-subunit complexes that assemble on specialized chromatin domains, the centromeres, that are able to enrich nucleosomes containing the histone H3 variant centromeric protein A (CENP-A). A group of several additional CENPs, collectively known as constitutive centromere associated network (CCAN), establish the inner kinetochore, whereas a ten-subunit assembly known as the KMN network creates a microtubule-binding site in the outer kinetochore. Interactions between CENP-A and two CCAN subunits, CENP-C and CENP-N, have been previously described, but a comprehensive understanding of CCAN organization and of how it contributes to the selective recognition of CENP-A has been missing. Here we use biochemical reconstitution to unveil fundamental principles of kinetochore organization and function. We show that cooperative interactions of a seven-subunit CCAN subcomplex, the CHIKMLN complex, determine binding selectivity for CENP-A over H3-nucleosomes. The CENP-A:CHIKMLN complex binds directly to the KMN network, resulting in a 21-subunit complex that forms a minimal high-affinity linkage between CENP-A nucleosomes and microtubules in vitro. This structural module is related to fungal point kinetochores, which bind a single microtubule. Its convolution with multiple CENP-A proteins may give rise to the regional kinetochores of higher eukaryotes, which bind multiple microtubules. Biochemical reconstitution paves the way for mechanistic and quantitative analyses of kinetochores.
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