KNL1 and the CENP-H/I/K Complex Coordinately Direct Kinetochore Assembly in Vertebrates

Abstract

Chromosome segregation during mitosis requires the assembly of a large proteinaceous structure termed the kinetochore. In Caenorhabditis elegans, KNL-1 is required to target multiple outer kinetochore proteins. Here, we demonstrate that the vertebrate KNL1 counterpart is essential for chromosome segregation and is required to localize a subset of outer kinetochore proteins. However, unlike in C. elegans, depletion of vertebrate KNL1 does not abolish kinetochore localization of the microtubule-binding Ndc80 complex. Instead, we show that KNL1 and CENP-K, a subunit of a constitutively centromere-associated complex that is missing from C. elegans, coordinately direct Ndc80 complex localization. Simultaneously reducing both hKNL1 and CENP-K function abolishes all aspects of kinetochore assembly downstream of centromeric chromatin and causes catastrophic chromosome segregation defects. These findings explain discrepancies in kinetochore assembly pathways between different organisms and reveal a surprising plasticity in the assembly mechanism of an essential cell division organelle.

The fate of metaphase kinetochores is weighed in the balance of SUMOylation during S phase

“…Mis12 is a member of another key outer kinetochore complex whose localization has been reported depend partially upon CENP-H/I/K. 66 Mis12 showed decreased localization on kinetochores, particularly those of unattached chromosomes, although the extent of this decrease was somewhat less than we observed for Hec1. 33 Notably, outer kinetochore components whose recruitment has been reported to be independent of CENP-H/I/K (CENP-E and -F 66 ) did not show significant changes after SENP6 depletion.…”

“…64 Quantitative immunofluorescence showed that the Hec1 complex was partially mis-localized in the absence of SENP6, 33 consistent with its pattern of mis-localization after CENP-H/I/K complex depletion. 66 Interestingly, we observed differential loading of Hec1 on the attached and unattached kinetochores within individual cells: Hec1 levels were reduced around 45% on the aligned kinetochores, whereas they were reduced around 75% on the unaligned kinetochores. This differential pattern was surprising because earlier reports had not found that Hec1 recruitment is sensitive to the attachment status of individual kinetochores.…”

“…Loss of the CENP-H/I/K complex also results in reduced levels of some outer kinetochore complexes, including the Hec1 and Mis12 complexes. 66 The CENP-O/P/Q/U/R complex recruits polo like kinase 1 (Plk1), which is responsible for phosphorylating many kinetochore proteins, 63,[67][68][69][70] suggesting that Plk1 localization should indirectly depend upon the CENP-H/I/K complex.…”

The RZZ Complex and the Spindle Assembly Checkpoint

“…Vertebrate KNL-1 is expected to function upstream of the Ndc80 complex . The KMN network is thus a key structural component of the kinetochore, but also directly binds to MTs via the Ndc80 subunit of the Ndc80 complex and KNL-1 (Cheeseman et al, 2006;Cheeseman et al, 2008). The intact KMN network is incorporated into the outer kinetochore plate to form the repeating MT-binding sites of eukaryotic kinetochores (Cheeseman et al, 2006;Przewloka et al, 2007).…”

The CCAN complex: Linking centromere specification to control of kinetochore–microtubule dynamics

“…Table 1 Parts list -Constitutive Centromere Associated Network (CCAN). kinetochores was reduced by ∼25% following depletion of CENP-H [32] or CENP-K [33]. However, it is important to be aware that depletion of CENP-H, which prevents binding of all CCAN subunits except CENP-T/W/C, does not affect the ability of kinetochore to form stable end-on bipolar attachments [17].…”