Abstract
To form functional kinetochores, CENP-C and CENP-T independently recruit the KMN (Knl1C, Mis12C, and Ndc80C) network onto the kinetochores. To clarify the functions of the KMN network on CENP-T, we evaluated its roles in chicken DT40 cell lines lacking the CENP-C-KMN network interaction. By analyzing mutants lacking both CENP-T-Mis12C and CENP-C-Mis12C interactions, we demonstrated that Knl1C and Mis12C (KM) play critical roles in the cohesion of sister chromatids or the recruitment of spindle checkpoint proteins onto kinetochores. Two copies of Ndc80C (N-N) exist on CENP-T via Mis12C or direct binding. Analyses of cells specifically lacking the Mis12C-Ndc80C interaction revealed that N-N is needed for proper kinetochore-microtubule interactions. However, using artificial engineering to directly bind the two copies of Ndc80C to CENP-T, we demonstrated that N-N functions without direct Mis12C binding to Ndc80C in native kinetochores. This study demonstrated the mechanisms by which complicated networks play roles in native kinetochores.
Highlights
To form functional kinetochores, centromere protein (CENP)-C and CENP-T independently recruit the KMN (Knl1C, Mis12 complex (Mis12C), and Ndc80 complex (Ndc80C)) network onto the kinetochores
Since the Ndc80C-Mis12C interaction is thought to be critical for the kinetochore functions7,42,43, our observations provide new insights for kinetochore studies and explain how the KMN network plays an essential role in kinetochore functions via the CENP-T pathway
Mis12C binds to both CENP-C and CENP-T, and we have previously shown that the CENP-T-Mis12C interaction is much more critical than the CENP-C-Mis12C interaction in chicken DT40 cells35
Summary
To form functional kinetochores, CENP-C and CENP-T independently recruit the KMN (Knl1C, Mis12C, and Ndc80C) network onto the kinetochores. The spindle microtubules bind to a large protein complex called kinetochore, which is formed on the centromere of each sister chromatid, to ensure the accurate segregation of the chromosomes. From the late G2 phase to mitosis, another large complex starts to associate with CCAN to form a fully functional kinetochore This complex contains the Knl complex (Knl1C), Mis complex (Mis12C), and Ndc complex (Ndc80C), which form the KMN network. Among the CCAN proteins, CENP-C and the CENPT-W-S-X complex bind to both centromeric chromatin and the KMN network1,33–35 Based on these studies and the results of artificial tethering of CENP-C or CENP-T into a noncentromeric region, two parallel pathways for the recruitment of the KMN network onto the kinetochores in vertebrate cells have been proposed: the CENP-C and CENP-T pathways. To evaluate the precise role of each complex or protein in native kinetochores, we must characterize a narrow functional domain in each protein and remove the redundancies to assess the function of an individual protein (complex)
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