Molecular details and phospho-regulation of the CENP-T-Mis12 complex interaction during mitosis in DT40 cells

To establish bi-polar attachments of microtubules to sister chromatids, an inner kinetochore subcomplex, the constitutive centromere-associated network (CCAN), is assembled on centromeric chromatin and recruits the microtubule-binding subcomplex called the KMN network. Although it is important to characterize the interaction between CCAN and KMN, it is difficult to evaluate the significance of each interaction in cells, since CCAN proteins CENP-C and CENP-T independently bind to the Mis12 complex (Mis12C) of KMN. Here, we demonstrate molecular details of the CENP-T-Mis12C interaction using chicken DT40 cells lacking CENP-C-Mis12C interaction. Based on AlphaFold2 predictions, we identified two binding surfaces for the CENP-T-Mis12C interaction, demonstrating that each surface is critical for Mis12C recruitment and cell viability. This interaction via two interaction surfaces is cooperatively regulated by dual phosphorylation of Dsn1 (a Mis12C component) and CENP-T, ensuring robust CENP-T-Mis12C interaction and proper mitotic progression. These findings deepen our understanding of kinetochores assembly in cells.