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

Yusuke Takenoshita; Masatoshi Hara; Reiko Nakagawa; Mariko Ariyoshi; Tatsuo Fukagawa

doi:10.1016/j.isci.2024.111295

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

iScience. 2024 Nov 1;27(12):111295. doi: 10.1016/j.isci.2024.111295. eCollection 2024 Dec 20.

Authors

Yusuke Takenoshita¹, Masatoshi Hara¹, Reiko Nakagawa², Mariko Ariyoshi¹, Tatsuo Fukagawa¹

Affiliations

¹ Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan.
² RIKEN Center for Biosystems Dynamics Research, Kobe, Hyogo 650-0047, Japan.

Abstract

To establish bipolar 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. Since CCAN proteins CENP-C and CENP-T independently bind to the Mis12 complex (Mis12C) of KMN, it is difficult to evaluate the significance of each interaction in cells. Here, we demonstrate the molecular details of the CENP-T-Mis12C interaction using chicken DT40 cells lacking the CENP-C-Mis12C interaction. Using AlphaFold predictions combined with cell biological and biochemical analyses, we identified three binding surfaces of the CENP-T-Mis12C interaction, demonstrating that each interface is important for recruiting Mis12C to CENP-T in cells. This interaction, via three interaction surfaces, is cooperatively regulated by dual phosphorylation of Dsn1 (a Mis12C component) and CENP-T, ensuring a robust CENP-T-Mis12C interaction and proper mitotic progression. These findings deepen our understanding of kinetochore assembly in cells.

Keywords: Biological sciences; Biophysics; Cell biology.