Mobility of kinetochore proteins measured by FRAP analysis in living cells

Abstract

The kinetochore is essential for faithful chromosome segregation during mitosis and is assembled through dynamic processes involving numerous kinetochore proteins. Various experimental strategies have been used to understand kinetochore assembly processes. Fluorescence recovery after photobleaching (FRAP) analysis is also a useful strategy for revealing the dynamics of kinetochore assembly. In this study, we introduced fluorescence protein-tagged kinetochore protein cDNAs into each endogenous locus and performed FRAP analyses in chicken DT40 cells. Centromeric protein (CENP)-C was highly mobile in interphase, but immobile during mitosis. CENP-C mutants lacking the CENP-A-binding domain became mobile during mitosis. In contrast to CENP-C, CENP-T and CENP-H were immobile during both interphase and mitosis. The mobility of Dsn1, which is a component of the Mis12 complex and directly binds to CENP-C, depended on CENP-C mobility during mitosis. Thus, our FRAP assays provide dynamic aspects of how the kinetochore is assembled.

The centromere/kinetochore is assembled through CENP-C oligomerization

“…We found that Mini-CENP-C was sufficient for CENP-C function. This further supports our previous observations that two CENP-C functional domains, the Mis12- and CENP-A-binding domains, are dispensable for CENP-C function in chicken DT40 cells (Hara et al, 2018; Watanabe et al, 2019; Watanabe et al, 2022). Given that the Mis12- and CENP-A-binding domains are related to mitotic kinetochore functions, our results lead to an intriguing idea that CENP-C plays no direct essential roles in kinetochore function during mitosis in tissue culture cells.…”

Structure of the human inner kinetochore CCAN complex and its significance for human centromere organization

“…Depletion of CENP-C has been shown to be compatible with short-term retention of very significant levels of CCAN subunits in DT40 cells ( Hori et al., 2008 ). Furthermore, CENP-C displays slow but significant turnover at interphase kinetochores in DT40 chicken cells, contrary to CCAN subunits CENP-H and CENP-T, which are essentially immobile ( Watanabe et al., 2022 ). Thus, CCAN may depend on CENP-A for its localization in addition to CENP-C, and previous work points to CENP-N as a plausible mediator of these additional interactions ( Carroll et al., 2009 , 2010 ).…”

The cryo-EM structure of the CENP-A nucleosome in complex with ggKNL2

“…We have previously demonstrated that CENP-C–CENP-A nucleosome interaction does not occur in interphase chicken DT40 cells, but CENP-C binds to the CENP-A nucleosome upon CDK1-mediated CENP-C phosphorylation during mitosis (Ariyoshi et al ., 2021; Watanabe et al ., 2019; Watanabe et al , 2022). We have also demonstrated that CDK1 mediated CENP-C phosphorylation facilitates the CENP-C–CENP-A interaction in human mitotic cells (Watanabe et al ., 2019).…”