Mobility of kinetochore proteins measured by FRAP analysis in living cells

Reito Watanabe,Yasushi Hiraoka,Yasuhiro Hirano,Tatsuo Fukagawa,Masatoshi Hara

doi:10.1007/s10577-021-09678-x

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.

Highlights

Chromosomes harboring all the genetic information are duplicated during the S-phase and segregated into daughter cells during mitosis
We previously showed that CENPC dynamically changes its binding partners during cell cycle progression (Fukagawa et al 2001; Kwon et al 2007; Nagpal et al 2015), suggesting that the Centromeric protein (CENP)-A binding domain in the CENP-C C-terminal region is not used during interphase but rather that CENP-C binds to the CENP-A nucleosome during mitosis (Nagpal et al 2015)
We demonstrated that CENP-C phosphorylation by cyclin-dependent kinase 1 (CDK1) facilitates the binding of CENP-C to the CENP-A nucleosome during mitosis (Watanabe et al 2019; Ariyoshi et al 2021)

Summary

Introduction

Chromosomes harboring all the genetic information are duplicated during the S-phase and segregated into daughter cells during mitosis. As CENP-C binds to both the CENP-A nucleosome in centromeric chromatin (Fachinetti et al 2013; Kato et al 2013; Falk et al 2015; Guo et al 2017; Watanabe et al 2019; Ariyoshi et al 2021) and the Mis complex of the KMN network at the outer kinetochores (Klare et al 2015; Hara and Fukagawa 2017; Hara et al 2018), CENP-C bridges the centromeric chromatin and outer kinetochore, which is associated with spindle microtubules. Using FRAP analyses, we examined the mobility of kinetochore proteins in interphase and mitotic cells by replacing endogenous proteins with fluorescently tagged proteins that are functional at the native expression level and demonstrated various mobilities of each kinetochore component, which provides dynamic information of how the kinetochore is assembled

Materials and methods

C Pre- Postbleach bleach 48s 120s 264s CENP-CGFP

Results and discussion