Mobility of kinetochore proteins measured by FRAP analysis in living cells

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...

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Autores principales: Watanabe, Reito, Hirano, Yasuhiro, Hara, Masatoshi, Hiraoka, Yasushi, Fukagawa, Tatsuo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Netherlands 2022
Materias:
Original Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8942963/
https://www.ncbi.nlm.nih.gov/pubmed/34997387
http://dx.doi.org/10.1007/s10577-021-09678-x
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author Watanabe, Reito
Hirano, Yasuhiro
Hara, Masatoshi
Hiraoka, Yasushi
Fukagawa, Tatsuo
author_facet Watanabe, Reito
Hirano, Yasuhiro
Hara, Masatoshi
Hiraoka, Yasushi
Fukagawa, Tatsuo
author_sort Watanabe, Reito
collection PubMed
description 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10577-021-09678-x.
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spelling pubmed-89429632022-04-07 Mobility of kinetochore proteins measured by FRAP analysis in living cells Watanabe, Reito Hirano, Yasuhiro Hara, Masatoshi Hiraoka, Yasushi Fukagawa, Tatsuo Chromosome Res Original Article 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10577-021-09678-x. Springer Netherlands 2022-01-08 2022 /pmc/articles/PMC8942963/ /pubmed/34997387 http://dx.doi.org/10.1007/s10577-021-09678-x Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Original Article
Watanabe, Reito
Hirano, Yasuhiro
Hara, Masatoshi
Hiraoka, Yasushi
Fukagawa, Tatsuo
Mobility of kinetochore proteins measured by FRAP analysis in living cells
title Mobility of kinetochore proteins measured by FRAP analysis in living cells
title_full Mobility of kinetochore proteins measured by FRAP analysis in living cells
title_fullStr Mobility of kinetochore proteins measured by FRAP analysis in living cells
title_full_unstemmed Mobility of kinetochore proteins measured by FRAP analysis in living cells
title_short Mobility of kinetochore proteins measured by FRAP analysis in living cells
title_sort mobility of kinetochore proteins measured by frap analysis in living cells
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8942963/
https://www.ncbi.nlm.nih.gov/pubmed/34997387
http://dx.doi.org/10.1007/s10577-021-09678-x
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