{"title":"FRAP法测定活细胞中着丝点蛋白的迁移率。","authors":"Reito Watanabe, Yasuhiro Hirano, Masatoshi Hara, Yasushi Hiraoka, Tatsuo Fukagawa","doi":"10.1007/s10577-021-09678-x","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":50698,"journal":{"name":"Chromosome Research","volume":null,"pages":null},"PeriodicalIF":2.4000,"publicationDate":"2022-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8942963/pdf/","citationCount":"9","resultStr":"{\"title\":\"Mobility of kinetochore proteins measured by FRAP analysis in living cells.\",\"authors\":\"Reito Watanabe, Yasuhiro Hirano, Masatoshi Hara, Yasushi Hiraoka, Tatsuo Fukagawa\",\"doi\":\"10.1007/s10577-021-09678-x\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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.</p>\",\"PeriodicalId\":50698,\"journal\":{\"name\":\"Chromosome Research\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.4000,\"publicationDate\":\"2022-03-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8942963/pdf/\",\"citationCount\":\"9\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Chromosome Research\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1007/s10577-021-09678-x\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2022/1/8 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q3\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Chromosome Research","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1007/s10577-021-09678-x","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2022/1/8 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
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 (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.
期刊介绍：
Chromosome Research publishes manuscripts from work based on all organisms and encourages submissions in the following areas including, but not limited, to:
· Chromosomes and their linkage to diseases;
· Chromosome organization within the nucleus;
· Chromatin biology (transcription, non-coding RNA, etc);
· Chromosome structure, function and mechanics;
· Chromosome and DNA repair;
· Epigenetic chromosomal functions (centromeres, telomeres, replication, imprinting,
dosage compensation, sex determination, chromosome remodeling);
· Architectural/epigenomic organization of the genome;
· Functional annotation of the genome;
· Functional and comparative genomics in plants and animals;
· Karyology studies that help resolve difficult taxonomic problems or that provide
clues to fundamental mechanisms of genome and karyotype evolution in plants and animals;
· Mitosis and Meiosis;
· Cancer cytogenomics.
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