CTCF is essential for proper mitotic spindle structure and anaphase segregation
Mitosis is an essential process in which the duplicated genome is segregated equally into two daughter cells. CTCF has been reported to be present in mitosis and has a role in localizing CENP-E, but its importance for mitotic fidelity remains to be determined. To evaluate the importance of CTCF in m...
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| Veröffentlicht in: | Chromosoma 2024-07, Vol.133 (3), p.183-194 |
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| creator | Chiu, Katherine Berrada, Yasmin Eskndir, Nebiyat Song, Dasol Fong, Claire Naughton, Sarah Chen, Tina Moy, Savanna Gyurmey, Sarah James, Liam Ezeiruaku, Chimere Capistran, Caroline Lowey, Daniel Diwanji, Vedang Peterson, Samantha Parakh, Harshini Burgess, Ayanna R. Probert, Cassandra Zhu, Annie Anderson, Bryn Levi, Nehora Gerlitz, Gabi Packard, Mary C. Dorfman, Katherine A. Bahiru, Michael Seifu Stephens, Andrew D. |
| description | Mitosis is an essential process in which the duplicated genome is segregated equally into two daughter cells. CTCF has been reported to be present in mitosis and has a role in localizing CENP-E, but its importance for mitotic fidelity remains to be determined. To evaluate the importance of CTCF in mitosis, we tracked mitotic behaviors in wild-type and two different CTCF CRISPR-based genetic knockdowns. We find that knockdown of CTCF results in prolonged mitoses and failed anaphase segregation via time-lapse imaging of SiR-DNA. CTCF knockdown did not alter cell cycling or the mitotic checkpoint, which was activated upon nocodazole treatment. Immunofluorescence imaging of the mitotic spindle in CTCF knockdowns revealed disorganization via tri/tetrapolar spindles and chromosomes behind the spindle pole. Imaging of interphase nuclei showed that nuclear size increased drastically, consistent with failure to divide the duplicated genome in anaphase. Long-term inhibition of CNEP-E via GSK923295 recapitulates CTCF knockdown abnormal mitotic spindles with polar chromosomes and increased nuclear sizes. Population measurements of nuclear shape in CTCF knockdowns do not display decreased circularity or increased nuclear blebbing relative to wild-type. However, failed mitoses do display abnormal nuclear morphologies relative to successful mitoses, suggesting that population images do not capture individual behaviors. Thus, CTCF is important for both proper metaphase organization and anaphase segregation which impacts the size and shape of the interphase nucleus likely through its known role in recruiting CENP-E. |
| doi_str_mv | 10.1007/s00412-023-00810-w |
| format | Article |
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CTCF has been reported to be present in mitosis and has a role in localizing CENP-E, but its importance for mitotic fidelity remains to be determined. To evaluate the importance of CTCF in mitosis, we tracked mitotic behaviors in wild-type and two different CTCF CRISPR-based genetic knockdowns. We find that knockdown of CTCF results in prolonged mitoses and failed anaphase segregation via time-lapse imaging of SiR-DNA. CTCF knockdown did not alter cell cycling or the mitotic checkpoint, which was activated upon nocodazole treatment. Immunofluorescence imaging of the mitotic spindle in CTCF knockdowns revealed disorganization via tri/tetrapolar spindles and chromosomes behind the spindle pole. Imaging of interphase nuclei showed that nuclear size increased drastically, consistent with failure to divide the duplicated genome in anaphase. Long-term inhibition of CNEP-E via GSK923295 recapitulates CTCF knockdown abnormal mitotic spindles with polar chromosomes and increased nuclear sizes. Population measurements of nuclear shape in CTCF knockdowns do not display decreased circularity or increased nuclear blebbing relative to wild-type. However, failed mitoses do display abnormal nuclear morphologies relative to successful mitoses, suggesting that population images do not capture individual behaviors. Thus, CTCF is important for both proper metaphase organization and anaphase segregation which impacts the size and shape of the interphase nucleus likely through its known role in recruiting CENP-E.</description><identifier>ISSN: 0009-5915</identifier><identifier>ISSN: 1432-0886</identifier><identifier>EISSN: 1432-0886</identifier><identifier>DOI: 10.1007/s00412-023-00810-w</identifier><identifier>PMID: 37728741</identifier><language>eng</language><publisher>Berlin/Heidelberg: Springer Berlin Heidelberg</publisher><subject>Anaphase ; Animal Genetics and Genomics ; Biochemistry ; Biomedical and Life Sciences ; CCCTC-Binding Factor - genetics ; CCCTC-Binding Factor - metabolism ; Cell Biology ; Cell division ; Chromosome Segregation ; Chromosomes ; CRISPR ; Developmental Biology ; Eukaryotic Microbiology ; fluorescent antibody technique ; Gene Knockdown Techniques ; genome ; Genomes ; HeLa Cells ; Human Genetics ; Humans ; Immunofluorescence ; Interphase ; Life Sciences ; Metaphase ; Mitosis ; mitotic spindle apparatus ; Nocodazole ; Nuclei ; Spindle Apparatus - metabolism ; Spindles</subject><ispartof>Chromosoma, 2024-07, Vol.133 (3), p.183-194</ispartof><rights>The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.</rights><rights>2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c452t-3d1a8d155ce5aa34613e62c02aabf063fab88edc2bed393d57072a9166ef62753</citedby><cites>FETCH-LOGICAL-c452t-3d1a8d155ce5aa34613e62c02aabf063fab88edc2bed393d57072a9166ef62753</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00412-023-00810-w$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00412-023-00810-w$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,776,780,27903,27904,41467,42536,51298</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/37728741$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Chiu, Katherine</creatorcontrib><creatorcontrib>Berrada, Yasmin</creatorcontrib><creatorcontrib>Eskndir, Nebiyat</creatorcontrib><creatorcontrib>Song, Dasol</creatorcontrib><creatorcontrib>Fong, Claire</creatorcontrib><creatorcontrib>Naughton, Sarah</creatorcontrib><creatorcontrib>Chen, Tina</creatorcontrib><creatorcontrib>Moy, Savanna</creatorcontrib><creatorcontrib>Gyurmey, Sarah</creatorcontrib><creatorcontrib>James, Liam</creatorcontrib><creatorcontrib>Ezeiruaku, Chimere</creatorcontrib><creatorcontrib>Capistran, Caroline</creatorcontrib><creatorcontrib>Lowey, Daniel</creatorcontrib><creatorcontrib>Diwanji, Vedang</creatorcontrib><creatorcontrib>Peterson, Samantha</creatorcontrib><creatorcontrib>Parakh, Harshini</creatorcontrib><creatorcontrib>Burgess, Ayanna R.</creatorcontrib><creatorcontrib>Probert, Cassandra</creatorcontrib><creatorcontrib>Zhu, Annie</creatorcontrib><creatorcontrib>Anderson, Bryn</creatorcontrib><creatorcontrib>Levi, Nehora</creatorcontrib><creatorcontrib>Gerlitz, Gabi</creatorcontrib><creatorcontrib>Packard, Mary C.</creatorcontrib><creatorcontrib>Dorfman, Katherine A.</creatorcontrib><creatorcontrib>Bahiru, Michael Seifu</creatorcontrib><creatorcontrib>Stephens, Andrew D.</creatorcontrib><title>CTCF is essential for proper mitotic spindle structure and anaphase segregation</title><title>Chromosoma</title><addtitle>Chromosoma</addtitle><addtitle>Chromosoma</addtitle><description>Mitosis is an essential process in which the duplicated genome is segregated equally into two daughter cells. CTCF has been reported to be present in mitosis and has a role in localizing CENP-E, but its importance for mitotic fidelity remains to be determined. To evaluate the importance of CTCF in mitosis, we tracked mitotic behaviors in wild-type and two different CTCF CRISPR-based genetic knockdowns. We find that knockdown of CTCF results in prolonged mitoses and failed anaphase segregation via time-lapse imaging of SiR-DNA. CTCF knockdown did not alter cell cycling or the mitotic checkpoint, which was activated upon nocodazole treatment. Immunofluorescence imaging of the mitotic spindle in CTCF knockdowns revealed disorganization via tri/tetrapolar spindles and chromosomes behind the spindle pole. Imaging of interphase nuclei showed that nuclear size increased drastically, consistent with failure to divide the duplicated genome in anaphase. Long-term inhibition of CNEP-E via GSK923295 recapitulates CTCF knockdown abnormal mitotic spindles with polar chromosomes and increased nuclear sizes. Population measurements of nuclear shape in CTCF knockdowns do not display decreased circularity or increased nuclear blebbing relative to wild-type. However, failed mitoses do display abnormal nuclear morphologies relative to successful mitoses, suggesting that population images do not capture individual behaviors. Thus, CTCF is important for both proper metaphase organization and anaphase segregation which impacts the size and shape of the interphase nucleus likely through its known role in recruiting CENP-E.</description><subject>Anaphase</subject><subject>Animal Genetics and Genomics</subject><subject>Biochemistry</subject><subject>Biomedical and Life Sciences</subject><subject>CCCTC-Binding Factor - genetics</subject><subject>CCCTC-Binding Factor - metabolism</subject><subject>Cell Biology</subject><subject>Cell division</subject><subject>Chromosome Segregation</subject><subject>Chromosomes</subject><subject>CRISPR</subject><subject>Developmental Biology</subject><subject>Eukaryotic Microbiology</subject><subject>fluorescent antibody technique</subject><subject>Gene Knockdown Techniques</subject><subject>genome</subject><subject>Genomes</subject><subject>HeLa Cells</subject><subject>Human Genetics</subject><subject>Humans</subject><subject>Immunofluorescence</subject><subject>Interphase</subject><subject>Life Sciences</subject><subject>Metaphase</subject><subject>Mitosis</subject><subject>mitotic spindle apparatus</subject><subject>Nocodazole</subject><subject>Nuclei</subject><subject>Spindle Apparatus - metabolism</subject><subject>Spindles</subject><issn>0009-5915</issn><issn>1432-0886</issn><issn>1432-0886</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2024</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU1LwzAYx4Mobk6_gAcpePFSfZI0Lz1KcSoMdpnnkLVPZ0fX1qRl-O3N3FTwoIcQ8uT3_PPyI-SSwi0FUHceIKEsBsZjAE0h3h6RMU14KGktj8kYANJYpFSMyJn3692SSTglI64U0yqhYzLPFtk0qnyE3mPTV7aOytZFnWs7dNGm6tu-yiPfVU1RY-R7N-T94DCyTRGG7V6tD2VcOVzZvmqbc3JS2trjxWGekJfpwyJ7imfzx-fsfhbniWB9zAtqdUGFyFFYyxNJOUqWA7N2WYLkpV1qjUXOlljwlBdCgWI2pVJiKZkSfEJu9rnhpm8D-t5sKp9jXdsG28EbTgVXNFVS_YsyLaUSmrFd6vUvdN0OrgkPMRz07tek5IFieyp3rfcOS9O5amPdu6FgdmbM3owJZsynGbMNTVeH6GG5weK75UtFAPge8GGrWaH7OfuP2A9oFJin</recordid><startdate>20240701</startdate><enddate>20240701</enddate><creator>Chiu, Katherine</creator><creator>Berrada, Yasmin</creator><creator>Eskndir, Nebiyat</creator><creator>Song, Dasol</creator><creator>Fong, Claire</creator><creator>Naughton, Sarah</creator><creator>Chen, Tina</creator><creator>Moy, Savanna</creator><creator>Gyurmey, Sarah</creator><creator>James, Liam</creator><creator>Ezeiruaku, Chimere</creator><creator>Capistran, Caroline</creator><creator>Lowey, Daniel</creator><creator>Diwanji, Vedang</creator><creator>Peterson, Samantha</creator><creator>Parakh, Harshini</creator><creator>Burgess, Ayanna R.</creator><creator>Probert, Cassandra</creator><creator>Zhu, Annie</creator><creator>Anderson, Bryn</creator><creator>Levi, Nehora</creator><creator>Gerlitz, Gabi</creator><creator>Packard, Mary C.</creator><creator>Dorfman, Katherine A.</creator><creator>Bahiru, Michael Seifu</creator><creator>Stephens, Andrew D.</creator><general>Springer Berlin Heidelberg</general><general>Springer Nature B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QP</scope><scope>7QR</scope><scope>7TK</scope><scope>7TM</scope><scope>8FD</scope><scope>FR3</scope><scope>K9.</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>7S9</scope><scope>L.6</scope></search><sort><creationdate>20240701</creationdate><title>CTCF is essential for proper mitotic spindle structure and anaphase segregation</title><author>Chiu, Katherine ; Berrada, Yasmin ; Eskndir, Nebiyat ; Song, Dasol ; Fong, Claire ; Naughton, Sarah ; Chen, Tina ; Moy, Savanna ; Gyurmey, Sarah ; James, Liam ; Ezeiruaku, Chimere ; Capistran, Caroline ; Lowey, Daniel ; Diwanji, Vedang ; Peterson, Samantha ; Parakh, Harshini ; Burgess, Ayanna R. ; Probert, Cassandra ; Zhu, Annie ; Anderson, Bryn ; Levi, Nehora ; Gerlitz, Gabi ; Packard, Mary C. ; Dorfman, Katherine A. ; Bahiru, Michael Seifu ; Stephens, Andrew D.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c452t-3d1a8d155ce5aa34613e62c02aabf063fab88edc2bed393d57072a9166ef62753</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2024</creationdate><topic>Anaphase</topic><topic>Animal Genetics and Genomics</topic><topic>Biochemistry</topic><topic>Biomedical and Life Sciences</topic><topic>CCCTC-Binding Factor - 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CTCF has been reported to be present in mitosis and has a role in localizing CENP-E, but its importance for mitotic fidelity remains to be determined. To evaluate the importance of CTCF in mitosis, we tracked mitotic behaviors in wild-type and two different CTCF CRISPR-based genetic knockdowns. We find that knockdown of CTCF results in prolonged mitoses and failed anaphase segregation via time-lapse imaging of SiR-DNA. CTCF knockdown did not alter cell cycling or the mitotic checkpoint, which was activated upon nocodazole treatment. Immunofluorescence imaging of the mitotic spindle in CTCF knockdowns revealed disorganization via tri/tetrapolar spindles and chromosomes behind the spindle pole. Imaging of interphase nuclei showed that nuclear size increased drastically, consistent with failure to divide the duplicated genome in anaphase. Long-term inhibition of CNEP-E via GSK923295 recapitulates CTCF knockdown abnormal mitotic spindles with polar chromosomes and increased nuclear sizes. 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| ispartof | Chromosoma, 2024-07, Vol.133 (3), p.183-194 |
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| language | eng |
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| subjects | Anaphase Animal Genetics and Genomics Biochemistry Biomedical and Life Sciences CCCTC-Binding Factor - genetics CCCTC-Binding Factor - metabolism Cell Biology Cell division Chromosome Segregation Chromosomes CRISPR Developmental Biology Eukaryotic Microbiology fluorescent antibody technique Gene Knockdown Techniques genome Genomes HeLa Cells Human Genetics Humans Immunofluorescence Interphase Life Sciences Metaphase Mitosis mitotic spindle apparatus Nocodazole Nuclei Spindle Apparatus - metabolism Spindles |
| title | CTCF is essential for proper mitotic spindle structure and anaphase segregation |
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