Biomaterial Database

CTCF is essential for proper mitotic spindle structure and anaphase segregation. 2023

Katherine Chiu, and Yasmin Berrada, and Nebiyat Eskndir, and Dasol Song, and Claire Fong, and Sarah Naughton, and Tina Chen, and Savanna Moy, and Sarah Gyurmey, and Liam James, and Chimere Ezeiruaku, and Caroline Capistran, and Daniel Lowey, and Vedang Diwanji, and Samantha Peterson, and Harshini Parakh, and Ayanna R Burgess, and Cassandra Probert, and Annie Zhu, and Bryn Anderson, and Nehora Levi, and Gabi Gerlitz, and Mary C Packard, and Katherine A Dorfman, and Michael Seifu Bahiru, and Andrew D Stephens

Biology Department, University of Massachusetts Amherst, Amherst, MA, 01003, USA.

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.