CTCF is essential for proper mitotic spindle structure and anaphase segregation.
CTCF is essential for proper mitotic spindle structure and anaphase segregation.
复制标题
CTCF 对于适当的有丝分裂纺锤体结构和后期分离至关重要。
DOI：
10.1101/2023.01.09.523293
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Parakh,Har
中科院分区：
文献类型：
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作者：
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,Har
有丝分裂是一个重要的过程，在这个过程中，复制的基因组被均等地分离成两个子细胞。据报道，CTCF存在于有丝分裂中，并在定位CENP-E中发挥作用，但其对有丝分裂保真度的重要性仍有待确定。为了评估CTCF在有丝分裂中的重要性，我们跟踪了野生型和两种不同的基于CTCF CRISPR的遗传敲减中的有丝分裂行为。我们发现，敲除CTCF的结果在延长有丝分裂和失败的后期分离，通过延时成像的SiR-DNA。CTCF敲除不改变细胞周期或有丝分裂检查点，其在诺考达唑处理后被激活。CTCF敲除中的有丝分裂纺锤体的免疫荧光成像显示通过三/四极纺锤体和纺锤体极后面的染色体的解体。间期细胞核的成像显示，细胞核的大小急剧增加，与分裂后期复制的基因组失败相一致。通过GSK 923295对CNEP-E的长期抑制再现了CTCF敲低异常有丝分裂纺锤体与极性染色体和增加的核大小。在CTCF敲低中细胞核形状的群体测量相对于野生型不显示圆形度降低或核起泡增加。然而，相对于成功的有丝分裂，失败的有丝分裂确实显示出异常的核形态，这表明群体图像不能捕获个体行为。因此，CTCF对于适当的中期组织和后期分离都很重要，这可能通过其在募集CENP-E中的已知作用影响间期核的大小和形状。
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.