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Abstract
Chromosome segregation requires coordinated separation of sister chromatids following biorientation of all chromosomes on the mitotic spindle. Chromatid separation at the metaphase-to-anaphase transition is accomplished by cleavage of the cohesin complex that holds chromatids together. Here we show using live-cell imaging that extending the metaphase bioriented state using five independent perturbations (expression of non-degradable Cyclin B, expression of a Spindly point mutant that prevents spindle checkpoint silencing, depletion of the anaphase inducer Cdc20, treatment with a proteasome inhibitor, or treatment with an inhibitor of the mitotic kinesin CENP-E) leads to eventual scattering of chromosomes on the spindle. This scattering phenotype is characterized by uncoordinated loss of cohesion between some, but not all sister chromatids and subsequent spindle defects that include centriole separation. Cells with scattered chromosomes persist long-term in a mitotic state and eventually die or exit. Partial cohesion loss-associated scattering is observed in both transformed cells and in karyotypically normal human cells, albeit at lower penetrance. Suppressing microtubule dynamics reduces scattering, suggesting that cohesion at centromeres is unable to resist dynamic microtubule-dependent pulling forces on the kinetochores. Consistent with this view, strengthening cohesion by inhibiting the two pathways responsible for its removal significantly inhibits scattering. These results establish that chromosome scattering due to uncoordinated partial loss of chromatid cohesion is a common outcome following extended arrest with bioriented chromosomes in human cells. These findings have important implications for analysis of mitotic phenotypes in human cells and for development of anti-mitotic chemotherapeutic approaches in the treatment of cancer.
Highlights
Accurate passage through mitosis is a highly orchestrated process that relies on the timely coordination of multiple events to ensure proper segregation of genetic material
We describe an aberrant mitotic state that is induced in karyotypically normal and abnormal human cells by multiple perturbations that prevent normal progression into anaphase
Entry into this state is triggered by partial and uncoordinated loss of sister chromatid cohesion on chromosomes aligned at the metaphase plate
Summary
Accurate passage through mitosis is a highly orchestrated process that relies on the timely coordination of multiple events to ensure proper segregation of genetic material. A majority of the cohesin resides on the chromosome arms and is removed at the beginning of mitosis, whereas centromeric cohesin remains bound until the metaphase-to-anaphase transition [3]. Separase is activated at anaphase onset when the anaphase promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase, targets its inhibitor securin for degradation and reduces Cdk activity [10,11]. The APC/C activity targeting securin is inhibited by the spindle assembly checkpoint until all chromosomes are fully aligned on the metaphase plate. When the last pair of chromatids properly aligns, the checkpoint is turned off, which leads to APC/C-mediated degradation of securin, and in turn activates separase. Separase cleaves the centromeric cohesin in a coordinated manner so that cohesin is lost from all sister chromatids as the cell enters anaphase. Previous studies have investigated the consequences of uncoupling these regulated events and have shown how important their coordination is for proper chromosome segregation and progression through mitosis [12,13,14]
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