Abstract
The metaphase-anaphase transition is orchestrated through proteolysis of numerous proteins by a ubiquitin protein ligase called the anaphase-promoting complex or cyclosome (APC/C). A crucial aspect of this process is sister chromatid separation, which is thought to be mediated by separase, a thiol protease activated by the APC/C. Separase cleaves cohesin, a ring-shaped complex that entraps sister DNAs. It is a matter of debate whether cohesin-independent forces also contribute to sister chromatid cohesion. Using 4D live-cell imaging of Drosophila melanogaster syncytial embryos blocked in metaphase (via APC/C inhibition), we show that artificial cohesin cleavage is sufficient to trigger chromosome disjunction. This is nevertheless insufficient for correct chromosome segregation. Kinetochore-microtubule attachments are rapidly destabilized by the loss of tension caused by cohesin cleavage in the presence of high Cdk1 (cyclin-dependent kinase 1) activity, as occurs when the APC/C cannot destroy mitotic cyclins. Metaphase chromosomes undergo a bona fide anaphase when cohesin cleavage is combined with Cdk1 inhibition. We conclude that only two key events, opening of cohesin rings and downregulation of Cdk1, are sufficient to drive proper segregation of chromosomes in anaphase.
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📊 Figures
Figure 1
Injection of Mad2 L13Q or UbcH10 C114S prevents anaphase onset in Drosophila syncytial embryos
Embryos expressing His2A-mRFP1 were (a) not injected, (b) injected with 25 mg/ml of active Mad2 (Mad2 L13Q ) or (c) injected with 30 mg/ml of a dominant negative form of the human E2 ubiquitin-conjuga...
Figure 2
Cleavage of cohesin is sufficient to trigger sister chromatid disjunction
Embryos containing TEV-cleavable Rad21 (Rad21 TEV ) as their sole source of Rad21 were either unperturbed (control) or arrested in metaphase by injection of Mad2 L13Q , followed by injection of either...
Figure 3
Loss of cohesin leads to unstable kinetochore-microtubule attachments
Rad21 TEV embryos were injected with TEV protease to trigger sister centromere disjunction in Mad2 L13Q -induced metaphase-arrested embryos. a) Selected stills from a movie showing His2Av-mRFP1 (red) ...
Figure 4
Localization of Aurora B and BubRI after TEV-induced sister chromatid disjunction
Early embryos surviving on Rad21 TEV and expressing His2A-mRFP1 (red) were injected with mRNA encoding Aurora B-EGFP (a) or EGFP-BubRI (b) (shown in green); a) upper panel - Aurora B localization in a...
Figure 5
Cleavage of cohesin and Cdk inhibition trigger formation of daughter nuclei
Embryos surviving on Rad21 TEV were either uninjected (control) or arrested in metaphase with Mad2 L13Q or UbcH10 C114S and subsequently co-injected with TEV protease and the CDK inhibitor human p27. ...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
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