Mammalian mad2 and bub1/bubR1 recognize distinct spindle-attachment and kinetochore-tension checkpoints
D A Skoufias1, P R Andreassen, F B Lacroix
1Institut de Biologie Structurale J.-P. Ebel (Commissariat à l'Energie Atomique-Centre National de la Recherche Scientifique), 41 Rue Jules Horowitz, 38027 Grenoble Cedex 1, France.
Abstract:
Metaphase checkpoint controls sense abnormalities of chromosome alignment during mitosis and prevent progression to anaphase until proper alignment has been attained. A number of proteins, including mad2, bub1, and bubR1, have been implicated in the metaphase checkpoint control in mammalian cells. Metaphase checkpoints have been shown, in various systems, to read loss of either spindle tension or microtubule attachment at the kinetochore. Characteristically, HeLa cells arrest in metaphase in response to low levels of microtubule inhibitors that leave an intact spindle and a metaphase plate. Here we show that the arrest induced by nanomolar vinblastine correlates with loss of tension at the kinetochore, and that in response the checkpoint proteins bub1 and bubR1 are recruited to the kinetochore but mad2 is not. mad2 remains competent to respond and is recruited at higher drug doses that disrupt spindle association with the kinetochores. Further, although mad2 forms a complex with cdc20, it does not associate with bub1 or bubR1. We conclude that mammalian bub1/bubR1 and mad2 operate as elements of distinct pathways sensing tension and attachment, respectively.
Insights
Mammalian cells use distinct pathways to ensure proper chromosome alignment during mitosis. The study reveals that bub1/bubR1 proteins sense tension, while mad2 detects microtubule attachment at kinetochores.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Metaphase checkpoint controls ensure accurate chromosome segregation during mitosis.
- Key proteins like mad2, bub1, and bubR1 are involved in mammalian cell metaphase checkpoint control.
- These checkpoints monitor spindle tension and microtubule attachment at kinetochores.
Purpose of the Study:
- To investigate the distinct roles of checkpoint proteins in sensing kinetochore tension versus microtubule attachment.
- To elucidate the molecular mechanisms underlying metaphase arrest in response to microtubule inhibitors.
Main Methods:
- Utilized nanomolar vinblastine treatment in HeLa cells to induce metaphase arrest.
- Monitored recruitment of checkpoint proteins (bub1, bubR1, mad2) to kinetochores.
- Analyzed protein complex formation, including mad2-cdc20 interactions.
Main Results:
- Vinblastine-induced arrest at low doses correlates with loss of kinetochore tension.
- Bub1 and bubR1 are recruited to kinetochores upon tension loss, but mad2 is not.
- Mad2 is recruited only at higher drug concentrations that disrupt spindle-kinetochore attachment.
- Mad2 forms complexes with cdc20 but not with bub1 or bubR1.
Conclusions:
- Mammalian cells employ distinct pathways for metaphase checkpoint control.
- Bub1/bubR1 proteins act in a pathway sensing spindle tension.
- Mad2 operates in a separate pathway that detects microtubule attachment to kinetochores.
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