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Multiple roles for separase auto-cleavage during the G2/M transition

Nature Cell Biology volume 7, pages 1029–1035 (2005)Cite this article

Abstract

The cysteine protease separase triggers anaphase onset by cleaving chromosome-bound cohesin. In humans, separase also cleaves itself at multiple sites, but the biological significance of this reaction has been elusive. Here we show that preventing separase auto-cleavage, via targeted mutagenesis of the endogenous hSeparase locus in somatic cells, interferes with entry into and progression through mitosis. The initial delay in mitotic entry was not dependent on the G2 DNA damage checkpoint, but rather involved improper stabilization of the mitosis-inhibiting kinase Wee1. During M phase, cells deficient in separase auto-cleavage exhibited striking defects in spindle assembly and metaphase chromosome alignment, revealing an additional early mitotic function for separase. Both the G2 and M phase phenotypes could be recapitulated by separase RNA interference and corrected by re-expressing wild-type separase in trans. We conclude that separase auto-cleavage coordinates multiple aspects of the G2/M programme in human cells, thus contributing to the timing and efficiency of chromosome segregation.

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Figure 1: Generation of human cells deficient in separase auto-cleavage.
Figure 2: Preventing separase auto-cleavage retards mitotic entry.
Figure 3: NC3/NC2 cells are delayed in late G2 by a checkpoint-independent mechanism involving Wee1.
Figure 4: NC3/NC2 cells exhibit intra-mitotic defects in spindle assembly and metaphase chromosome alignment.
Figure 5: Rescue of mitotic entry and chromosome alignment defects by re-expression of wild-type separase.

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Acknowledgements

The authors thank A. Chestukhin for providing the XJ11-1B12 antibody and F. Weis-Garcia for expert assistance with hybridoma screening and monoclonal antibody production. M.P. was supported through an American-Italian Cancer Foundation postdoctoral fellowship. Additional support was provided to P.V.J. by the Pew Scholars Program in the Biomedical Sciences, the V Foundation for Cancer Research, and the National Cancer Institute, National Institutes of Health.

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  1. Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, 10021, NY, USA

    Maura Papi, Eli Berdougo, Catherine L. Randall, Sonali Ganguly & Prasad V. Jallepalli

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Papi, M., Berdougo, E., Randall, C. et al. Multiple roles for separase auto-cleavage during the G2/M transition. Nat Cell Biol 7, 1029–1035 (2005). https://doi.org/10.1038/ncb1303

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