Abstract
Separase proteolytically removes cohesin complexes from sister chromatid arms in meiosis I, which is essential for chromosome segregation. Regulation of separase activity is essential for proper cell cycle progression and correct chromosome segregation. Onset of endogenous separase activity has not yet been observed in live oocytes.
We describe here a method for detecting separase activity in mouse oocytes in vivo. This method utilizes a previously described cleavage sensor made up of H2B-mCherry fused with Scc1(107–268 aa)-YFP. The cleavage sensor is loaded on the chromosomes through its H2B-tag, and the signal from both mCherry and YFP is visible. Upon separase activation the Scc1 fragment is cleaved and YFP dissociates from the chromosomes. The change in the ratio between mCherry and YFP fluorescence intensity is a readout of separase activity.
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Acknowledgments
We thank Professor Olaf Stemmann for providing the cleavage sensor construct. This work was supported by a postdoctoral fellowship by the Fondation de la Recherche Médicale (SPF20150934093) and an EMBO short-term fellowship (ASTF 426-2015) for E.N., and the French Ministry of Research through the Doctoral School of Physiology, Physiopathology and Therapeutics for N.B. K. Wassmann acknowledges the financial support through grants by the Agence Nationale de la Recherche (ANR-16-CE92-0007-01), a grant “Equipe FRM” by the Fondation de la Recherche Médicale (Equipe DEQ20160334921), and core funding by UPMC and CNRS.
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Nikalayevich, E., Bouftas, N., Wassmann, K. (2018). Detection of Separase Activity Using a Cleavage Sensor in Live Mouse Oocytes. In: Verlhac, MH., Terret, ME. (eds) Mouse Oocyte Development. Methods in Molecular Biology, vol 1818. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8603-3_11
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DOI: https://doi.org/10.1007/978-1-4939-8603-3_11
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