Detection of Separase Activity Using a Cleavage Sensor in Live Mouse Oocytes
Elvira Nikalayevich1,2, Nora Bouftas3,4, Katja Wassmann5,6
1Sorbonne Universités, UPMC Université Paris 06, Institut de Biologie Paris Seine (IBPS), UMR7622, Paris, France. elvira.nikalayevich@upmc.fr.
Methods in Molecular Biology (Clifton, N.J.)
|July 2, 2018
Summary
Researchers developed a novel method to detect separase activity in live mouse oocytes. This technique visualizes cohesin cleavage, crucial for accurate chromosome segregation during meiosis I.
Area of Science:
- Cell Biology
- Molecular Biology
- Reproductive Biology
Background:
- Separase enzyme activity is critical for proper chromosome segregation during meiosis I.
- Regulating separase is essential for cell cycle progression and accurate cell division.
- Endogenous separase activity has not been directly observed in live oocytes previously.
Purpose of the Study:
- To establish a method for detecting endogenous separase activity in live mouse oocytes.
- To visualize the cleavage of cohesin complexes by separase in real-time within oocytes.
Main Methods:
- Development of a cleavage sensor using H2B-mCherry fused with Scc1(107-268 aa)-YFP.
- Loading the sensor onto chromosomes via the H2B-tag for fluorescence detection (mCherry and YFP).
- Monitoring the change in the mCherry/YFP fluorescence ratio as a readout for separase-mediated cleavage.
Main Results:
- Successfully detected separase activity in live mouse oocytes in vivo.
- Demonstrated that separase activation leads to the cleavage of the Scc1 fragment, causing YFP dissociation.
- Quantified separase activity by measuring the alteration in the fluorescence intensity ratio.
Conclusions:
- The developed method allows for in vivo detection and monitoring of separase activity in mouse oocytes.
- This advancement provides a new tool to study chromosome segregation regulation during oogenesis.
- Understanding separase dynamics is key to ensuring proper reproductive cell division.
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