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Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET
Published on: January 27, 2012
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Live Cell Monitoring of Separase Activity, a Key Enzymatic Reaction for Chromosome Segregation, with Chimeric
Md Shazadur Rahman1,2, Yutaka Shindo3, Kotaro Oka3,4
1Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.
Biosensors
|April 26, 2024
Summary
Researchers developed novel FRET-based molecular sensors to monitor separase activity in live cells, crucial for understanding chromosome segregation and aneuploidy in cancer. These sensors enable precise, real-time analysis of separase function during mitosis.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Separase is a critical cysteine protease regulating sister chromatid separation by cleaving cohesin, essential for the metaphase-anaphase transition.
- Separase activity is tightly controlled by securin and cyclinB-CDK1, preventing premature chromosome segregation.
- Chromosome missegregation and aneuploidy are hallmarks of cancer, yet studying separase activity in live cells is challenging due to its instability and limited monitoring tools.
Purpose of the Study:
- To develop and validate Förster Resonance Energy Transfer (FRET)-based molecular sensors for monitoring live separase activity.
- To assess the utility of these sensors for both specific and statistical analyses of separase function in live cells.
- To overcome limitations in biochemical examination and spatiotemporal resolution of separase activity.
Main Methods:
- Development of FRET-based molecular sensors utilizing GFP variants as donors and fluorescent dyes as acceptors, with separase-cleavable sequences.
- Application of sensors for live cell imaging and flow cytometry, leveraging efficient cellular uptake.
- Investigation of sensor performance with and without nuclear localization under controlled cell cycle conditions.
Main Results:
- Successful development of FRET-based sensors applicable to conventional live cell imaging and flow cytometry.
- Demonstrated significant detection of separase activity during synchronized cell cycle progression using both sensor types.
- Validated sensor performance through fluorescent microscopy, showing consistent results for localized sensors.
Conclusions:
- Established effective live cell monitoring systems for separase activity.
- These systems enable specific and statistical analyses, facilitating detailed elucidation of separase properties.
- The developed sensors offer a valuable tool for studying chromosome segregation dynamics and related pathologies like aneuploidy.
Related Concept Videos
02:17Separation of Sister Chromatids
At the transition from prophase to metaphase, there is a reduction in cohesion along the chromosomal arms, resulting in the resolution of sister chromatids. However, residual cohesin connections remain to hold the sister chromatids together until the transition from metaphase to anaphase. The residual connection prevents any premature separation of sister chromatids, blocking the risks of aneuploidy within the daughter cells.
At the onset of anaphase, separase, a proteolytic enzyme, is...
At the onset of anaphase, separase, a proteolytic enzyme, is...
02:19The Spindle Assembly Checkpoint
The spindle assembly checkpoint is a molecular surveillance mechanism ensuring the fidelity of chromosome segregation during anaphase. The checkpoint monitors the completion of all the prerequisite steps before chromosome segregation to determine whether the segregation process should proceed or be delayed.
Many proteins function together to control the spindle assembly checkpoint. Mutations affecting these proteins may allow cells to proceed into anaphase prematurely, resulting in the...
Many proteins function together to control the spindle assembly checkpoint. Mutations affecting these proteins may allow cells to proceed into anaphase prematurely, resulting in the...