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Quantitative and Dynamic Imaging of ATM Kinase Activity.
Shyam Nyati1,2, Grant Young3, Brian Dale Ross4,5
1Center for Molecular Imaging, University of Michigan, Ann Arbor, MI, 48109, USA. shyamnya@med.umich.edu.
Methods in Molecular Biology (Clifton, N.J.)
|March 16, 2017
Summary
We developed a novel bioluminescent biosensor to measure Ataxia telangiectasia mutated (ATM) kinase activity in live cells. This tool offers a noninvasive method to track DNA damage response, crucial for understanding cellular health and disease.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Ataxia telangiectasia mutated (ATM) is a key kinase in DNA damage response.
- Traditional kinase assays are invasive and require significant cell numbers.
- Live-cell imaging offers a noninvasive alternative for studying kinase activity.
Purpose of the Study:
- To develop a genetically encoded biosensor for real-time monitoring of ATM kinase activity.
- To enable noninvasive assessment of the cellular DNA damage response.
Main Methods:
- Genetically engineered a hybrid protein utilizing a split luciferase system.
- Incorporated a CHK2 target sequence and a phospho-serine-binding domain (FHA2).
- Utilized bioluminescence imaging to quantify ATM activity in cultured cells and mouse xenografts.
Main Results:
- The biosensor's bioluminescent activity is inversely correlated with ATM phosphorylation.
- Demonstrated quantitative measurement of ATM kinase activity in a noninvasive, dynamic manner.
- Successfully imaged reporter-expressing cells in vitro and in vivo.
Conclusions:
- The developed biosensor provides a sensitive and quantitative surrogate for ATM kinase activity.
- This tool facilitates noninvasive monitoring of the DNA damage response in biological systems.
- Enables dynamic studies of cellular responses to DNA damage.
