Related Experiment Video
Updated: Oct 27, 2025
Visualization of DNA Repair Proteins Interaction by Immunofluorescence
Published on: June 26, 2020
A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced
Eric Moeglin1, Dominique Desplancq1, Audrey Stoessel1
1Biotechnologie et Signalisation Cellulaire, UMR 7242, CNRS/Université de Strasbourg, Boulevard S. Brant, 67412 Illkirch, France.
Abstract:
Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.
Insights
Researchers developed novel nanobodies to detect phosphorylated H2AX (γ-H2AX), a marker of DNA damage, in living cells. This breakthrough enables real-time monitoring of DNA repair dynamics and drug screening in cancer research.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Phosphorylated H2AX (γ-H2AX) is a critical marker for DNA double-strand breaks and replication stress in mammalian cells.
- Current methods for γ-H2AX detection are limited to fixed cells, hindering real-time analysis of DNA damage responses.
Purpose of the Study:
- To develop novel tools for detecting γ-H2AX in living cells.
- To enable dynamic studies of chromatin modifications and facilitate genotoxic drug screening.
Main Methods:
- Isolation of γ-H2AX-specific nanobodies using immune libraries and phage display.
- Crystal structure determination of nanobody-phosphopeptide complex.
- Engineering of bivalent nanobodies and fluorescent fusion proteins.
- Development of a transduction strategy for live-cell imaging.
Main Results:
- Successfully isolated and characterized nanobodies with high specificity for γ-H2AX.
- Demonstrated quantitative visualization of γ-H2AX in fixed cells using bivalent nanobodies.
- Achieved sensitive detection of γ-H2AX foci in intact living cells using fluorescent nanobody fusion proteins.
Conclusions:
- Developed a novel nanobody-based tool for sensitive and precise detection of γ-H2AX in both fixed and living cells.
- This tool facilitates rapid screening of genotoxic drugs and allows in-depth study of DNA damage dynamics in individual cells.
