Related Experiment Video
Updated: Feb 15, 2026
10:34
FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors
Published on: August 20, 2012
23.8K
Directed Evolution to Engineer Monobody for FRET Biosensor Assembly and Imaging at Live-Cell Surface
Praopim Limsakul1, Qin Peng1, Yiqian Wu1
1Department of Bioengineering, Institute of Engineering in Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
Cell Chemical Biology
|February 4, 2018
Summary
Researchers developed a novel hybrid biosensor for monitoring cell surface enzyme activity. This new tool visualizes matrix metalloproteinase activity at intercellular junctions, offering insights into cell-cell contact dynamics.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Engineering
Background:
- Monitoring cell surface enzymatic activities is difficult due to limitations with traditional fluorescence resonance energy transfer (FRET)-based biosensors.
- Existing biosensors face challenges with transport efficiency and membrane integration.
- A novel approach is needed to overcome these limitations for real-time cell surface monitoring.
Purpose of the Study:
- To engineer a novel hybrid FRET biosensor for monitoring enzymatic activities at the cell surface.
- To visualize the dynamic formation and separation of intercellular junctions.
- To investigate the activity of membrane-type-1 matrix metalloproteinase (MT1-MMP) at cell-cell contacts.
Main Methods:
- Developed a hybrid biosensor with in situ assembly of separate donor and acceptor components.
- Utilized directed evolution and sequence-function analysis to engineer a monobody variant (PEbody) that binds R-phycoerythrin (R-PE).
- Fused PEbody with enhanced CFP and an enzyme-specific peptide for MT1-MMP activity monitoring.
Main Results:
- Successfully visualized dynamic intercellular junction formation and separation using the engineered PEbody.
- The hybrid FRET biosensor enabled monitoring of MT1-MMP activities upon R-PE capture.
- Revealed asymmetric MT1-MMP activity distribution, with higher activity at loose cell-cell contacts and lower activity at stable contacts.
Conclusions:
- Directed evolution and rational design are effective strategies for engineering molecular binders and hybrid FRET biosensors.
- The developed biosensor allows for monitoring molecular regulations at the surface of living cells.
- This technology provides new insights into the spatial and temporal regulation of cell surface enzymes like MT1-MMP.
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