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Genetically encoded fluorescent biosensors for live-cell imaging of MT1-MMP protease activity
Mingxing Ouyang1, Shaoying Lu, Yingxiao Wang
1Department of Bioengineering, University of Illinois at Urbana-Champaign, Champaign, IL, USA.
Abstract:
The proteolytic activity of Membrane-type 1 Matrix Metalloproteinase (MT1-MMP) is crucial for cancer cell invasion and metastasis. To visualize the protease activity of MT1-MMP with high spatiotemporal resolution at the extracellular plasma membrane surface of live cancer cells, a genetically encoded fluorescent biosensor of MT1-MMP has been developed. Here we describe the design principles of the MT1-MMP biosensor, the characterization of the MT1-MMP biosensor in vitro, and the live-cell imaging protocol used to visualize MT1-MMP activity in mammalian cells. We also provide brief guidelines for observing MT1-MMP subcellular activity by fluorescence resonance energy transfer (FRET) in a cell migration assay.
Insights
A new fluorescent biosensor visualizes Membrane-type 1 Matrix Metalloproteinase (MT1-MMP) activity in live cancer cells. This tool aids in understanding cancer cell invasion and metastasis at the cell surface.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Membrane-type 1 Matrix Metalloproteinase (MT1-MMP) is a key enzyme in cancer cell invasion and metastasis.
- Visualizing MT1-MMP activity in real-time is essential for understanding cancer progression.
Purpose of the Study:
- To develop a genetically encoded fluorescent biosensor for MT1-MMP.
- To enable high spatiotemporal resolution imaging of MT1-MMP activity on live cancer cell surfaces.
Main Methods:
- Design and in vitro characterization of the MT1-MMP biosensor.
- Live-cell imaging protocols for mammalian cells.
- Fluorescence resonance energy transfer (FRET) for observing subcellular MT1-MMP activity.
Main Results:
- Successful development of a functional MT1-MMP fluorescent biosensor.
- Demonstration of the biosensor's utility in live-cell imaging.
- Guidelines provided for FRET-based observation of MT1-MMP activity.
Conclusions:
- The developed biosensor provides a novel tool for studying MT1-MMP.
- This technology facilitates research into cancer cell invasion and metastasis mechanisms.
- Enables detailed analysis of MT1-MMP's role in cellular processes.
