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FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors
Published on: August 20, 2012
Detection of MMP activity in living cells by a genetically encoded surface-displayed FRET sensor
Jie Yang1, Zhihong Zhang, Juqiang Lin
1The Key Laboratory of Biomedical Photonics of Ministry of Education, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, PR China.
Abstract:
Matrix metalloproteinases (MMPs) are secretory endopeptidases. They have been associated with invasion by cancer-cell and metastasis. Previous studies have demonstrated that proteolytic activity could be detected using fluorescence resonance energy transfer (FRET) with mutants of GFP. To monitor MMP activity, we constructed vectors that encoded a MMP Substrate Site (MSS) between YFP and CFP. In vitro, YFP-MSS-CFP can be used to detect MMP activity and 1,10-phenathroline inhibition of MMP activity. In living cells, MMPs are secreted proteins and act outside of the cell, and therefore YFP-MSS-CFPdisplay was anchored on the cellular surface to detect extracellular MMP. A pDisplay-YC vector expressing the YFP-MSS-CFPdisplay on the cellular surface was transfected into MCF-7 cells that expressed low levels of MMP. Efficient transfer of energy from excited CFP to YFP within the YFP-MSS-CFPdisplay molecule was observed, and real-time FRET was declined when MCF-7 was incubated with MMP2. However, no such transfer of energy was detected in the YFP-MSS-CFPdisplay expressing MDA-MB 435s cells, in which high secretory MMP2 were expressed. The FRET sensor YFP-MSS-CFPdisplay can sensitively and reliably monitor MMP activation in living cells and can be used for high-throughput screening of MMP inhibitors for anti-cancer treatments.
Insights
Researchers developed a novel FRET sensor to detect matrix metalloproteinase (MMP) activity in living cells. This tool enables sensitive monitoring of MMPs, crucial for understanding cancer metastasis and screening potential anti-cancer drugs.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Matrix metalloproteinases (MMPs) are enzymes linked to cancer invasion and metastasis.
- Fluorescence resonance energy transfer (FRET) with GFP mutants can detect proteolytic activity.
- Monitoring extracellular MMP activity in living cells is challenging.
Purpose of the Study:
- To develop and validate a novel FRET-based sensor for detecting extracellular MMP activity in living cells.
- To assess the sensor's ability to monitor MMP activity and inhibition in real-time.
- To evaluate the sensor's utility for high-throughput screening of MMP inhibitors.
Main Methods:
- Constructed a YFP-MSS-CFP vector for MMP substrate detection.
- Anchored the YFP-MSS-CFP sensor to the cellular surface (YFP-MSS-CFPdisplay).
- Transfected MCF-7 and MDA-MB 435s cells with the sensor and analyzed FRET signals before and after MMP2 treatment.
Main Results:
- The YFP-MSS-CFPdisplay sensor detected MMP activity in vitro and in living cells.
- Real-time FRET signal decreased in MCF-7 cells upon MMP2 incubation, indicating MMP activity.
- No significant FRET signal change was observed in MDA-MB 435s cells, correlating with high endogenous MMP2 levels.
Conclusions:
- The YFP-MSS-CFPdisplay FRET sensor reliably monitors extracellular MMP activation in living cells.
- This sensor is suitable for sensitive, real-time detection of MMP activity.
- The developed sensor can be applied for high-throughput screening of anti-cancer drugs targeting MMPs.
