Collagen-Immobilized Extracellular FRET Reporter for Visualizing Protease Activity Secreted by Living Cells
- Title
- Collagen-Immobilized Extracellular FRET Reporter for Visualizing Protease Activity Secreted by Living Cells
- Author
- 김영필
- Keywords
- protease activity; MMP; FRET; collagen; intein; ECM
- Issue Date
- 2020-02
- Publisher
- AMER CHEMICAL SOC
- Citation
- ACS SENSORS, v. 5, no. 3, page. 655-664
- Abstract
- Despite the diverse roles of cell-secreted proteases in the extracellular matrix (ECM), classical methods to analyze protease activity have not been explored at the cell culture site. Here, we report a stable, matrix-sticky, and protease-sensitive extracellular reporter that comprises a collagen-binding protein and a Forster resonance energy transfer (FRET) coupler of an enhanced green fluorescent protein and a small dye molecule. The extracellular FRET reporter via split intein-mediated protein trans-splicing is able to adhere to collagen matrices, leading to fluorescence changes by matrix metalloproteinase-2 (MMP2) activity during living cell culture without impeding cell viability. When a proMMP2 mutant (Y581A) with altered protease secretion and activity was transfected into cancer cells, the reporter revealed a dramatic reduction in MMP2 activity in both two- and three-dimensional culture systems, compared with cells transfected with wild-type proMMP2. Our reporter is immediately amenable to monitor protease activity in diverse ECM-resident cells as well as to study protease-related extracellular signaling and tissue remodeling.
- URI
- https://pubs.acs.org/doi/10.1021/acssensors.9b01456https://repository.hanyang.ac.kr/handle/20.500.11754/161342
- ISSN
- 2379-3694
- DOI
- 10.1021/acssensors.9b01456
- Appears in Collections:
- COLLEGE OF NATURAL SCIENCES[S](자연과학대학) > LIFE SCIENCE(생명과학과) > Articles
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