Collagen-Immobilized Extracellular FRET Reporter for Visualizing Protease Activity Secreted by Living Cells
- Authors
- Lee, Hawon; Kim, Se-jeong; Shin, Heungsoo; Kim, Young-Pil
- Issue Date
- Mar-2020
- Publisher
- AMER CHEMICAL SOC
- Citation
- ACS SENSORS, v.5, no.3, pp.655 - 664
- Indexed
- SCIE
SCOPUS
- Journal Title
- ACS SENSORS
- Volume
- 5
- Number
- 3
- Start Page
- 655
- End Page
- 664
- URI
- https://scholarworks.bwise.kr/hanyang/handle/2021.sw.hanyang/10672
- DOI
- 10.1021/acssensors.9b01456
- ISSN
- 2379-3694
- 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.
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