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Fluorescent Protein-Based Biosensors

Volume 1071 of the series Methods in Molecular Biology pp 163-174

Date:

Genetically Encoded Fluorescent Biosensors for Live-Cell Imaging of MT1-MMP Protease Activity

  • Mingxing OuyangAffiliated withDepartment of Bioengineering, University of Illinois at Urbana-Champaign
  • , Shaoying LuAffiliated withDepartment of Bioengineering, University of Illinois at Urbana-Champaign
  • , Yingxiao WangAffiliated withDepartment of Bioengineering, Neuroscience Program, Center of Biophysics and Computational Biology, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-ChampaignDepartment of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign

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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.

Key words

Fluorescent biosensor FRET Live-cell imaging MT1-MMP Matrix Metalloproteinase Protease Cancer cell