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

  • Mingxing Ouyang
  • Shaoying Lu
  • Yingxiao Wang
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1071)

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 

Notes

Acknowledgment

This work is supported by grants from NIH HL098472, CA139272, NS063405, NSF CBET0846429 (Y.W., S. L.), and the Wallace H. Coulter Foundation and Beckman Laser Institute, Inc. (Y.W.). The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Mingxing Ouyang
    • 1
  • Shaoying Lu
    • 1
  • Yingxiao Wang
    • 2
    • 3
  1. 1.Department of BioengineeringUniversity of Illinois at Urbana-ChampaignChampaignUSA
  2. 2.Department of Bioengineering, Neuroscience Program, Center of Biophysics and Computational Biology, Beckman Institute for Advanced Science and TechnologyUniversity of Illinois at Urbana-ChampaignChampaignUSA
  3. 3.Department of Molecular and Integrative PhysiologyUniversity of Illinois at Urbana-ChampaignChampaignUSA

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