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.
Mingxing Ouyang and Shaoying Lu contributed equally to this work.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544
Kunkel MT et al (2005) Spatio-temporal dynamics of protein kinase B/Akt signaling revealed by a genetically encoded fluorescent reporter. J Biol Chem 280(7):5581–5587
Miyawaki A et al (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388(6645):882–887
Mochizuki N et al (2001) Spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Nature 411(6841):1065–1068
Pertz O et al (2006) Spatiotemporal dynamics of RhoA activity in migrating cells. Nature 440(7087):1069–1072
Ting AY et al (2001) Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells. Proc Natl Acad Sci U S A 98(26):15003–15008
Violin JD et al (2003) A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J Cell Biol 161(5):899–909
Wang Y et al (2005) Visualizing the mechanical activation of Src. Nature 434(7036):1040–1045
Zhang J et al (2005) Insulin disrupts beta-adrenergic signalling to protein kinase A in adipocytes. Nature 437(7058):569–573
Zhang J et al (2001) Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc Natl Acad Sci U S A 98(26):14997–15002
Ouyang M et al (2008) Determination of hierarchical relationship of Src and Rac at subcellular locations with FRET biosensors. Proc Natl Acad Sci U S A 105(38):14353–14358
Shaner NC et al (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22(12):1567–1572
Sabeh F et al (2004) Tumor cell traffic through the extracellular matrix is controlled by the membrane-anchored collagenase MT1-MMP. J Cell Biol 167(4):769–781
Itoh Y, Seiki M (2006) MT1-MMP: a potent modifier of pericellular microenvironment. J Cell Physiol 206(1):1–8
Deryugina EI et al (2001) MT1-MMP initiates activation of pro-MMP-2 and integrin alphavbeta3 promotes maturation of MMP-2 in breast carcinoma cells. Exp Cell Res 263(2):209–223
Seiki M (2003) Membrane-type 1 matrix metalloproteinase: a key enzyme for tumor invasion. Cancer Lett 194(1):1–11
Seiki M, Yana I (2003) Roles of pericellular proteolysis by membrane type-1 matrix metalloproteinase in cancer invasion and angiogenesis. Cancer Sci 94(7):569–574
Kinoshita T et al (1996) Processing of a precursor of 72-kilodalton type IV collagenase/gelatinase A by a recombinant membrane-type 1 matrix metalloproteinase. Cancer Res 56(11):2535–2538
Sato H et al (1994) A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 370(6484):61–65
Lehti K et al (2005) An MT1-MMP-PDGF receptor-beta axis regulates mural cell investment of the microvasculature. Genes Dev 19(8):979–991
Ouyang M et al (2010) Simultaneous visualization of protumorigenic Src and MT1-MMP activities with fluorescence resonance energy transfer. Cancer Res 70(6):2204–2212
Ouyang M et al (2008) Visualization of polarized membrane type 1 matrix metalloproteinase activity in live cells by fluorescence resonance energy transfer imaging. J Biol Chem 283(25):17740–17748
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Ouyang, M., Lu, S., Wang, Y. (2014). Genetically Encoded Fluorescent Biosensors for Live-Cell Imaging of MT1-MMP Protease Activity. In: Zhang, J., Ni, Q., Newman, R. (eds) Fluorescent Protein-Based Biosensors. Methods in Molecular Biology, vol 1071. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-622-1_13
Download citation
DOI: https://doi.org/10.1007/978-1-62703-622-1_13
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-621-4
Online ISBN: 978-1-62703-622-1
eBook Packages: Springer Protocols