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J. Biol. Chem., Vol. 283, Issue 25, 17740-17748, June 20, 2008

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Visualization of Polarized Membrane Type 1 Matrix Metalloproteinase Activity in Live Cells by Fluorescence Resonance Energy Transfer Imaging^*

Mingxing Ouyang, Shaoying Lu, Xiao-Yan Li, Jing Xu, Jihye Seong^¶, Ben N. G. Giepmans^||, John Y.-J. Shyy^**, Stephen J. Weiss, and Yingxiao Wang^¶1

From the Department of Bioengineering and the Beckman Institute for Advanced Science and Technology, the ^¶Neuroscience Program, the Department of Molecular and Integrative Physiology, Center for Biophysics and Computational Biology, Institute of Genomic Biology, University of Illinois, Urbana-Champaign, Illinois 61801, the Department of Internal Medicine & Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, the ^||Department of Cell Biology, University Medical Center Groningen, 9713 AV Groningen, The Netherlands, and the ^**Division of Biomedical Science Program, University of California, Riverside, California 92521-0121

Membrane type 1 matrix metalloproteinase (MT1-MMP) plays a critical role in cancer cell biology by proteolytically remodeling the extracellular matrix. Utilizing fluorescence resonance energy transfer (FRET) imaging, we have developed a novel biosensor, with its sensing element anchoring at the extracellular surface of cell membrane, to visualize MT1-MMP activity dynamically in live cells with subcellular resolution. Epidermal growth factor (EGF) induced significant FRET changes in cancer cells expressing MT1-MMP, but not in MT1-MMP-deficient cells. EGF-induced FRET changes in MT1-MMP-deficient cells could be restored after reconstituting with wild-type MT1-MMP, but not MMP-2, MMP-9, or inactive MT1-MMP mutants. Deletion of the transmembrane domain in the biosensor or treatment with tissue inhibitor of metalloproteinase-2, a cell-impermeable MT1-MMP inhibitor, abolished the EGF-induced FRET response, indicating that MT1-MMP acts at the cell surface to generate FRET changes. In response to EGF, active MT1-MMP was directed to the leading edge of migrating cells along micropatterned fibronectin stripes, in tandem with the local accumulation of the EGF receptor, via a process dependent upon an intact cytoskeletal network. Hence, the MT1-MMP biosensor provides a powerful tool for characterizing the molecular processes underlying the spatiotemporal regulation of this critical class of enzymes.

Received for publication, December 4, 2007 , and in revised form, April 21, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants CA71699 (to S. J. W.) and HL77448 (Y.-J. S.). This work was also supported by grants from Wallace H. Coulter Foundation and the Beckman Laser Institute, Inc. (to Y. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental text, Movies S1-S5, Table S1, and Figs. S1-S9.

¹ To whom correspondence should be addressed: 4261 Beckman Institute, 405 N. Mathews Ave., Urbana, IL 61801. Tel.: 217-333-6727; Fax: 217-265-0246; E-mail: yingxiao{at}uiuc.edu.

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