Visualization of polarized membrane type 1 matrix metalloproteinase activity in live cells by fluorescence resonance energy transfer imaging.
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.
Ouyang
Mingxing
M
Department of Bioengineering and the Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana-Champaign, Illinois 61801, USA.
Lu
Shaoying
S
Li
Xiao-Yan
XY
Xu
Jing
J
Seong
Jihye
J
Giepmans
Ben N G
BN
Shyy
John Y-J
JY
Weiss
Stephen J
SJ
Wang
Yingxiao
Y
D015374:Biosensing Techniques; D002462:Cell Membrane / Q000201:enzymology; D002465:Cell Movement; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D053511:Matrix Metalloproteinase 14 / Q000378:metabolism*; D008954:Models, Biological; D010957:Plasmids / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism
D005353:Fibronectins; D019716:Tissue Inhibitor of Metalloproteinase-2; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D053511:Matrix Metalloproteinase 14