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,Yingxiao Wang

doi:10.1074/jbc.m709872200

Abstract

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.

Highlights

Extracellular matrix macromolecules present cancer cells with a structural barrier that serves to limit their unregulated growth and movement [1, 2]
We reasoned that active MT1-matrix metalloproteinases (MMPs) would cleave the biosensor substrate peptide and separate ECFP and YPet, resulting in a change in fluorescence resonance energy transfer (FRET) that could be tracked by an increase in the emission ratio of ECFP/YPet
These results indicate that Membrane type 1 matrix metalloproteinase (MT1-MMP) hydrolyzes the biosensor at the designed cleavage site, which leads to the expected FRET change in vitro

Summary

Introduction

Extracellular matrix macromolecules present cancer cells with a structural barrier that serves to limit their unregulated growth and movement [1, 2]. In Vitro Characterization of the MT1-MMP Biosensor—To generate a sensitive biosensor for detecting MT1-MMP activity, a substrate peptide (31CPKESCNLFVLKD43) derived from the MT1-MMP cleavage site in the propeptide sequence of MMP-2 [24] was flanked by a fluorescence protein pair, ECFP and YPet [22] for FRET (Fig. 1A).

Results

Conclusion