Visualization of Polarized Membrane Type 1 Matrix Metalloproteinase Activity in Live Cells by Fluorescence Resonance Energy Transfer Imaging
2008; Elsevier BV; Volume: 283; Issue: 25 Linguagem: Inglês
10.1074/jbc.m709872200
ISSN1083-351X
AutoresMingxing Ouyang, Shaoying Lu, Xiaoyan Li, Jing Xu, Jihye Seong, Ben N. G. Giepmans, John Y.‐J. Shyy, Stephen J. Weiss, Yingxiao Wang,
Tópico(s)Peptidase Inhibition and Analysis
ResumoMembrane 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. 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. Extracellular matrix macromolecules present cancer cells with a structural barrier that serves to limit their unregulated growth and movement (1Seiki M. Cancer Lett. 2003; 194: 1-11Crossref PubMed Scopus (354) Google Scholar, 2Visse R. Nagase H. Circ. Res. 2003; 92: 827-839Crossref PubMed Scopus (3568) Google Scholar). In turn, cancer cells have been postulated to use matrix metalloproteinases (MMPs), 2The abbreviations used are: MMP, matrix metalloproteinase; MT1-MMP, membrane type 1 MMP; FRET, fluorescence resonance energy transfer; TIMP-2, tissue inhibitor of metalloproteinase-2; EGF, epidermal growth factor; EGFR, EGF receptor; PDGFR, platelet-derived growth factor receptor; GFP, green fluorescent protein; EGFP, enhanced GFP; Cyto D, cytochalasin D. 2The abbreviations used are: MMP, matrix metalloproteinase; MT1-MMP, membrane type 1 MMP; FRET, fluorescence resonance energy transfer; TIMP-2, tissue inhibitor of metalloproteinase-2; EGF, epidermal growth factor; EGFR, EGF receptor; PDGFR, platelet-derived growth factor receptor; GFP, green fluorescent protein; EGFP, enhanced GFP; Cyto D, cytochalasin D. a class of zinc-dependent proteolytic enzymes, as a means to dissolve these extracellular matrix barriers during neoplastic progression (1Seiki M. Cancer Lett. 2003; 194: 1-11Crossref PubMed Scopus (354) Google Scholar, 2Visse R. Nagase H. Circ. Res. 2003; 92: 827-839Crossref PubMed Scopus (3568) Google Scholar, 3Deryugina E.I. Quigley J.P. Cancer Metastasis Rev. 2006; 25: 9-34Crossref PubMed Scopus (1609) Google Scholar). Although the human MMP family is comprised of 16 secreted and seven membrane-tethered enzymes, increasing evidence suggests that a subclass of the membrane-anchored proteinases, termed the membrane type (MT) MMPs, plays dominant roles in controlling cancer cell behavior (1Seiki M. Cancer Lett. 2003; 194: 1-11Crossref PubMed Scopus (354) Google Scholar, 4Deryugina E.I. Ratnikov B. Monosov E. Postnova T.I. DiScipio R. Smith J.W. Strongin A.Y. Exp. Cell Res. 2001; 263: 209-223Crossref PubMed Scopus (329) Google Scholar, 5Itoh Y. Takamura A. Ito N. Maru Y. Sato H. Suenaga N. Aoki T. Seiki M. EMBO J. 2001; 20: 4782-4793Crossref PubMed Scopus (335) Google Scholar, 6Remacle A.G. Rozanov D.V. Baciu P.C. Chekanov A.V. Golubkov V.S. Strongin A.Y. J. Cell Sci. 2005; 118: 4975-4984Crossref PubMed Scopus (60) Google Scholar, 7Sabeh F. Ota I. Holmbeck K. Birkedal-Hansen H. Soloway P. Balbin M. Lopez-Otin C. Shapiro S. Inada M. Krane S. Allen E. Chung D. Weiss S.J. J. Cell Biol. 2004; 167: 769-781Crossref PubMed Scopus (476) Google Scholar, 8Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2361) Google Scholar). The MT-MMPs are expressed either as type I transmembrane proteins (i.e. MT1, 2, 3, and 5 MMPs) or in a glycosylphosphatidyl-inositol-anchored format (i.e. MT4- and MT6-MMP) (1Seiki M. Cancer Lett. 2003; 194: 1-11Crossref PubMed Scopus (354) Google Scholar). Among these enzymes, MT1-MMP is considered to be the family member most closely linked to neoplastic cell behavior (6Remacle A.G. Rozanov D.V. Baciu P.C. Chekanov A.V. Golubkov V.S. Strongin A.Y. J. Cell Sci. 2005; 118: 4975-4984Crossref PubMed Scopus (60) Google Scholar, 8Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2361) Google Scholar). Indeed, recent studies have demonstrated that MT1-MMP plays a direct and essential role in allowing tumor cells to degrade and invade multiple connective tissue barriers through a mechanism independent of MMP-2, an effector protease that operates downstream of MT1-MMP (4Deryugina E.I. Ratnikov B. Monosov E. Postnova T.I. DiScipio R. Smith J.W. Strongin A.Y. Exp. Cell Res. 2001; 263: 209-223Crossref PubMed Scopus (329) Google Scholar, 7Sabeh F. Ota I. Holmbeck K. Birkedal-Hansen H. Soloway P. Balbin M. Lopez-Otin C. Shapiro S. Inada M. Krane S. Allen E. Chung D. Weiss S.J. J. Cell Biol. 2004; 167: 769-781Crossref PubMed Scopus (476) Google Scholar). Synthesized as a catalytically inactive proenzyme, the MT1-MMP precursor undergoes proteolytic processing in the transGolgi complex wherein the prodomain is removed by members of the proprotein convertase family (9Pei D. Weiss S.J. Nature. 1995; 375: 244-247Crossref PubMed Scopus (527) Google Scholar). Subsequently, the mature enzyme traffics to the plasma membrane via a process controlled by Rab8 and the microtubular apparatus (6Remacle A.G. Rozanov D.V. Baciu P.C. Chekanov A.V. Golubkov V.S. Strongin A.Y. J. Cell Sci. 2005; 118: 4975-4984Crossref PubMed Scopus (60) Google Scholar, 10Bravo-Cordero J.J. Marrero-Diaz R. Megias D. Genis L. Garcia-Grande A. Garcia M.A. Arroyo A.G. Montoya M.C. EMBO J. 2007; 26: 1499-1510Crossref PubMed Scopus (189) Google Scholar). Little is known with regard to the signaling molecules that mobilize MT1-MMP to the cell surface, but EGF, an extracellular ligand of EGF receptor family members (EGFRs; also known as ERBB/HERs), has been reported to induce cancer invasion by modulating MT1-MMP expression (11Sato T. Iwai M. Sakai T. Sato H. Seiki M. Mori Y. Ito A. Br. J. Cancer. 1999; 80: 1137-1143Crossref PubMed Scopus (43) Google Scholar, 12Van Meter T.E. Broaddus W.C. Rooprai H.K. Pilkington G.J. Fillmore H.L. Neuro-oncol. 2004; 6: 188-199Crossref PubMed Scopus (35) Google Scholar). Once delivered to the cell surface, the short cytoplasmic tail of MT1-MMP regulates its internalization and turnover at the plasma membrane, possibly through its interactions with adaptor protein 2, which resides in clathrin-coated pits (13Jiang A. Lehti K. Wang X. Weiss S.J. Keski-Oja J. Pei D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13693-13698Crossref PubMed Scopus (221) Google Scholar, 14Uekita T. Itoh Y. Yana I. Ohno H. Seiki M. J. Cell Biol. 2001; 155: 1345-1356Crossref PubMed Scopus (213) Google Scholar). Migrating cells are then thought to localize MT1-MMP to various cellular domains by complex processes involving caveolar proteins, the actin network, and extracellular binding partners (5Itoh Y. Takamura A. Ito N. Maru Y. Sato H. Suenaga N. Aoki T. Seiki M. EMBO J. 2001; 20: 4782-4793Crossref PubMed Scopus (335) Google Scholar, 10Bravo-Cordero J.J. Marrero-Diaz R. Megias D. Genis L. Garcia-Grande A. Garcia M.A. Arroyo A.G. Montoya M.C. EMBO J. 2007; 26: 1499-1510Crossref PubMed Scopus (189) Google Scholar, 15Nakahara H. Howard L. Thompson E.W. Sato H. Seiki M. Yeh Y. Chen W.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7959-7964Crossref PubMed Scopus (357) Google Scholar, 16Sato T. del Carmen Ovejero M. Hou P. Heegaard A.M. Kumegawa M. Foged N.T. Delaisse J.M. J. Cell Sci. 1997; 110: 589-596Crossref PubMed Google Scholar, 17Galvez B.G. Matias-Roman S. Albar J.P. Sanchez-Madrid F. Arroyo A.G. J. Biol. Chem. 2001; 276: 37491-37500Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Src kinase has also been shown to phosphorylate the cytoplasmic tail of MT1-MMP and regulate its functions (18Nyalendo C. Michaud M. Beaulieu E. Roghi C. Murphy G. Gingras D. Beliveau R. J. Biol. Chem. 2007; 282: 15690-15699Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Despite these insights, however, few of these principles have been evaluated directly because techniques have not yet been developed for monitoring MT1-MMP activity with subcellular resolution in live cells. Recent advances in fluorescent probes have enabled the study of protein activity and distribution in live cells (19Pertz O. Hahn K.M. J. Cell Sci. 2004; 117: 1313-1318Crossref PubMed Scopus (83) Google Scholar, 20Giepmans B.N. Adams S.R. Ellisman M.H. Tsien R.Y. Science. 2006; 312: 217-224Crossref PubMed Scopus (2272) Google Scholar). Along these lines, we previously developed a biosensor based on fluorescence resonance energy transfer (FRET) to monitor Src activity in real time at the single cell level (21Wang Y. Botvinick E.L. Zhao Y. Berns M.W. Usami S. Tsien R.Y. Chien S. Nature. 2005; 434: 1040-1045Crossref PubMed Scopus (566) Google Scholar). We report here the development and characterization of a new, FRET-based MT1-MMP biosensor (GenBank™ accession number EU545473) anchored at the plasma membrane wherein a MT1-MMP-sensitive substrate has been concatenated between the high efficiency FRET pair, ECFP and YPet (22Nguyen A.W. Daugherty P.S. Nat. Biotechnol. 2005; 23: 355-360Crossref PubMed Scopus (499) Google Scholar). Utilizing this biosensor, MT1-MMP activity and distribution have been resolved in live cells with high spatiotemporal resolution. Further, new mechanistic insights have been developed into the means by which the local activity of MT1-MMP is coordinated at the surface of migrating cancer cells. Protein Expression, in Vitro Spectroscopy, and Cleavage Assays—Chimeric proteins were expressed with N-terminal His6 tags in Escherichia coli and purified by nickel chelation chromatography as described (21Wang Y. Botvinick E.L. Zhao Y. Berns M.W. Usami S. Tsien R.Y. Chien S. Nature. 2005; 434: 1040-1045Crossref PubMed Scopus (566) Google Scholar). Emission ratios of ECFP/YPet (476 nm/526 nm) were measured by a fluorescence plate reader (TECAN, Sapphire II) before and after adding the recombinant catalytic domain of human MT1-, MT2-, or MT3-MMP (2 μg/ml; Calbiochem) or of human MMP-2 or MMP-9 (6 μg/ml; Calbiochem) into the proteolysis assay buffer (50 mm HEPES, 10 mm CaCl2, 0.5 mm MgCl2, 50 μm ZnCl2, and 0.01% Brij-35, pH 6.8) at 37 °C (23Rozanov D.V. Deryugina E.I. Monosov E.Z. Marchenko N.D. Strongin A.Y. Exp. Cell Res. 2004; 293: 81-95Crossref PubMed Scopus (63) Google Scholar). The samples were separated by 10% SDS-PAGE gels followed by Coomassie Blue staining. After destaining (50% v/v methanol in water with 10% acetic acid), the protein was visualized, and the image was recorded by digital camera (Olympus). Gene Construction and DNA Plasmids—The substrate peptide sequence CPKESCNLFVLKD was derived from the MT1-MMP cleavage site identified in proMMP-2 (24Kinoshita T. Sato H. Takino T. Itoh M. Akizawa T. Seiki M. Cancer Res. 1996; 56: 2535-2538PubMed Google Scholar) and used for the MT1-MMP FRET biosensor. The YPet cDNA was amplified by PCR with a sense primer containing a BglII site and a reverse primer containing a SacI site. ECFP was amplified by PCR with a sense primer containing a SacI site and the sequence of the MT1-MMP substrate peptide and a reverse primer containing a PstI site, a stop codon, and a HindIII site. The PCR products were fused together and cloned into pRSETb (Invitrogen) using BglII/HindIII sites for bacterial expression and into pDisplay (Invitrogen) using BglII/PstI sites for mammalian cell expression (see Fig. 1A). The pDisplay vector contains an N-terminal murine Ig κ-chain leader sequence, which directs the biosensor protein to the secretory pathway, and a C-terminal transmembrane domain of the platelet-derived growth factor receptor β (i.e. PDGFRβ)), which targets the biosensor protein to the plasma membrane. The cytosolic MT1-MMP biosensor for mammalian cell expression was constructed by PCR amplification of the fused full-length gene encoding the MT1-MMP biosensor and subcloned into pcDNA3.1 (Invitrogen) with BamHI and EcoRI sites. A mutant MT1-MMP biosensor was constructed by replacing the sequence encoding NL with IV in the sense primer used for YPet amplification. The mCherry-conjugated MT1-MMP was constructed by PCR amplification of the cDNA encoding MT1-MMP with a sense primer containing a HindIII site and a reverse primer containing a gene sequence encoding GGS as a linker and an EcoRI site. The PCR product of cDNA encoding mCherry was fused at the C-terminal of MT1-MMP with GGS and cloned into pcDNA3.1 with HindIII/XhoI sites. The GFP-fused EGFR construct was a gift from Dr. Sorkin at the Department of Pharmacology, University of Colorado (25Carter R.E. Sorkin A. J. Biol. Chem. 1998; 273: 35000-35007Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). The constructs for MT1-MMP and various other plasmids: control vector (PCR3.1 Uni; Invitrogen); human MT1-MMP; catalytically inactive human MT1-MMP (E/A mutant, Glu240 → Ala); transmembrane-deleted human MT1-MMP (MT1ΔTM); cytoplasmic tail-deleted human MT1-MMP (MT1ΔCT); and human MMP-2, MMP-9, and their convertase-activable forms (MMP-2RXKR and MMP-9RXKR) have been previously published (26Hotary K.B. Allen E.D. Brooks P.C. Datta N.S. Long M.W. Weiss S.J. Cell. 2003; 114: 33-45Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar). Micropatterning and Migration Assay—Glass coverslips (Fisher) were cleaned with a solution containing H2SO4 and H2O2 prior to the silanization in 2% dimethyl dichlorosilane (Aldrich) in dichlorobenzene for 10 s. The treated slips were rinsed with acetone, ethanol, and water, blown dry, and oxidized by UV-generated ozone (UVO Cleaner; Jelight, Irvine, CA) for 1 min (27Tan J.L. Liu W. Nelson C.M. Raghavan S. Chen C.S. Tissue Eng. 2004; 10: 865-872Crossref PubMed Scopus (190) Google Scholar). These treated coverslips were stored at 4 °C before usage. The polydimethylsiloxane microchannel mold was created by soft lithography. Negative photo-resist Epon SU8 2015 was coated on a silicon wafer, which was then exposed to UV light through a transparency mask with parallel lines and spacing before being developed. Polydimethylsiloxane prepared by mixing two liquid components (Sylgard 184 kit; Dow Corning) was poured onto the developed wafer and cured. After solidification, the polydimethylsiloxane mold with microgrooves was peeled off and sealed on treated coverslips to create microfluidic channels. Fibronectin solution (40 μg/ml) was perfused through the channel driven by pressure gradient to coat the coverslips with defined parallel lines (10 μm in width with 30-μm spacing). The coverslips were then backfilled with pluronic acid (F127) (BASF Corporation; 0.5% in phosphate-buffered) to prevent the cell adhesion outside of the patterned areas. After co-transfection with wild-type biosensor and MT1-MMP for 36-48 h, HeLa cells were detached with 4 mm EDTA (pH 7.4) in phosphate-buffered and seeded on fibronectin-coated glass-bottomed dishes or fibronectin stripes for 3 h before EGF stimulation. Microscopy, Image Acquisition, and Analysis—Cells expressing various exogenous proteins were starved in 0.5% fetal bovine serum for 36-48 h before EGF (50 ng/ml) treatment. During the imaging process, the cells were maintained in serum-free CO2-independent medium (Invitrogen) at 37 °C. The images were collected with a Zeiss axiovert inverted microscope equipped with a cooled charge-coupled device camera (Cascade 512B; Photometrics) using MetaFluor 6.2 software (Universal Imaging). The parameters of dichroic mirrors, excitation and emission filters for FRET, and different fluorescence proteins are shown in supplemental Table S1. The fluorescence intensity of nontransfected cells were quantified as the background signal and subtracted from the ECFP and YPet (FRET) signals on transfected cells. There is minimal cross-talk between ECFP and YPet channels (i.e. the direct excitation of YPet with ECFP excitation wavelength and the ECFP emission bleed-through into YPet channel) with our filter settings (data not shown). Hence, the pixel-by-pixel ratio images of ECFP/YPet were directly calculated based on the background-subtracted fluorescence intensity images of ECFP and YPet by the MetaFluor software to represent the FRET efficiency and activation levels of biosensor. Emission ratios of ECFP/YPet were averaged on chosen regions of interest to allow the quantification and statistical analysis by Excel (Microsoft) and Matlab (The MathWorks). Statistical Analysis—For statistical analysis, we used the Bonferroni multiple comparison test of means at 95% confidence interval, which is provided by the multcompare function in the MATLAB statistics toolbox. 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 (24Kinoshita T. Sato H. Takino T. Itoh M. Akizawa T. Seiki M. Cancer Res. 1996; 56: 2535-2538PubMed Google Scholar) was flanked by a fluorescence protein pair, ECFP and YPet (22Nguyen A.W. Daugherty P.S. Nat. Biotechnol. 2005; 23: 355-360Crossref PubMed Scopus (499) Google Scholar) for FRET (Fig. 1A). In this construct, we reasoned that active MT1-MMP would cleave the biosensor substrate peptide and thus separate ECFP and YPet, resulting in a change in FRET that could be tracked by an increase in the emission ratio of ECFP/YPet. Indeed, following incubation of the engineered biosensor with the catalytic domain of MT1-MMP (CAT), a significant decrease in the YPet spectrum max (526 nm) was detected along with a concomitant increase in the ECFP spectrum max (476 nm), indicative of FRET attenuation (Fig. 1B). Analysis by gel electrophoresis further revealed that the biosensor can be cleaved by CAT (Fig. 1C). If, however, the critical cleavage site NL was mutated to IV (24Kinoshita T. Sato H. Takino T. Itoh M. Akizawa T. Seiki M. Cancer Res. 1996; 56: 2535-2538PubMed Google Scholar), the FRET response of the biosensor was abolished (Fig. 1D). Taken together, these results indicate that MT1-MMP hydrolyzes the biosensor at the designed cleavage site, which leads to the expected FRET change in vitro. The specificity of biosensor was further examined by comparing the catalytic domains of MT1-MMP with other MMP family membranes. MT1-MMP displayed a much stronger activity toward the biosensor in comparison with MT2-MMP, MMP-2, and MMP-9, although MT3-MMP can also efficiently induce FRET change of the biosensor (Fig. 1E). Gel electrophoresis results on biosensor cleavage are consistent with the FRET signals (data not shown). These results indicate that the biosensor is sensitive to the catalytic domains of both MT1- and MT3-MMP in vitro, but not to MT2-MMP, MMP-2, and MMP-9. Targeting the MT1-MMP Biosensor to the Extracellular Face of the Cell Plasma Membrane—Because the catalytic domain of MT1-MMP is directed toward the extracellular face of the plasma membrane (1Seiki M. Cancer Lett. 2003; 194: 1-11Crossref PubMed Scopus (354) Google Scholar), the cDNA encoding the MT1-MMP biosensor was subcloned into pDisplay vector which contains an N-terminal murine Ig κ-chain leader sequence for secretory pathway targeting and a C-terminal transmembrane domain of PDGFRβ for plasma membrane targeting. Because both MT1-MMP and PDGFRβ have been demonstrated previously to colocalize at the cell surface (28Lehti K. Allen E. Birkedal-Hansen H. Holmbeck K. Miyake Y. Chun T.H. Weiss S.J. Genes Dev. 2005; 19: 979-991Crossref PubMed Scopus (98) Google Scholar), the functional domain of the biosensor protrudes outward from the surface of plasma membrane in proximity to the MT1-MMP catalytic domain (Fig. 2A). Following transfection of HeLa cells with the biosensor, the full-length construct was oriented properly on the cell surface as evidenced by the strong staining observed with an ECFP/YPet-reactive antibody in nonpermeabilized cells (Fig. 2B, panels i-iii). By contrast, when cells were transfected with a biosensor construct lacking PDGFR_TM, ECFP/YPet could only be immunodetected after the membrane was permeabilized (Fig. 2B, panels iv-ix). These results confirmed that the MT1-MMP biosensor is correctly targeted to the cell surface. Of note, a portion of the membrane-targeted biosensor was localized to perinuclear regions, possibly reflecting biosensors trapped in an endo/exocytosis recycling route before being transported to the plasma membrane (29Wiley H.S. Burke P.M. Traffic. 2001; 2: 12-18Crossref PubMed Scopus (204) Google Scholar). Functional Characterization of the MT1-MMP Biosensor in Live Cells—To begin characterizing the utility of the MT1-MMP biosensor, HeLa cells (a cell type that expresses minimal levels of endogenous MT1-MMP) (30Zhai Y. Hotary K.B. Nan B. Bosch F.X. Munoz N. Weiss S.J. Cho K.R. Cancer Res. 2005; 65: 6543-6550Crossref PubMed Scopus (97) Google Scholar) were co-transfected with wild-type MT1-MMP and the membrane-targeted biosensor. In the presence of EGF, a significant FRET change was induced over a 60-min monitoring period (Fig. 3A and supplemental Movie S1). By contrast, the FRET response was ablated when the NL → IV cleavage-resistant (24Kinoshita T. Sato H. Takino T. Itoh M. Akizawa T. Seiki M. Cancer Res. 1996; 56: 2535-2538PubMed Google Scholar) biosensor was coexpressed with MT1-MMP (Fig. 3B). As expected, deletion of PDGFR_TM in the biosensor probe, a modification that precludes its trafficking to the cell surface, also abolished the EGF-induced FRET response (Fig. 3C), supporting the conclusion that EGF-induced MT1-MMP activation occurs preferentially at the cell surface. Quantification of FRET responses further confirmed that EGF-induced biosensor cleavage occurs only with the wild-type membrane-anchored construct but not in the cleavage-resistant mutant or cytosol-directed biosensors (Fig. 3D). Finally, in a fashion consistent with the extracellular proteolysis of the biosensor, tissue inhibitor of metalloproteinase-2 (TIMP-2), an endogenous, ∼25-kDa inhibitor of MT1-MMP(7Sabeh F. Ota I. Holmbeck K. Birkedal-Hansen H. Soloway P. Balbin M. Lopez-Otin C. Shapiro S. Inada M. Krane S. Allen E. Chung D. Weiss S.J. J. Cell Biol. 2004; 167: 769-781Crossref PubMed Scopus (476) Google Scholar), reversed the EGF-induced FRET change in the MT1-MMP-transfected HeLa cells (Fig. 3E). The observed decrease in the ECFP/YPet emission ratio that occurred in the presence of TIMP-2 is most likely attributed to an enhanced turnover rate of the biosensor as well as MT1-MMP at the plasma membrane upon EGF stimulation (see "Discussion"). To further delineate the structural characteristics that underlie the MT1-MMP-dependent hydrolysis of the biosensor, EGF-treated HeLa cells were co-transfected with MT1-MMP constructs harboring either an inactivating point mutation in the catalytic domain (14Uekita T. Itoh Y. Yana I. Ohno H. Seiki M. J. Cell Biol. 2001; 155: 1345-1356Crossref PubMed Scopus (213) Google Scholar) (i.e. MT1 (E/A)) or a transmembrane-deletion mutant that results in the secretion of a truncated, but active, form of the enzyme (6Remacle A.G. Rozanov D.V. Baciu P.C. Chekanov A.V. Golubkov V.S. Strongin A.Y. J. Cell Sci. 2005; 118: 4975-4984Crossref PubMed Scopus (60) Google Scholar) (i.e. MT1ΔTM). In either case, EGF was unable to induce a significant FRET response in HeLa cells (Fig. 4A and supplemental Fig. S1). Similarly, HeLa cells transfected with either MMP-2 or MMP-9 did not generate a significant FRET response following EGF treatment (Fig. 4A and supplemental Fig. S1). By contrast, when the cytosolic tail of MT1-MMP was deleted (MT1ΔCT), the membrane-tethered mutant generated a high ECFP/YPet ratio with or without EGF stimulation (Fig. 4A and supplemental Fig. S1). Western blot analysis of cell lysates further revealed that EGF triggered biosensor cleavage only in cells transfected with MT1-MMP, as evidenced by the generation of a ∼35-kDa fragment of the biosensor (supplemental Fig. S2), most likely reflecting the ECFP-PDGFR_TM fusion protein that remains tethered to the plasma membrane after cleavage (Fig. 2A). Interestingly, MT1ΔCT resulted in a very low level of both intact and cleaved biosensors (supplemental Fig. S2), possibly representing a sustained high level of cleavage and degradation of the biosensor. Serum-supplemented medium also induced a FRET change in biosensor-expressing cells transfected with wild-type MT1-MMP, but not MT1 (E/A) or the control vector (supplemental Fig. S3). Further examination revealed that in the presence of serum, the biosensor is sensitive to MT1-MMP, but not to wild-type or constitutively active forms of MMP-2 and MMP-9 (Fig. 4B). The expression of MMP-2 or MMP-9 with MT1-MMP did not cause significant increase in FRET changes when comparing with cells expressing MT1-MMP only. Interestingly, although the catalytic domain of MT3-MMP displayed a substantial activity toward the biosensor in vitro (Fig. 1E), in live cells, the biosensor is most sensitive to MT1-MMP as compared with MT2- or MT3-MMP (Fig. 4B). Hence, in live cells, the biosensor preferably monitors the activity of MT1-MMP, although MT2- and MT3-MMP may also contribute to the signal depending on their respective levels of activities. To monitor the ability of the biosensor to detect endogenous MT1-MMP activity, FRET imaging was assessed in (i) HT-1080 cells that are known to express high levels of the proteinase or (ii) mouse dermal fibroblasts isolated from either MT1MMP+/+ or MT1-MMP-/- mice (7Sabeh F. Ota I. Holmbeck K. Birkedal-Hansen H. Soloway P. Balbin M. Lopez-Otin C. Shapiro S. Inada M. Krane S. Allen E. Chung D. Weiss S.J. J. Cell Biol. 2004; 167: 769-781Crossref PubMed Scopus (476) Google Scholar). Among these cells, the ECFP/YPet emission ratio was highest in HT-1080 cells, modest in wild-type fibroblasts, and lowest in MT1-MMP-/- fibro-blasts (Fig. 4C). Western blot analyses confirmed that the expression of MT1-MMP in MT1-MMP-/- fibroblasts is ablated (supplemental Fig. S4). Although the wild-type fibro-blasts generate only a modest signal (consistent with their low level of MT1-MMP activity (7Sabeh F. Ota I. Holmbeck K. Birkedal-Hansen H. Soloway P. Balbin M. Lopez-Otin C. Shapiro S. Inada M. Krane S. Allen E. Chung D. Weiss S.J. J. Cell Biol. 2004; 167: 769-781Crossref PubMed Scopus (476) Google Scholar)), the addition of the synthetic MMP inhibitor, GM6001, caused strong FRET changes while exerting only small effects on MT1-MMP-/- cells (supplemental Fig. S5), presumably because of their MT3-MMP activity (31Hotary K.B. Yana I. Sabeh F. Li X.Y. Holmbeck K. Birkedal-Hansen H. Allen E.D. Hiraoka N. Weiss S.J. J. Exp. Med. 2002; 195: 295-308Crossref PubMed Scopus (179) Google Scholar). These results reinforced the note that MT1-MMP plays a more dominant role than MT3-MMP in biosensor cleavage. Because both MT1-MMP and EGFR are abundant in the MDA-MB-231 breast cancer cell line (32deFazio A. Chiew Y.E. Donoghue C. Lee C.S. Sutherland R.L. J. Biol. Chem. 1992; 267: 18008-18012Abstract Full Text PDF PubMed Google Scholar, 33Koshikawa N. Giannelli G. Cirulli V. Miyazaki K. Quaranta V. J. Cell Biol. 2000; 148: 615-624Crossref PubMed Scopus (545) Google Scholar), we further examined whether the biosensor can monitor the dynamic activation of endogenous MT1-MMP in response to EGF stimulation. Indeed, a significant FRET change was observed in MDA-MB-231 cells upon EGF stimulation with the highest level of activity found at the cell periphery (Fig. 4D). Time course analyses further revealed that this EGF-induced FRET change was inhibited by TIMP-2 and GM6001 (a general, synthetic MMP inhibitor (34Varon C. Tatin F. Moreau V. Van Obberghen-Schilling E. FernandezSauze S. Reuzeau E. Kramer I. Genot E. Mol. Cell Biol. 2006; 26: 3582-3594Crossref PubMed Scopus (134) Google Schol
Referência(s)