TC-MMP1-for-EX2 -39-ATGGCGCCCAGTGGAAGCTCT-59 and TC-MMP1-rev-EX3 -39-CTTCCCGTACAAAGCCTG- GAT-59. The resulting PCR amplification product was subse- quently cloned by use of the TA Expression Kit into the pCRT7/ CT-TOPO expression vector (Invitrogen). This construct allowed the production of Tribolium MMP-1 in Escherichia coli with a carboxy-terminal histidine tag. BL21(DE3) E. coli cells (Invitrogen, Carlsbad, CA) were used for production of recombinant Tribolium MMP-1. Optimal protein production was observed when cultures were grown at 32uC without IPTG to an optical density of 5–6 at 600 nm. The cells were harvested by centrifugation and cell pellets were stored at 220uC until use. We purified the recombinant protein under denaturating conditions using 8 M urea and 100 mM sodium phosphate buffer at pH 8 since all Tribolium MMP-1 protein was found in insoluble inclusion bodies. All purification steps were monitored by SDS-PAGE analysis using 15%-Tris–tricine gels. The bacterial cell pellet was resuspended in saline buffer and disintegrated by french press. After centrifugation of the cell lysate, proteins from inclusion bodies were solubilized in 8 M urea and 100 mM sodium phosphate buffer, pH 8. After a subsequent centrifugation step Tribolium MMP-1 was purified from the supernatant using immobilized metal affinity chromatography on ProtinoH Ni-IDA resin (Macherey Nagel, Du¨ ren, Germany) according to the instructions of the manufacturer. The protein was desalted, refolded, and concentrated using AmiconH Ultra-4 centrifugal filter devices (Millipore, Billerica, MA). In brief, the protein solution was concentrated by ultrafiltration to a volume of about 100 ml and diluted in 5 mM NaH2PO4, pH 7, to a volume of 4 ml. This procedure was repeated five times. For over night auto-activation, recombinant MMP was incubated in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, 1 mM 2- mercaptoethanol, 10 mM ZnCl2, pH 7) for 16 h at 25uC. Protein concentrations were measured using the method of Bradford with bovine serum albumin as a standard. The concentration of purified Tribolium MMP-1 was determined in 20 mM sodium phosphate buffer (pH 6.5) containing 6 M guanidium hydrochlo- ride using UV spectroscopy ( 280 = 34,380 mM21 cm21). References 1. Nagase H, Woessner JF (1999) Matrix metalloproteinases. J Biol Chem 274: 21491–21494. 2. Page-McCaw A, Ewald AJ, Werb Z (2007) Matrix metalloproteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Biol 8: 221–233. 3. Sternlicht MD, Werb Z (2001) How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol 17: 463–516. 4. Vu TH, Werb Z (2000) Matrix metalloproteinases: effectors of development and normal physiology. Genes Dev 14: 2123–2133. 5. Bode W, Maskos K (2003) Structural basis of the matrix metalloproteinases and their physiological inhibitors, the tissue inhibitors of metalloproteinases. Biol Chem 384: 863–872. 6. 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(1998) Regulation of matrix metalloproteinase-9 and inhibition of tumor invasion by In Vivo MMP Functions Collagen IV degrading activity assay Collagen IV degrading activity of recombinant Tribolium MMP- 1 was monitored using 100 mg/ml DQTM collagen (type IV from human placenta, fluorescein conjugate; Invitrogen) in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, 10 mM ZnCl2, pH 7) at 30uC, according to the recommendations of the manufacturer. Fluorescence was measured using the real-time PCR system Mx3000P (Stratagene, La Jolla, CA, USA) as a fluorescence reader with filter settings for excitation at 492 nm and emission at 516 nm. The broad-spectrum MMP inhibitor GM6001 (Merck KGaA, Darmstadt, Germany) was used to inhibit MMP activity. Ex vivo microscopic analysis of Tribolium hemocytes Larvae were punctured with a sterile needle, and obtained hemolymph samples were dropped on microscope slides and immediately mixed with buffer (NaH2PO4, pH 7) including no substrate, 100 mg/ml DQTM collagen (type IV from human placenta, fluorescein conjugate; Invitrogen, Carlsbad, CA), and 500 mM fluorogenic MMP-1/MMP-9 substrate (Calbiochem, Merck KGaA, Darmstadt, Germany), respectively, and were monitored with an Axioplan 2 microscope (Zeiss, Jena, Germany) with a fluorescence filter according to manufacturers’ recommendations. Acknowledgments We thank Meike Fischer for excellent technical assistance and Katja Altincicek for critical reading of the manuscript. We are grateful to Nicole Gerado (Department of Biology, Emory University) for helpful comments and assistance with language, Gregor Bucher (Georg August University of Go¨ttingen, Germany) for kindly providing us with Tribolium beetles and Radislav Sedlacek (Institute of Molecular Genetics, Prague, Czech Republic) for kindly providing us with recombinant human MMP-19 and helpful comments on the manuscript. We thank Zentrale Biotech- nische Betriebseinheit of Justus-Liebig-University Giessen for using electron microscopes and Dr. Martin Hardt for his helpful EM assistance. Author Contributions Conceived and designed the experiments: AV BA. Performed the experiments: EK HS BA. Analyzed the data: EK BA. Contributed reagents/materials/analysis tools: BA. Wrote the paper: EK AV BA. the membrane-anchored glycoprotein RECK. Proc Natl Acad Sci U S A 95: 13221–13226. 13. Noda M, Takahashi C (2007) Recklessness as a hallmark of aggressive cancer. Cancer Sci 98: 1659–1665. 14. 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