Video-Rate Bioluminescence Imaging of MMP-2 Figure 5. Identification of MMP2-GLase being secreted and bound on the cell surface of a HeLa cell using bioluminescence imaging. Bioluminescence imaging of HeLa cells transiently expressing MMP2-GLase. Objective lens; 406. Luminescence signals recording was started after 20 s from the addition of HBSS buffer containing coelenterazine with an exposure time of 500 ms for 75 s (Video image is in Video S4). (A) The bright- field image (the left panel), and luminescence images acquire at 0, 6, and 25 s. Two continuous luminescence spots (spot 1 and 2) are indicated by yellow arrowheads labeled with 1 and 2, respectively, and two transient diffusive luminescence spots (spot 3 and 4) are indicated by yellow arrowheads labeled with 3 and 4, respectively. (B) Magnified luminescence images of spot 1–4 indicated by arrowheads labeled with 1–4 in (A). (C) Time-dependent changes of the average luminescence intensity of spot 1–4 indicated by arrowheads labeled with 1–4 in (A). doi:10.1371/journal.pone.0025243.g005 MMP-2 in various types of cells including cancer cells. As the polarized distribution of other proteins including metalloprotei- nases and integrins have been investigated in migrating cells [17,33,34], the video-rate bioluminescence imaging might be useful to investigate the secretion and binding of these proteins in living cells. In conclusion, we have demonstrated for the first time the visualization of the exocytotic secretion of MMP-2 from a migrating cell in real time using bioluminescence video imaging. This method showed the repeated secretion of MMP-2 from specific sites at the leading edge of the cell. The bioluminescence video imaging is a powerful approach to investigate distribution and dynamics of proteins on the whole surface of polarized cells in real time. Materials and Methods Plasmids The secreted Gaussia Luciferase (GLase, K18 to D185) with the signal peptide sequence (Figure 1D) was expressed in HeLa cells (ATCC CCL-2) using pcDNA3-GLuc (Prolume Ltd., Pinetop, AZ). To express the fused protein of hMMP-2 to GLase (MMP2- GLase), the vector pcDNA3-hMMP2-GLuc was constructed as follows. The HindIII-EcoRI cDNA fragment coding human MMP- 2 preproprotein was obtained from IMAGE cDNA clone 3161383 by PCR amplification (25 cycles; 15 sec at 98uC, 15 sec at 55uC, 2 min at 68uC) using KOD-plus-DNA polymerase (Toyobo Co., Osaka, Japan) with a primer set of hMMP2-P1 (59 ggc AAGCTT AGCCACC ATG GAG GCG CTA ATG GCC C 39; HindIII site underlined, methionine bolded) and hMMP2-P2 (59 ggc GAATTC GCA GCC TAG CCA GTC GGA T 39; EcoRI site underlined). The PCR fragment was digested with HindIII and EcoRI, and then inserted into pcDNA3-GLuc-BE [26] to give pcDNA3-hMMP2-GLuc. The protein consisting of the signal peptide sequence of MMP-2, pro-MMP-2 and GLase was expressed (Figure 1D). To express wild type human MMP-2 and FLAG (DYKD- DDDK) tagged human MMP-2 (MMP2-FLAG) at the carboxyl terminus in HeLa cells, the vectors of pcDNA3-hMMP2 and pcDNA3-hMMP2-Flag were constructed as follows. For pcDNA3- hMMP2, the EcoRI-XbaI fragment of human MMP-2 cDNA was obtained from IMAGE cDNA clone 3161383 by PCR amplifica- tion (25 cycles; 15 sec at 98uC, 15 sec at 55uC, 2 min at 68uC) using KOD-plus-DNA polymerase with a primer set of hMMP2- P1 and hMMP2-P3 (59 ccc TCTAGA TTA GCA GCC TAG CCA GTC GGA TTT GAT 39; XbaI site underlined, stop codon bolded). The PCR fragment obtained was digested with HindIII and XbaI, and then was inserted into HindIII/XbaI sites of pcDNA3 to give pcDNA3-hMMP2. For pcDNA3-hMMP2-Flag, the EcoRI-Flag-TAA-NotI linker (GAC TAC AAA GAC GAT GAC GAC AAG for Flag sequence) was replaced with the EcoRI/ NotI fragment of GLuc in pcDNA3-hMMP2-GLuc to give pcDNA3-hMMP2-Flag. Cell culture and transfection HeLa cells were cultured in DMEM (D5796; Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). For transient expression, HeLa cells were transfected with an expression vector using Fugene HD (Roche Applied Science, Mannheim, Germany). The transformed cells were cultured for 24 h at 37uC in a humidified 5% CO2 incubator. Measurement of GLase activity using a luminometer HeLa cells (26105 cells) cultured in a Falcon 6 well-plate (BD Bioscience, Bedford, MA) were transfected with 2 mg of GLase- PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e25243