Figure 3. Time-dependent decrease of luminescence intensity of MMP2-GLase with coelenterazine. Luminescence intensity of MMP2-GLase with coelenterazine (3 mg/ml) is recorded for 60 s. HeLa cells were transfected with pcDNA3-hMMP2-GLuc and cultured for 24 h. After removing the medium, the cells were incubated with HBSS for 60 min at 37uC and the luminescence activity in HBSS was determined using a luminometer, as described in Materials and Methods. doi:10.1371/journal.pone.0025243.g003 GLase was decreased in a time-dependent manner (Figure 3) as previously reported [26,29,30]. MMP2-GLase expressed in HeLa cells was identified by Western blot analyses using anti-MMP-2 and anti-GLase antibodies (Figure 4A and B). On SDS-PAGE analysis under reducing conditions, pro- and processed forms of MMP2-GLase were detected in the cell lysate with both antibodies (Figure 4A), indicating that MMP2-GLase expressed and processed correctly. In the conditioned medium, pro-form of MMP2-GLase was only detected as well as exogenous MMP-2 and MMP2-FLAG (Figure 4B lane 4, 6 and 8). These results suggested that MMP2- GLase, as well as exogenous MMP-2 and MMP2-FLAG, was secreted as pro-form and then a processed form was bound on cell surface. Further, the co-localization of MMP2-GLase with MMP2-FLAG on the cell surface was confirmed by immunoflu- orescence analysis using anti-GLase antibody and anti-Flag antibody. As a result, MMP2-GLase was co-localized with MMP2-FLAG (Figure 2C), supporting that MMP2-GLase ex- pressed in HeLa cells was secreted as pro-form, and was processed for activation on the cell surface. Identification of both secretory and membrane- associated forms of MMP-2 on the cell surface in a migrating HeLa cell by video-rate bioluminescence imaging To visualize the secretion and localization of MMP-2 on the cell surface, video-rate bioluminescence imaging of MMP2-GLase expressed in HeLa cells was performed using the improved imaging system with an EM-CCD camera. With an exposure time of 500 ms per image, the luminescence video image of a migrating HeLa cell was obtained using the 406objective lens. The transient luminescence spots and continuous luminescence spots were detected on the cell (Figure 5A; Video S4). The continuous luminescence spots within 3 mm in diameter retained and gradually disappeared in 1 min after adding of coelenterazine (Figure 5B and C). This slow decrease of the continuous luminescence signals seemed to be time-dependent decrease of luminescence activity of MMP2-GLase (Figure 3). The results of Western blot analyses suggested that the pro-form and processed form of MMP2-GLase could bind on the cell surface (Figure 4B), Video-Rate Bioluminescence Imaging of MMP-2 and the video images supported that the continuous luminescence spots represented the signals of MMP2-GLase binding on the cell surface. On the other hand, the transient luminescence spots could be interpreted as MMP2-GLase being secreted from the cell. The individual luminescence signals for secreted GLase were rapidly diffused from cells (Figure 2B and video S1) and the luminescence signals of MMP2-GLase were remained as spots for a few seconds on the cells (spot 3 and 4 in Figure 5B, and also see transient luminescence signals in Video S4). These results indicated that MMP2-GLase could be used as a reporter for MMP-2 secretion. Thus, we could visualize two distinct types of MMP-2, secreted as well as membrane-associated MMP-2, on the cell surface in real time. Characterization of exocytotic MMP-2 secretion in a migrating HeLa cell After the disappearance of continuous luminescence signals on the cell surface of HeLa cells, the luminescence signals from exocytotic secretion of MMP2-GLase were mainly observed at the leading edge (Figure 6A and B; Video S4) and were diffused in a few seconds. This result indicated that the leading edge is the major domain to supply MMP-2 into the extracellular space of a migrating cell. Occasionally, individual MMP2-GLase secretion sequentially occurred within a few seconds along the leading edge (Figure 6B). The luminescence image of maximum luminescence intensity superimposed on the bright-field image clearly showed that the secretion sites of MMP2-GLase are close to the leading edge (Figure 6C). Further, the luminescence signal of MMP2- GLase was also observed at the trailing edge of a migrating cell (Figure 6C; Video S4). The frequency of MMP-2 secretion in each hot spot (ca. 2.5 mm in diameter) was estimated by calculating the maximum luminescence intensities of MMP2-GLase in the video image (Figure 6D). Thus, the repeated secretion of MMP-2 was observed in the specific area towards the leading and the trailing edges of the migration cell. Presumably, microtubule may define the sites for MMP-2 secretion, as previously reported [31,32]. The secretion of MMP2-GLase was not stimulated by elevated K+ (data not shown), suggesting that the depolarization-independent regulatory mechanism for the sequential exocytosis of vesicles containing MMP-2 might be present. To obtain high-resolution images of luminescence spots with the EM-CCD camera (pixel size = 16 mm), we used a 1006 objective lens (NA1.45) instead of the 406 objective lens. With the 1006 objective lens, each luminescence spot of MMP2-GLase secretion at the leading edge was observed more clearly (Figure 6E; Video S5) and the size of luminescence spots of MMP2-GLase secretion was as small as in 1 mm (Figure 6F). Previously, the sizes of vesicle containing MMP-2 in lammelipodia of melanoma cells were estimated to be 0.5–1 mm using the fluorescence imaging method [31]. Thus, the luminescence spots derived from secreted MMP2- GLase were interpreted as a single exocytotic vesicle. An image of maximum luminescence intensity made from successive lumines- cence images showed the localized sites of MMP2-GLase secretion on the leading edge with a high spatial resolution (Figure 6G). During observation for 100 s, the numbers of transient lumines- cence spots in the upper and lower area defined in Figure 5G were estimated to be at least 70 and 20, respectively (Video S5). Thus, the high-resolution video image of luminescence spot showed frequent vesicle fusion for MMP-2 secretion at the leading edge. In our imaging procedure, the focus of luminescence spots for MMP2-GLase secretion was within ,1 mm from the cover slip (Figure 7), and both luminescence spots of secreted MMP2-GLase and the membrane-associated MMP2-GLase were presumably present in the bottom side of a cell membrane (Figure 5 and Video PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e25243