cence images of Alexa Fluor 488 and Alexa Fluor 647 were acquired and processed using an Apotome microscope system (Carl Zeiss, Jena, Germany) with AxioVision4.6 software. Bioluminescence imaging For live cell luminescence imaging, HeLa cells cultured on a 35 mm glass-bottom dish (Asahi Glass Co., Tokyo, Japan) were transfected with GLase expressing vectors and cultured for 24 h. To obtain the luminescence image, the cells were washed 3 times with 3 ml of PBS and then were soaked with 1 ml of Hank’s balanced sodium solution (HBSS; Invitrogen) containing coelen- terazine (3 mg/ml). Luminescence signals were monitored at 37uC using a model IX71 or IX81-ZDC microscopes (Olympus Co., Tokyo, Japan) equipped with a thermostat incubator (Tokai Hit, Shizuoka, Japan) and with a back-thinned EM-CCD camera (model C9100-13; 5126512 pixels, pixel size = 16 mm; Hama- matsu photonics, K.K., Hamamastu, Japan) in a dark box. In an IX81-ZDC Zero Drift microscope system, a camera lens adaptor with an IR cut filter was attached between the microscope and the EM-CCD camera. High numerical aperture (NA) objective lens of UPLFLN 406O (NA1.30), UPLSAPO 606O (NA1.35), and PlanApo 1006OTIRFM (NA1.45) (Olympus) were used. Data of bioluminescence signals were captured on a computer hard disk using AQUACOSMOS software version 2.6 (Hamamatsu pho- tonics, K.K.) with acquisition mode of 161 binning, fast scanning, EM gain level = 255, and photon-counting level = 1 in the software. The luminescence signals were acquired with an exposure time of 30.5, 100, or 500 ms with an interval time of approximately 0.78 ms per image. Captured images were processed and calculation of luminescence signals was performed using the AQUACOSMOS software. In some cases, the maximum luminescence intensities in the successive luminescence images were used (using ‘‘MaxTrace’’ method in ‘‘sequential calculation’’ menu in the software) to generate an image, which consisted of maximum of luminescence intensities from successive video images at a time resolution of 500 ms. The luminescence signals were converted to pseudo-colored images (cyan or green), and then superimposed on the bright-field image to show localization of luminescence signals on a cell. To analyze time-dependent changes in luminescence intensity in a video image, average and maximum of luminescence intensities in the defined areas were calculated. References 1. Chen WT, Wang JY (1999) Ann N Y Acad Sci 878: 361–371. 2. Nabeshima K, Inoue T, Shimao Y, Okada Y, Itoh Y, et al. (2000) Cancer Res 60: 3364–3369. 3. Suetsugu S, Yamazaki D, Kurisu S, Takenawa T (2003) Dev Cell 5: 595–609. 4. Ogier C, Bernard A, Chollet AM, Le Diguardher T, Hanessian S, et al. (2006) Glia 54: 272–284. 5. VanSaun MN, Matrisian LM (2006) Birth Defects Res Part C Embryo Today 78: 69–79. 6. Seiki M (2002) Curr Opin Cell Biol 14: 624–632. 7. Egeblad M, Werb Z (2002) Nat Rev Cancer 2: 161–174. 8. Lafleur MA, Handsley MM, Edwards DR (2003) Expert Rev Mol Med 5: 1–39. 9. Overall CM, Kleifeld O (2006) Nat Rev Cancer 6: 227–239. 10. Candelario-Jalil E, Yang Y, Rosenberg GA (2009) Neuroscience 158: 983–994. 11. Tonti GA, Mannello F, Cacci E, Biagioni S (2009) Int J Dev Biol 53: 1–17. 12. Stetler-Stevenson WG, Krutzsch HC, Liotta LA (1989) J Biol Chem 264: 17374–17378. 13. Strongin AY, Collier I, Bannikov G, Marmer BL, Grant GA, et al. (1995) J Biol Chem 270: 5331–5338. 14. Atkinson SJ, Crabbe T, Cowell S, Ward RV, Butler MJ, et al. (1995) J Biol Chem 270: 30479–30485. 15. Brooks PC, Stro¨mblad S, Sanders LC, von Schalscha TL, Aimes RT, et al. (1996) Cell 85: 683–693. 16. Deryugina EI, Ratnikov B, Monosov E, Postnova TI, DiScipio R, et al. (2001) Exp Cell Res 263: 209–223. 17. Hood JD, Cheresh DA (2002) Nat Rev Cancer 2: 91–100. Video-Rate Bioluminescence Imaging of MMP-2 Supporting Information Video S1 Video-rate bioluminescence imaging of GLase secretion from a single HeLa cell with an exposure time of 500 ms. Luminescence signals were recorded for 75 s with an exposure time of 500 ms and a reading time of 0.78 ms per image. Luminescence signals were converted to pseudo-colored images (cyan) and then superimposed on the bright-field image. Elapsed time from the start of recording is shown in the movie. (MOV) Video S2 Video-rate bioluminescence imaging of GLase secretion from a single HeLa cell with an exposure time of 100 ms. Luminescence signals were recorded for 15 s with an exposure time of 100 ms and a reading time of 0.78 ms per image. (MOV) Video S3 Video-rate bioluminescence imaging of GLase secretion from a single HeLa cell with an exposure time of 30.5 ms. Luminescence signals were recorded for 4.7 s with an exposure time of 30.5 ms and a reading time of 0.78 ms per image. (MOV) Video S4 Video-rate bioluminescence imaging of MMP2-GLase being secreted and bound on the cell surface in a migrating HeLa cell with a 406 objective lens. Luminescence signals were recorded for 75 s with an exposure time of 500 ms and a reading time of 0.78 ms per image using a 406 objective lens. Video S4 was obtained after the addition of coelenterazine for 20 s. (MOV) Video S5 Video-rate bioluminescence imaging of MMP2-GLase secretion from a migrating HeLa cell with a 1006 objective lens. Luminescence signals were recorded for 100 s with an exposure time of 500 ms and a reading time of 0.78 ms per image using a 1006 objective lens. After the acquisition of Video S4, Video S5 was immediately obtained. (MOV) Author Contributions Conceived and designed the experiments: TS CK SI. Performed the experiments: TS CK. Analyzed the data: TS CK SI. Contributed reagents/materials/analysis tools: TS SI. Wrote the paper: TS TK SI. 18. Oheim M, Loerke D, Chow RH, Stu¨ hmer W (1999) Philos Trans R Soc Lond B Biol Sci 354: 307–318. 19. Steyer JA, Almers W (2001) Nat Rev Mol Cell Biol 2: 268–275. 20. Axelrod D (2003) Methods Enzymol 361: 1–33. 21. Schmoranzer J, Kreitzer G, Simon SM (2003) J Cell Sci 116: 4513–4519. 22. Jaiswal JK, Simon SM (2007) Nat Chem Biol 3: 92–98. 23. Inouye S, Ohmiya Y, Toya Y, Tsuji FI (1992) Proc Natl Acad Sci U S A 89: 9584–9587. 24. Thompson EM, Adenot P, Tsuji FI, Renard JP (1995) Proc Natl Acad Sci U S A 92: 1317–1321. 25. Miesenbo¨ck G, Rothman JE (1997) Proc Natl Acad Sci U S A 94: 3402–3407. 26. Suzuki T, Usuda S, Ichinose H, Inouye S (2007) FEBS Lett 581: 4551–4556. 27. Ma Z, Chang MJ, Shah R, Adamski J, Zhao X, et al. (2004) J Biol Chem 279: 46326–46334. 28. Zhai Y, Hotary KB, Nan B, Bosch FX, Mu n˜oz N, et al. (2005) Cancer Res 65: 6543–6550. 29. Verhaegen M, Christopoulos TK (2002) Anal Chem 74: 4378–4385. 30. Tannous BA, Kim DE, Fernandez JL, Weissleder R, Breakefield XO (2005) Mol Ther 11: 425–443. 31. Schnaeker EM, Ossig R, Ludwig T, Dreier R, Oberleithner H, et al. (2004) Cancer Res 64: 8924–8931. 32. Sbai O, Ferhat L, Bernard A, Gueye Y, Ould-Yahoui A, et al. (2008) Mol Cell Neurosci 39: 549–568. 33. Bretscher MS (1996) Cell 87: 601–606. 34. Jones MC, Caswell PT, Norman JC (2006) Curr Opin Cell Biol 18: 549–557. PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e25243