Video-Rate Bioluminescence Imaging of Matrix Metalloproteinase-2 Secreted from a Migrating Cell Takahiro Suzuki1*, Chihiro Kondo1, Takao Kanamori1, Satoshi Inouye2* 1 Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan, 2 Yokohama Research Center, JNC Corporation, Yokohama, Japan Abstract Background: Matrix metalloproteinase-2 (MMP-2) plays an important role in cancer progression and metastasis. MMP-2 is secreted as a pro-enzyme, which is activated by the membrane-bound proteins, and the polarized distribution of secretory and the membrane-associated MMP-2 has been investigated. However, the real-time visualizations of both MMP-2 secretion from the front edge of a migration cell and its distribution on the cell surface have not been reported. Methodology/Principal Findings: The method of video-rate bioluminescence imaging was applied to visualize exocytosis of MMP-2 from a living cell using Gaussia luciferase (GLase) as a reporter. The luminescence signals of GLase were detected by a high speed electron-multiplying charge-coupled device camera (EM-CCD camera) with a time resolution within 500 ms per image. The fusion protein of MMP-2 to GLase was expressed in a HeLa cell and exocytosis of MMP-2 was detected in a few seconds along the leading edge of a migrating HeLa cell. The membrane-associated MMP-2 was observed at the specific sites on the bottom side of the cells, suggesting that the sites of MMP-2 secretion are different from that of MMP-2 binding. Conclusions: We were the first to successfully demonstrate secretory dynamics of MMP-2 and the specific sites for polarized distribution of MMP-2 on the cell surface. The video-rate bioluminescence imaging using GLase is a useful method to investigate distribution and dynamics of secreted proteins on the whole surface of polarized cells in real time. Citation: Suzuki T, Kondo C, Kanamori T, Inouye S (2011) Video-Rate Bioluminescence Imaging of Matrix Metalloproteinase-2 Secreted from a Migrating Cell. PLoS ONE 6(9): e25243. doi:10.1371/journal.pone.0025243 Editor: Matthew Bogyo, Stanford University, United States of America Received May 23, 2011; Accepted August 29, 2011; Published September 28, 2011 Copyright: 2011 Suzuki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science (JSPS) and by Strategic Research AGU-Platform Formation (2008–2012). No additional external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: SI is an employee of JNC Corporation. This does not alter the authors’ aherence to all the PLoS ONE policies on sharing data and materials. The authors have declared that no competing interests exist. * E-mail: tsuzuki@dpc.agu.ac.jp (TS); sinouye@jnc-corp.co.jp (SI) Introduction Matrix metalloproteinases degrade extracellular matrix proteins and regulate cell adhesion and migration. The polarized distribution of these proteinases has been demonstrated in migrating cells [1–5]. MMP-2 is one of the enzymes in degradation of basement membrane collagen and has a major role in cancer cell invasion. Regulatory mechanisms and inhibitors on MMP-2 protease activity have been extensively studied in cancer research [5–9]. Up-regulations of gene expression and secretion of MMP-2 in both cancer cells and surrounding stromal cells have been shown to promote cancer progression and metastasis [7]. In addition, MMP-2 plays important roles in immune and neural cells under physiological and pathological conditions [5,7,9–11]. On the cell surface, the inactive form of MMP-2 (pro-MMP-2) binds to tissue inhibitor of metalloproteinase-2 (TIMP-2) [12], which associated with the membrane type 1-matrix metallopro- teinase (MT1-MMP; also called MMP-14) [13], and then the amino terminal peptide of pro-MMP-2 is cleaved by MT1-MMP to give intermediate form [14]. The intermediate form binds to integrin avb3 at the cell surface, and full active MMP-2 is produced [15–17]. The polarized localization of MMP-2 on lamellipodia and invadopodia of a cell [1–4] were shown by the immunohistochemical studies using a fluorescence-labeled anti- body, and the activated MMP-2 is considered to be localized in front of a migrating cell with protease activity. However, the regulatory mechanism of MMP-2 secretion is still poorly understood, and exocytotic secretion of MMP-2 from the migrating cells has not been visualized in real time. To visualize an individual exocytotic event in a single living cell, total internal reflection fluorescence (TIRF) imaging has been mainly applied and can only visualize within the evanescent field [18–22]. For example, exocytosis of secretory vesicles possessing the fusion protein of low-density lipoprotein receptor with green fluorescence protein was polarized toward the leading edge in migrating fibroblasts [21]. However, some exocytotic events around the leading edge of cell, especially within 1,2 mm from the cell edge, could not be detected clearly by TIRF imaging, because lamellipodia in a migrating cell are often wavering and are detached from the cover slip [21]. This limitation of fluorescence imaging for protein secretion prompted us to apply video-rate bioluminescence imaging for the whole surface of a cell [23–26] (Figure 1A and B). Here, we introduced an electron multiplying charge-coupled device (EM-CCD) camera as a sensitive detector and established PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e25243