MT3-MMP in Matrix-Dependent Melanoma Invasion which is ineffective type I collagenase, suppressed MT1-MMP- dependent collagen invasion upon co-expression (Fig. 5A), a result consistent with the conclusion that MT3-MMP interfered with the collagen-invasive activity of MT1-MMP in melanoma cells. In COS-1 cells cultured on either fibrin or collagen the effects of MT3-MMP co-expression on total MT1-MMP protein levels were minor (Fig. 5B). In these co-transfected cells, MT1-MMP instead reduced the levels of full-length MT3-MMP with a concomitant increase in the processed, 37 kDa fragment of the protease via a process that was blocked by GM6001 (Fig. 5B). The catalytically- inactive MT1E/A did not induce MT3-MMP processing (Fig. 5B), indicating that the increased MT3-MMP cleavage required MT1- MMP activity. Furthermore, MT3-MMP slightly enhanced the processing of MT1E/A in the cells cultured on collagen (Fig. 5B). Figure 5. MT3-MMP regulates cell invasion in a matrix-composition dependent manner by driving fibrin invasion, while reducing the cell surface levels and collagen invasive activity of MT1-MMP. (A) COS-1 cells transiently transfected to express MT1-MMP (MT1), MT3- MMP (MT3), or both in combination, were allowed to invade 3D fibrin or collagen gels for 5 d. Quantitative results are expressed as the number of invasive foci per microscopic field (n = 3). (B–C) MT1-MMP, inactive MT1-MMP (MT1E/A), MT3-MMP, and MT3E/A were expressed alone or in different combinations in COS-1 cells for 48 h followed by immunoblotting and immunoprecipitation as indicated. GM6001 (GM, 10 mM) was added where indicated. Ponceau-staining and tubulin served as loading controls (n = 3). Asterix marks a non-specific band. (D) Cell surface MT1-MMP was detected from the transfected cells by surface biotinylation followed by immunoprecipitation. Asterix marks a non-specific band. (E) The transfected cells were fixed and stained for MT1-MMP with an antibody against its catalytic domain. Arrows indicate MT1-MMP staining on the cell surface in the cells expressing MT1-MMP alone or with inactive MT3E/A, as opposed to the perinuclear staining (arrowheads) in the cells co-expressing MT1-MMP and MT3-MMP. (F) The transfected cells were embedded in 3D collagen or fibrin as single cell suspension and allowed to grow for 5 d. Relative sizes of invasive areas per colony were calculated using ImageJ software (n = 3). (G) Fluorescence micrographs visualize representative colonies of COS-1 cells cultured inside collagen or fibrin and stained for filamentous actin. doi:10.1371/journal.pone.0028325.g005 PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e28325