Figure 4.

The TM/cytoplasmic and catalytic domains of MT-MMP are required for activation of ECM-bound MMP-2, local ECM degradation and subsequent invasion by melanoma cells. Gelatinase activities of MMP-2 released into the medium by the cells transfected with pSG5 lacking an insert (Mock), pSG5 plasmids containing MT-MMP (MT-MMP), truncated mutant lacking the TM domain (ΔTM_MT-MMP), chimeric ΔTM_MT-MMP/TM_IL-2R, TIMP-1, chimeric TIMP-1/TM_IL-2R, or chimeric TIMP-1/TM_MT-MMP, were analyzed by gelatin zymography. (B) Gelatinase activities of MMP-2 associated with the ECM and invadopodial membranes of cells transfected with chimeric MT-MMP molecules. Cells cultured on crosslinked gelatin films were fractionated as described (24) and ECM/invadopodial fractions were examined by gelatin zymography. (C) The fibronectin-degrading activities of transfectants were measured with Optimas image analyzer after 6 h. Each value represents the mean of three separate determinations (among 100 cells) ± SD. (D) The invasiveness of transfectants after 12 h was analyzed in terms of the percentage of cells forming invadopodial extensions in the crosslinked gelatin film. Each value represents the mean of three separate determinations (among 100 cells) ± SD.