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Files in this Data Supplement:
Movie 1. A375 cells transfected with GFP-tagged mt1-mmp were cultured in glass bottom microwell dishes (Mattek, Ashland, MA) coated with rhodamine-conjugated gelatin in the presence of 5 mM BB94 to block MMP activity. After 16 hours cells were washed 3 times to remove BB94 and incubated in DMEM/F12 containing 20 mM HEPES pH 7.4 under an Olympus microscope (Olympus Italia, Segrate, Italy) fitted with a CCD camera. Images were acquired at 1 frame/minute for 1.5 hours.
Fig. S1. Effect of 20°C block on A375 melanoma cells. (A) Subcellular distribution of MT1-MMP and TGN 46 in A375 melanoma cells by immunofluorescence. A375 cells transfected with MT1-MMP were plated on coverlips for 16 hours, incubated at 20°C for 30 minutes, where indicated, and then fixed and analyzed at the confocal microscope. Cells were double-labeled with immunopurified polyclonal anti-MT1-MMP antibody MMR2 (MT1) and with polyclonal sheep antibody directed against TGN46 (TGN46), Merged staining is also shown. Bars: 20 mm. (B,C) MT1-MMP-transfected A375 cells were incubated for 30 minutes at 20°C, where indicated, and then fixed and prepared for cryo-immunogold labeling as decribed in Materials and Methods by using differently sized gold particles, as indicated. (B) Subcellular colocalization of MT1-MMP with endogenous TGN46 by cryo-immuno electron microscopy. MT1-MMP-transfected A375 cells at steady state or incubated for 30 minutes at 20°C where indicated, were fixed and prepared for cryo-immunogold labeling as decribed in Materials and Methods by using differently sized gold particles, as indicated: at 37°C, 15 nm, anti-TGN46; 10 nm, anti-MT1-MMP (immunopurified antibody MMR2); at 20°C, 5 nm, anti-TGN46; 10 nm, anti-MT1-MMP. Arrows mark the positions of specific areas containing TGN46, arrowheads mark MT1-MMP. Asterisks mark proteins detected at 20°C in Golgi cisternae. Of note in these conditions, MT1-MMP and furin colocalization appears to increase. Bar, 200 nm (C) Subcellular colocalization of MT1-MMP with endogenous furin by cryo-immuno electron microscopy. MT1-MMP-transfected A375 cells at steady state or incubated for 30 minutes at 20°C where indicated, were fixed and prepared for cryo-immunogold labeling as decribed in Materials and Methods by using differently sized gold particles, as indicated: 15 nm, anti-furin; 10 nm, anti-MT1-MMP (immunopurified antibody MMR2). Arrows mark the positions of specific areas containing both furin and MT1-MMP. Asterisks mark proteins detected at 20°C in Golgi cisternae. Bar, 150 nm. (D) Effect of the 20°C temperature block of furin membrane partitioning. Untransfected (NT) and furinGFP-transfected A375 cells were pulse-labeled with [35S]methionine for 5 minutes and, after the indicated chase times, incubated with TNE containing 1% Triton X-100 on ice. Soluble (S) and insoluble (P) fractions were separated by ultracentrifugation, immunoprecipitated with polyclonal polyclonal anti-GFP antibody, subjected to SDS-PAGE, transferred to nitrocellulose membrane and visualized by autoradiography.
Fig. S2. Effect of BFA on MT1-MMP processing and cell surface presentation. Untransfected (NT) and MT1-MMP-transfected A375 cells were biotinylated at 4°C and then lysed with a TNE containing 1% Triton X-100, as described in Materials and Methods. After centrifugation at 4°C (13,000 g, 10 minutes), intracellular proteins (Int) were separated from biotinylated plasma membrane proteins (PM) by capture with streptavidine-conjugated agarose beads. Each fraction was subjected to SDS-PAGE and transferred on nitrocellulose membrane. The MT1-MMP distribution profile was analyzed with polyclonal anti-MT1-MMP antibody M3927 (Sigma). The black arrowheads indicate the 65 and 63 kDa forms. Where indicated, cells were treated with BFA (10 mg/ml) for 3 hours prior to biotinylation.
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