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First published online 23 November 2004
doi: 10.1242/jcs.01563

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Intracellular processing and activation of membrane type 1 matrix metalloprotease depends on its partitioning into lipid domains

¹ Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, S. Maria Imbaro, 66030, Chieti, Italy
² Department of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794, USA
³ Department of Veterans Affairs Medical Center, Northport, NY 11768, USA

View larger version (47K): [in a new window]   Fig. 1. MT1-MMP processing and localization in low-density Optiprep-gradient fractions from A375 melanoma cells. (A) Untransfected (NT) and MT1-MMP-transfected (MT1-MMP) A375 cells were pulse-labeled with [35S]methionine for 2 hours and their cleared lysates were immunoprecipitated with different antibodies, as indicated. (B) MT1-MMP-transfected A375 cells were pulse-labeled with [35S]methionine for 5 minutes and chased for the indicated times: these MT1-MMP immunoprecipitates were obtained with polyclonal anti-MT1-MMP antiserum MMR2. Where indicated, transfected cells were preincubated with 5 µM BB94 overnight and throughout the chase. MT1-MMP immunoprecipitates in A and B were subjected to SDS-PAGE, transferred to nitrocellulose membrane and visualized by autoradiography. The black arrowheads mark the positions of the MT1-MMP forms detected: calculated molecular masses are, respectively, 65, 63 and 60 kDa. The white arrowheads mark an unspecific band visible in all lanes that should not be confused with the 43 kDa form shown to be a MT1-MMP degradation product in some reports. (C) MT1-MMP-transfected A375 cells were lysed with 1% Triton X-100-containing TNE buffer, as described in Materials and Methods. Insoluble (P) and soluble (S) fractions were separated by ultracentrifugation at 4°C (120,000 g, 1 hour), resuspended in sample buffer and subjected to SDS-PAGE. Proteins were then transferred to nitrocellulose membrane and probed with polyclonal anti-MT1-MMP antiserum MMR2. MT1-MMP molecular forms in each fraction were quantified with the public domain ImageJ v.1.3 software and plotted on the reported chart as a percentage of total MT1-MMP. (D) MT1-MMP-transfected A375 cells were labeled with [35S]methionine for 2 hours and lysed with a 1% Triton X-100-containing buffer, as described in Materials and Methods. Lysates were fractionated on a discontinuous Optiprep-density gradient, as reported in the scheme on the bottom. MT1-MMP immunoprecipitates from each fraction and a sample of the initial preparation (Inp) were subjected to SDS-PAGE, transferred to nitrocellulose membrane and visualized by autoradiography. The amount of 63 kDa form in each fraction was determined with Fuji BAS software and plotted on the reported chart. A small sample of each fraction was tested by western blotting with a polyclonal anti-caveolin-1 antibody as a bona-fide marker for the DRM preparation.

View larger version (31K): [in a new window]   Fig. 2. Time-course analysis of MT1-MMP processing in A375 melanoma cells and association with different membrane fractions. Untransfected (NT) and MT1-MMP-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 anti-MT1-MMP antiserum MMR2, subjected to SDS-PAGE, transferred to nitrocellulose membrane and visualized by autoradiography. The chart shows the distribution in time of immature and mature MT1-MMP and association with different membrane fractions as determined with the Fuji BAS software.

View larger version (89K): [in a new window]   Fig. 3. 1-PDX blocks gelatin degradation and proMT1-MMP processing in MT1-MMP-transfected A375 melanoma cells. (A) A375 cells transfected with MT1-MMP and pcDNA 3.0 (MT1) or MT1-MMP and FLAG-tagged 1-PDX (MT1/1-PDX) were cultured on surfaces coated with rhodamine-conjugated gelatin. After 16 hours of incubation, cells were fixed and labeled with specific primary antibodies: polyclonal immunopurified anti-MT1-MMP antibody MMR2 (MT1-MMP) and monoclonal anti-FLAG antibody M2 (1-PDX). Degradation of the underlying fluorescent gelatin by the MT1-MMP/pcDNA3.0 and MT1-MMP/1-PDX cotransfectants is shown (Gelatin). Merged staining (MT1-MMP/Gelatin) for the MT1-MMP/pcDNA3.0 cotransfectants is also shown. Transfected cells were visualized by wide-field fluorescence microscopy and acquired images were deconvoluted as described in Materials and Methods. Bars, 20 µm. (B) Steady-state distribution profile of MT1-MMP in A375 melanoma cells: A375 cells transfected with MT1-MMP and pcDNA3.0 or MT1-MMP and 1-PDX were biotinylated and incubated with TNE containing 1% Triton X-100 on ice. Soluble (S) and insoluble (P) fractions were separated by ultracentrifugation. Intracellular fractions (Int) were separated from plasma membrane fractions (PM) by capturing the biotinylated proteins with streptavidin-conjugated agarose beads. Each fraction was subjected to SDS-PAGE and transferred on nitrocellulose membrane. The MT1-MMP distribution profile was analyzed with immunopurified polyclonal anti-MT1-MMP antibody MMR2. The white arrowheads mark the positions of the pro- and mature (63 and 60 kDa) forms in the MT1-MMP/pcDNA cotransfectants, the black arrowheads indicate the 65 and 63 kDa immature forms in the MT1-MMP/1-PDX cotransfectants. Staining for caveolin-1 in the same experiment is reported as a bona-fide marker for the DRM preparation.

View larger version (115K): [in a new window]   Fig. 4. Subcellular distribution of MT1-MMP in A375 melanoma cells by immunofluorescence. A375 cells transfected with MT1-MMP and pcDNA3.0 (A-I) or MT1-MMP and furin-GFP (J-L) were plated on coverlips for 16 hours, then fixed and analyzed at the confocal microscope. Cells were double-labeled with immunopurified polyclonal anti-MT1-MMP antibody MMR2 (A,D,G,J) and with antibodies directed against TGN46 (B), transferrin receptor (E) and mannose 6-phosphate receptor (H). Merged staining is also shown (C,F,I,L). Bars, 10 µm.

View larger version (48K): [in a new window]   Fig. 5. MT1-MMP processing takes place in an intracellular, post-Golgi compartment. (A) MT1-MMP-transfected A375 cells were pulse-labeled with [35S]methionine for 5 minutes. Labeled proteins were accumulated in the trans-Golgi network by incubating the cells at 20°C for 30 minutes, then released at 37°C for different lengths of time, as indicated. Triton X-100-soluble (S) and insoluble (P) fractions were separated by ultracentrifugation, immunoprecipitated and analyzed by SDS-PAGE/autoradiography. Tannic acid 0.5% was added during the 20°C block, 10 minutes prior to release at 37°C for 60 minutes. (B) Tannic acid completely blocks arrival of proteins to plasma membrane. A375 cells were transfected with the ts045 temperature sensitive mutant of VSV-G-GFP chimera, incubated at 40°C overnight, blocked at 20°C for 30 minutes then chased at 32°C for the indicated time in the absence or presence of 0.5% tannic acid. Panels illustrate representative control (top) and treated (bottom) cells. VSV-G staining on PM detected from non permeabilized cells with an antibody raised against its extracellular domain is shown in red. Bar, 10 µm. The graph shows ratio of VSV-G on plasma membrane to total VSV-G at indicated chase time. (C) EGTA treatment: after incubating the cells at 20°C for 30 minutes, the MT1-MMP-transfected pulsed A375 cells were treated (lane 3) or not (lane 2) with 5 mM EGTA for 30 minutes at 37°C. Only the soluble fraction is shown. MT1-MMP molecular forms in lanes 2 and 3 were quantified with the public domain ImageJ v.1.3 software and plotted on the reported chart as a ratio of MT1-MMP processing.

View larger version (155K): [in a new window]   Fig. 6. Characterization of an MT1-MMP- and furin-positive post-Golgi compartment in A375 melanoma cells by cryo-immuno-electron microscopy. MT1-MMP-transfected A375 cells were fixed and prepared for cryo-immunogold labeling as decribed in Materials and Methods by using differently sized gold particles, as indicated. Subcellular colocalization of MT1-MMP with endogenous furin: (A-C): 10 nm, anti-furin; 15 nm, anti-MT1-MMP (immunopurified antibody MMR2). Arrows mark the positions of specific regions containing both MT1-MMP and furin. Subcellular and plasma membrane colocalization of MT1-MMP with endogenous caveolin (D): 10 nm, anti-caveolin-1; 15 nm, anti-MT1-MMP (immunopurified antibody MMR2). PM indicates plasma membrane rims. C indicates caveosome. Bars, 100 nm (A); 75 nm (B,C); and 150 nm (D).

View larger version (13K): [in a new window]   Fig. 7. Furin is excluded from detergent-resistant membranes. (A) Untransfected (NT) and furin-GFP-transfected (FUR) A375 cells were labeled with [35S]methionine for 2 hours and lysed with TNE buffer containing 1% Triton X-100 on ice. Soluble (S) and insoluble (P) fractions were separated by ultracentrifugation, immunoprecipitated with a polyclonal anti-GFP antibody and analyzed by SDS-PAGE/autoradiography. (B) A375 cells transfected with MT1-MMP (MT1-MMP) or with MT1-MMP and furin-GFP (MT1+FUR) were labeled with [35S]methionine for 2 hours and lysed with TNE containing 1% Triton X-100 on ice. Soluble fractions were collected after ultracentrifugation, immunoprecipitated with polyclonal anti-MT1-MMP antiserum MMR2 and analyzed by SDS-PAGE/autoradiography. The processing ratio of MT1-MMP (MT1-MMP/MT1-MMP+proMT1-MMP) was determined with the Fuji BAS software and plotted on the reported chart.