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First published online 12 August 2003
doi: 10.1242/jcs.00710

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Membrane type I-matrix metalloproteinase (MT1-MMP) is internalised by two different pathways and is recycled to the cell surface

¹ School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK
² Department of Oncology, Cambridge Institute for Medical Research, Hills Road, Cambridge CB2 2XY, UK

View larger version (40K): [in a new window]   Fig. 1. Active MT1-MMP is internalised in nonstimulated HT1080 cells. (A) The biotinylation experiment. Plasma membrane proteins were biotinylated on ice (black spots; biotinylation) and then internalised for 15 minutes at 37°C (uptake). Cells were cooled on ice and surface biotin was removed by treating the cells with MESNA for 25 minutes at 4°C as described in Materials and Methods (cleavage 1). Recycling of internalised biotinylated proteins was performed by placing the cells at 37°C for various times (chase), followed by a second MESNA treatment to cleave the biotin present on internalised proteins re-exposed at the cell surface (cleavage 2). (B) Active MT1-MMP is internalised in HT1080 cells. HT1080 cells were incubated on ice in the presence (lanes 2-5) or absence (lane 1) of biotin to label cell-surface proteins. Biotinylated HT1080 cells were kept for 15 minutes on ice (lanes 2 and 3) or warmed to 37°C (lanes 4 and 5) to allow internalisation and then treated with MESNA to remove cell-surface biotin (lanes 3 and 5). Biotinylated proteins were immunoprecipitated (IP) using an anti-biotin antibody, separated on an SDS-PAGE and analysed by immunoblotting using anti-MT1-MMP affinity-purified IgGs (IP). Unbound materials (non-IP) were also analysed. (C) bbHT1080 cells treated with (lanes 4, 5 and 6) or without (lanes 1, 2 and 3) PMA for 16 hours at 37°C were incubated with (lanes 2, 3, 5 and 6) or without biotin (lanes 1 and 4) on ice to label cell-surface proteins. Cells were warmed to 37°C to allow internalisation and treated with (lanes 3 and 6) or without (lanes 2 and 5) MESNA. Biotinylated proteins were immunoprecipitated, separated on an SDS-PAGE and analysed by immunoblotting.

View larger version (70K): [in a new window]   Fig. 2. Internalisation of IgG-bound MT1-MMP complexes in bbHT1080 cells visualised by immunofluorescence microscopy. Intact and nonstimulated bbHT1080 cells were incubated with affinity-purified anti-MT1-MMP IgGs for 1 hour at 4°C. Cells were then washed and fixed immediately with paraformaldehyde (A) or warmed to 37°C for 10 (B), 20 (C), 30 (D) or 60 minutes (E) before fixation. In F, bbHT1080 cells were warmed to 37°C for 60 minutes in the presence of nocodazole to depolymerise microtubules. IgG-bound MT1-MMP complexes were detected using fluorescently labelled secondary antibody in TX-100-permeabilised cells. Bar, 20 µm.

View larger version (73K): [in a new window]   Fig. 3. Internalised MT1-MMP colocalises with different endocytic markers. bbHT1080 cells that had internalised IgG-bound MT1-MMP complexes for 30 (A-F), 45 (G-I) or 60 minutes (J-L) were fixed, permeabilised and incubated with antibodies directed against EEA1 (B), Eps15 (E), Rab4 (H) and LAMP-1 (K). Arrowheads are examples of colocalising structures. The merged panels (C, F, I and L) depict colocalisation between MT1-MMP and the appropriate endocytic marker. Bar, 20 µm.

View larger version (16K): [in a new window]   Fig. 4. Active MT1-MMP is recycled to the cell surface in HT1080 cells. HT1080 cells were incubated on ice in the presence (lanes 2-9) or absence (lane 1) of biotin to label cell-surface proteins. Biotinylated cells were warmed to 37°C for 15 minutes to allow internalisation then cooled to 0°C and treated with (lane 3) or without (lane 2) MESNA. Cells were warmed to 37°C for 15 (lanes 4 and 5), 30 (lanes 6 and 7) and 45 minutes (lanes 8 and 9) to allow recycling and then treated with (lanes 5, 7 and 9) or without (lanes 4, 6 and 8) MESNA. Biotinylated proteins were immunoprecipitated, separated on an SDS-PAGE and analysed by immunoblotting using anti-MT1-MMP affinity-purified IgGs.

View larger version (58K): [in a new window]   Fig. 5. Active MT1-MMP is internalised in EGFP-EpsE95/295-expressing cells. bbHT1080 cells transiently transfected with a dynK44A dominant-negative dynamin mutant (A-D) or with an EGFPEpsE95/295 dominant-negative Eps15 mutant (E-H) were allowed to internalise anti-MT1-MMP pAb (A,B,E,F) or Texas red-conjugated transferrin (C,D,G,H) at 37°C. After fixation and TX-100 permeabilisation, IgG-bound MT1-MMP complexes (B,F) were revealed using a Texas-red conjugated secondary antibody. Bar, 20 µm.

View larger version (73K): [in a new window]   Fig. 6. MT1-MMP internalisation is greatly reduced in cells with perturbed caveolae function. bbHT1080 cells treated in the absence (A,C) or in the presence of 30 µg/ml nystatin (B) or 4 mM methyl-ß-cyclodextrin (D) were subjected to an antibody uptake assay using anti-MT1-MMP affinity-purified IgGs. After 15 minutes, cells were fixed and TX-100 permeabilised, and IgG-bound MT1-MMP complexes were revealed using a FITC-conjugated secondary antibody. Arrowheads are examples of internalised MT1-MMP-positive structures. Bar, 20 µm. (E) bbHT1080 cells were not treated (lanes 2 and 3) or treated for 1 hour with 30 µg/ml nystatin (lanes 4 and 5) or for 16 hours with 50 µg/ml concanavalin A (lanes 6 and 7) before cell-surface biotinylation as described in Materials and Methods. After 15 minutes internalisation, cells were treated with (lanes 3, 5 and 7) or without (lanes 2, 4 and 6) MESNA to remove cell-surface biotin. Biotinylated proteins were immunoprecipitated, separated on a SDS-PAGE and analysed by immunoblotting using anti-MT1-MMP affinity-purified IgGs.

View larger version (77K): [in a new window]   Fig. 7. Internalised MT1-MMP colocalised with caveolin-1 and clathrin heavy chain and is present in caveolin-1 containing fractions. bbHT1080 cells, which had internalised IgG-bound MT1-MMP complexes for 10 minutes (A,D), were fixed, permeabilised and incubated with antibodies directed against caveolin-1 (B) and clathrin heavy chain (E) followed by a fluorescently labelled secondary antibody. Merged panels (C,F) are shown in the right-hand column. Arrowheads are examples of colocalising structures. Bar, 20 µm. (G) After a detergent-free carbonate-based extraction, HT1080 cells were homogenised and subjected to subcellular fractionation. Fractions (1 ml) were collected from the top of the gradient, separated on a 10% SDS-PAGE gel and immunoblotted using anti-MT1-MMP affinity-purified IgG (upper panel) or with an anti-caveolin-1 pAb (lower panel).

View larger version (83K): [in a new window]   Fig. 8. TIMP-2-mediated MT1-MMP internalisation. HeLa cells transiently transfected with full-length (A,B) or with cytoplasmic tail-deleted MT1-MMP construct (MT1C; C,D) were subjected to an antibody uptake assay using anti-MT1-MMP affinity-purified IgGs, together with anti-TIMP2 67.4H11 antibodies. Internalised MT1-MMP-bound and TIMP2-bound IgG complexes were revealed at the cell surface of non-permeabilised cells (A,C) and in TX-100 permeabilised cells (B,D). All panels are z-projections of a stack or 10 images. Bar, 10 µm.