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1.

Figure 4. From: Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity.

Effect of site-directed mutations on MT1-MMP internalization. (A) Schematic representation of the MT1-MMP cytoplasmic sequence and mutations. (B) Transiently expressed products in CHO-K1 cells were analyzed by Western blotting using anti-FLAG M2 antibody. Amount of cell surface MT1-F and mutant proteins was calculated from the bound 125I-labeled anti-FLAG M2 antibody. Values indicate the mean ± SD of three experiments. (C) Internalization of MT1-F and its mutant proteins after a 30-min incubation was analyzed as in Fig. 1. Values are the mean ± SD of three experiments. The asterisks (*) indicate statistically significant differences (P < 0.001) between MT1-F and the mutant.

Takamasa Uekita, et al. J Cell Biol. 2001 December 24;155(7):1345-1356.
2.
Figure 9.

Figure 9. From: Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity.

Effect of the mutations on Matrigel invasion. CHO-K1 cells were transfected with the expression plasmids for MT1-F, its mutants, or empty vector together with a GFP plasmid. Cells were then subjected to the Matrigel invasion assay. After 48 h of incubation, the invaded GFP-positive cells on the lower surface of the filters were counted under fluorescence microscopy. Each assay was performed in triplicate and numbers shown are the mean ± SD. The asterisks (*) indicate statistically significant differences (P < 0.001) between the cells expressing wild-type and mutant MT1-MMP molecules.

Takamasa Uekita, et al. J Cell Biol. 2001 December 24;155(7):1345-1356.
3.

Figure 3. From: Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity.

Effect of deleting the cytoplasmic domain on MT1-MMP internalization. (A) Schematic representation of the MT1-MMP cytoplasmic domain deletion mutants. (B) Transiently expressed products in CHO-K1 cells were analyzed by Western blotting using anti-FLAG M2 antibody. Amount of cell surface MT1-F and mutant proteins was calculated from the bound 125I-labeled anti-FLAG M2 antibody. (C) Internalization of MT1-F and its mutant proteins after a 30-min incubation was analyzed as in Fig. 1. Values in B and C are the mean ± SD of three experiments. The asterisks (*) indicate statistically significant differences (P < 0.001) between MT1-F and the mutant.

Takamasa Uekita, et al. J Cell Biol. 2001 December 24;155(7):1345-1356.
4.
Figure 6.

Figure 6. From: Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity.

Effect of cytoplasmic mutations on cell surface events mediated by MT1-MMP. (A) CHO-K1 cells were transfected with the expression plasmids for various MT1-MMP mutants. The cells were incubated with purified proMMP-2 in serum-free culture medium. After 18 h, MMP-2 in the culture medium was analyzed by gelatin zymography (top). Cell lysates were subjected to Western blot analysis using the anti–MT1-MMP monoclonal antibody. (B) The cells expressing MT1-F or MT1-F(ΔCP) or mock-transfected cells were incubated with 125I–TIMP-2, and the amount of TIMP-2 bound to the cells was calculated from the radioactivity. (C) Internalization of TIMP-2 bound to the cells was analyzed similarly as described in Fig. 1. Internalization of TIMP-2 bound to the cells expressing MT1-F (▪) or MT1-F(ΔCP) (⋄) is plotted. Values in B and C are the mean ± SD of three experiments.

Takamasa Uekita, et al. J Cell Biol. 2001 December 24;155(7):1345-1356.
5.

Figure 2. From: Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity.

Identification of the domain involved in MT1-MMP internalization in CHO-K1 cells. (A) Schematic representation of MT1-MMP mutants used in this experiment. SP, signal peptide; Pro, propeptide; CAT, catalytic domain; H, hinge; PEX, hemopexin-like domain; TM, transmembrane domain; CP, cytoplasmic domain; FLAG, FLAG epitope; GPI, glycosylphosphatidylinositol anchor. (B) Western blot analysis of MT1-MMP mutants expressed in CHO-K1 cells. Molecules expressed in the transfected cells were detected by anti-FLAG M2 antibody. The asterisks (*) indicate the nonspecific band. (C) Amount of MT1-F and mutants expressed on the cell surface calculated from the bound 125I-labeled anti-FLAG M2 antibody. Values are the mean ± SD of three experiments. (D) Internalization of MT1-MMP mutants after a 30-min incubation. Experiments were performed as described in Fig. 1 and the Materials and methods. Values are the mean ± SD of three experiments. The asterisks (*) indicate statistically significant differences (P < 0.001) between MT1-F and the mutant.

Takamasa Uekita, et al. J Cell Biol. 2001 December 24;155(7):1345-1356.
6.
Figure 7.

Figure 7. From: Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity.

Localization of MT1-F at the adherent edge and its internalization. (A) CHO-K1 cells were cotransfected with the plasmids for either MT1-F or ΔCP mutant and that for GFP. The cells were incubated with anti-FLAG M2 antibody, washed, and then incubated at 37°C for the indicated periods of time. The M2 antibody was detected by immunostaining with Cy3-labeled anti–mouse IgG. Signals were observed by confocal laser microscopy. The internalized antibody was stained using the cells that had been acid washed. Adherent layer; confocal section at the adherent layer, Integrated layers; integrated confocal layers. Bar, 10 μm. (B) The number of cells that retained the signals at the adherent layer were counted. Transfected cells were identified by GFP expression. At time 0, almost 100% of GFP-positive cells expressed MT1-F or MT1-F(ΔCP) at the adherent layer. Cells that retained more than 30% of the original average fluorescence intensity at the adherent layer at each time point were counted as positive and presented. 100 cells were counted independently three times and the values presented are the mean ± SD.

Takamasa Uekita, et al. J Cell Biol. 2001 December 24;155(7):1345-1356.
7.

Figure 8. From: Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity.

Internalization-defective MT1-MMP mutants cannot stimulate cell migration. (A) CHO-K1 cells were transfected with the expression plasmids for MT1-F and its mutants. Cell motility was analyzed on colloidal gold–coated coverslips. Representative phagokinetic tracks of the migrating cell are shown after visualization under bright-field illumination. Transfected cells were visualized by immunostaining using anti-FLAG antibody. (B) The migration area of the cell was visualized under bright-field illumination and analyzed using NIH Image software v1.62. The average of 10 cells ± SEM is shown. The asterisks (*) indicate statistically significant differences (P < 0.001) between MT1-F and the mutant. (C) Internalization of MT1-F during cell migration was monitored. Transfected cells were seeded on coverslips without colloidal gold and incubated. Cell surface MT1-F and its mutant were incubated with Texas red–labeled FLAG M2 antibody. After a 5-min incubation to allow for internalization, surface FLAG M2 antibody was removed by acid wash and internalized FLAG M2 antibody was observed by confocal laser microscopy. Actin was stained with AlexaTM488-conjugated phalloidin. Bars, (A) 100 μm; (B) 10 μm.

Takamasa Uekita, et al. J Cell Biol. 2001 December 24;155(7):1345-1356.
8.

Figure 1. From: Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity.

Internalization of wild-type MT1-MMP in CHO-K1 cells. (A) CHO-K1 cells were transfected with expression plasmids for MT1-F or hTfnR. 48 h later, the internalization of cell surface–expressed MT1-F or hTfnR was examined using 125I-labeled anti-FLAG M2 antibody or 125I-labeled hTfn as tracer ligands. The radioactivity relative to the amount of tracers bound to the surface at time 0 is plotted. ▪, radioactivity spontaneously released into the culture medium (Medium); ♦, radioactivity released by acid wash (Cell surface); ○, acid wash–resistant radioactivity (Internalize). Mean values of three independent experiments are shown (mean ± SD). Transfected cells were also analyzed by Western blotting using anti-FLAG M2 or anti-hTfnR antibodies (inset). (B) CHO-K1 cells expressing MT1-F or mock-transfected cells were incubated with anti-FLAG M2 antibody, after which the cells were incubated at 37°C for the indicated periods of time. The cell surface and internalized MT1-F molecules were analyzed by immunolocalization using AlexaTM488-conjugated anti–mouse IgG. Internalized MT1-F molecules were observed by washing the cells with acid solution before fixation and permeabilization. Signals were observed by confocal laser microscopy. Bar, 10 μm.

Takamasa Uekita, et al. J Cell Biol. 2001 December 24;155(7):1345-1356.
9.
Figure 5.

Figure 5. From: Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity.

Internalization of MT1-MMP through clathrin-coated vesicles. (A) The interaction of the MT1-MMP cytoplasmic tail with the μ2 subunit of AP-2 was examined by yeast two-hybrid analysis. The cytoplasmic domain sequences derived from MT1-WT and MT1-LY/A were expressed as fusion proteins with the GAL4 DNA binding domain (bait), while the μ2 subunit was expressed as a protein fusion with the GAL4 transcription activation domain (prey). Protein–protein interaction was detected by the X-α-Gal assay. Three independent clones of transformed yeasts were analyzed for each plasmid combination. TGN38 was a positive control. (B) Colocalization of internalizing MT1-F and clathrin. Cells expressing MT1-F or its cytoplasmic mutants (L1Y/A and ΔCP) were incubated with Texas red–labeled anti-FLAG M2 antibody (red) on ice, washed, and then left at 37°C for 5 min to allow for internalization. Clathrin molecules were immunoreacted with goat anti-clathrin antibody and visualized with Alexa™488-conjugated anti–goat IgG (green). The colocalization of internalizing MT1-F and clathrin was observed by washing the cells with acid solution before their fixation and permeabilization. Signals were then examined by confocal laser microscopy. Bar, 5 μm.

Takamasa Uekita, et al. J Cell Biol. 2001 December 24;155(7):1345-1356.

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