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First published online 11 October 2005
doi: 10.1242/jcs.02610

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The transmembrane domain is essential for the microtubular trafficking of membrane type-1 matrix metalloproteinase (MT1-MMP)

The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA

View larger version (82K): [in a new window]   Fig. 1. MT1-MMP-positive vesicles in MCF-MT-WT cells. Two hours after Chariot delivery of the MT1-MMP antibody Ab815 (left panels) and 1 hour after the Ab815 uptake (middle and right panels), the cells were fixed, permeabilized and stained for -tubulin (red) and Ab815 (green). Where indicated, the cells were pre-treated for 1 hour with nocodazole (1 µg/ml). Enlarged areas (boxed in top panels) show the MT1-MMP-positive vesicles (indicated by arrows) localized alongside the microtubule cytoskeleton. DAPI nuclear staining is blue.

View larger version (52K): [in a new window]   Fig. 2. Internalized MT1-MMP colocalizes with endosomal and centrosomal markers in MCF-MT-WT cells. (A) Following a 30-minute and a 45-minute uptake of the MT1-MMP antibody Ab815, the cells were stained for Rab-4 and Rab-11, respectively (both in red) and for Ab815 (green). Boxed areas are enlarged in the small panels. (B) Following the MT1-MMP antibody Ab815 uptake for 45 minutes, the cells were stained for Ab815 (green) and -tubulin (red). Where indicated, cells were pre-treated for 1 hour with nocodazole (1 µg/ml) or Brefeldin A (5 µg/ml). Arrows indicate the centrosomes. DAPI nuclear staining is blue.

View larger version (76K): [in a new window]   Fig. 3. Internalized MT1-MMP does not colocalize with the Golgi marker in MCF-MT-WT cells. Following the MT1-MMP antibody Ab815 uptake for 45 minutes, the cells were stained for Ab815 (green) and golgin-97 (red). Where indicated, cells were pre-treated for 1 hour with Brefeldin A (5 µg/ml). DAPI nuclear staining is blue.

View larger version (48K): [in a new window]   Fig. 4. Chariot delivery of the MT1-MMP antibody Ab815 into MDCK-MT-WT, MCF-MT-E240A, U-MT-WT and MCF-MT-WT cells. 30 minutes after delivery, the cells were stained for -tubulin (red) and the Ab815 (green). Where indicated, the cells were pre-treated for 1 hour with nocodazole (1 µg/ml). Arrows indicate centrosomes. DAPI nuclear staining is blue.

View larger version (50K): [in a new window]   Fig. 5. The transmembrane domain is required for the trafficking of MT-MMP to the cell surface of CHO cells. (A) Domain structure of mutant MT1-MMP. PRO, prodomain; CAT, catalytic domain; H, hinge region; PEX, hemopexin domain; TM, transmembrane domain; CT, cytoplasmic tail. (B) Immunoblotting and gelatin zymography of CHO cells expressing mutant MT1-MMP. Cells were lysed in a RIPA buffer containing a proteinase inhibitor cocktail-Set III and phenylmethylsulfonyl fluoride (Calbiochem, San Diego, CA, USA). The cell lysates were analyzed by western blotting with the MT1-MMP antibody Ab815 (upper panel). The ability of cells to activate MMP-2 was analyzed by gelatin zymography (bottom panel). An arrowhead indicates the gelatinolytically active, soluble, short MT-TM/CT construct found in the medium. Self-proteolysis of MT1-MMP leads to this short species (Osenkowski et al., 2004; Rozanov and Strongin, 2003). (C) Confocal microscopy of CHO cells expressing mutant MT1-MMP. Permeabilized and non-permeabilized cells were stained with the Ab815 antibody. The XY and ZX projections of cells are shown at the bottom and on the right of each panel, respectively. Notice the absence of staining of the non-permeabilized TM/CT cells.

View larger version (61K): [in a new window]   Fig. 6. Antibody uptake in MCF-MT-CT cells and delivery of the soluble MT1-MMP constructs. (A) MCF-MT-CT cells were treated with the MT1-MMP antibody Ab815. The antibody was then delivered to the cells by Chariot delivery (upper panel) and by antibody uptake (lower panel). The cells were stained for -tubulin (red) and Ab815 (green) 2 hours after Chariot delivery and 45 minutes after antibody uptake. Notice that the mutant was transported to the plasma membrane but it was not efficiently internalized and transported to the centrosomes. (B) MCF-zeo cells were treated with the PEX-TM/CT and PEX-TM constructs. The constructs were then delivered to the cells by Chariot delivery. Two hours after delivery, the cells were stained for MT1-MMP (green), golgin-97 and -tubulin (both in red). Notice that the delivered constructs do not colocalize with -tubulin and golgin-97, and appear to accumulate in the trans-Golgi network. Arrows indicate centrosomes. DAPI nuclear staining is blue.

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