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The patterns of inhibition are consistent with the integrin binding profiles of the respective ADAM disintegrin domains on cells with activated β1 integrins (Bax et al., 2004) (Figure 1; our unpublished data). However, the mechanisms by which ADAMs function in cell migration remain unclear, both for ADAMs with (e.g., Xenopus ADAM13) and for those without (e.g., Caenorhabditis elegans ADAM14; unc71) proteolytic activity. ADAM (and ADAM-like) proteins have been implicated in several cell migration events (Nishiwaki et al., 2000; Alfandari et al., 2001; Martin et al., 2002; Huang et al., 2003; Seals and Courtneidge, 2003; White, 2003). In contrast, both the rate (Figure 7C) and persistence (Figure 7D) of migration of ADAM12-transfected cells were reduced and the cells showed little net translocation (Figure 7B).

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An experiment with cells transfected to express intracellular GFP showed that the biotinylation protocol did not label intracellular proteins (our unpublished data). Migration tracks of individual cells were analyzed using Openlab 2.2.5 software. Briefly, 35-mm glass bottom culture dishes (MatTek) were precoated with 2 μg/ml CS-1 overnight at 4°C, and 5 × 105 mock or ADAM12-transfected cells were plated on a dish for 2 h. After the incubation, cells in the top chambers were then removed with a cotton swab and membranes were fixed with 4% formaldehyde. Cells were resuspended in serum free F-12 medium (5 × 105 cells/ml), added to the top chambers, and incubated for 4 h (for CHOK1 and CHO-α4/GFP cells) or 15 h (for CHOB2-α4 and CHOB2-α5 cells) at 37°C. To take sequential https://aberturasaconcagua.com/ pictures and make movies, transfected cells were plated on 35-mm glass bottom culture dishes (MatTek, Ashland, MA) precoated with 2 μg/ml CS-1, scratch wounded, and placed on an open chamber with temperature controller (Harvard Apparatus, Holliston, MA).

We next used time-lapse microscopy to analyze the effect of ADAM12 on the ability of CHO-α4/GFP cells to migrate on the CS-1 region of FN after scratch wounding. Frames from the videos at 2-h postscratch wounding are shown for mock-(left) and ADAM12 (right)-transfected cells at the wound edges. (A) Images of cells from the 12-h time point of the experiment shown in Figure 3B. (C) CHO-α4/GFP cells were transfected as indicated and then analyzed in scratch wound assays, as described in the legend to Figure 2, except that 24-well dishes were coated with 10 μg/ml CCBD.