In contrast, although some of the ADAM12-transfected cells extended membrane protrusions, these protrusions were less robust and did not lead to effective cell body translocation (Movie 2B). As seen in Figure 7, mock-transfected cells migrated persistently at a rate of ∼10 μm/h. Twenty-four hours after transfection, cells were labeled with the membrane-impermeable EZ-Link NHS-LC-biotin (Pierce Chemical), and avidin precipitation and immunoblotting were conducted as described in Materials and Methods. CHO-α4/GFP cells were transfected with 4 μg of DNA (per 6-cm dish) encoding the indicated 6Xmyc-tagged constructs. Expression of wild-type and mutant full-length ADAMs on the surface of CHO-α4/GFP cells.
Rather, by titering the functions of specific integrins, the ADAM may influence which integrin is used for cell migration. Our finding that different ADAMs can selectively affect cell migration mediated by different integrins suggests that ADAMs could influence integrin-mediated migration events in interesting and complex ways. Although ADAMs and integrins may interact in neighboring cells (Eto et al., 2000), the fact that ADAM 12 inhibits α4β1-mediated migration of single cells (Figure 7) indicates that an ADAM can influence an integrin in the same cell. Our results suggest that ADAM12 does not inhibit α4β1-mediated cell migration by altering the expression level of α4β1 integrin (Table 1).
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Integrins, which interact with extracellular matrix (ECM) molecules, are key players in cell migration (Ridley et al., 2003). ADAMs were expressed in Chinese hamster ovary cells that express defined integrins (α4β1, α5β1, or both), and cell migration on full-length fibronectin or on its α4β1 or α5β1 binding fragments was studied. Odds, markets, and casino content match how local players bet and play. Founded in the 2020s, this licensed platform brings live sports betting, eSports, and casino entertainment for Canada. New players may also qualify for a crypto casino sign up bonus as per current terms.
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For example, if a cell expresses both α4β1 and α5β1 integrins, which bind to different regions of FN, expression of ADAM12 could promote use of α5β1 for migration. The ability of ADAM 12 to influence α4β1-mediated cell migration in this defined system was not altered by a point mutation that ablates its proteolytic activity, nor by mutations that prevent removal of its Prodomain, an obligate event for proteolytic activity. Results presented in this study suggest that ADAMs may also influence cell migration in a manner independent of proteolytic activity. For example, ADAM proteases may shed factors that stimulate or direct cell migration (Maheshwari et al., 2001; Mechtersheimer et al., 2001). 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.
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 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).
(B) Scratch wound assays were repeated as in panel A, except that α5-mediated cell migration was scored on the CCBD region of FN. Thus, strikingly, ADAM12 and ADAM17 demonstrate reciprocal effects on α4β1 and α5β1 mediated cell migration (Figure 8, A and B). Analysis of movies showed that mock transfected cells displayed broad lamellipodia at the leading edge and the cells moved persistently in the direction of the protrusion (Movie 2A).