Bax, a member of the Bcl-2 family, translocates to mitochondria during apoptosis, where it forms oligomers which are thought to release apoptogenic factors such as cytochrome c. Using anoikis as a model system, we have examined spatial and temporal changes in Bax distribution. Bax translocates to mitochondria within 15 min of detaching cells from extracellular matrix, but mitochondrial permeabilization does not occur for a number of hours. The formation of Bax oligomers and perimitochondrial clusters occurs concomitant with caspase activation and loss of mitochondrial membrane potential, before nuclear condensation. Cells can be rescued from apoptosis if they are replated onto extracellular matrix within an hour, whereas cells detached for longer could not. The loss of ability to rescue cells from anoikis occurs after Bax translocation, but before the formation of clusters and cytochrome c release. Our data suggest that Bax regulation occurs at several levels, with formation of clusters a late event, and with critical changes determining cell fate occurring earlier.
Tumor necrosis factor (TNF) ␣-converting enzyme (TACE/ADAM-17) has diverse roles in the proteolytic processing of cell surface molecules and, due to its ability to process TNF␣, is a validated therapeutic target for anti-inflammatory therapies. Unlike a number of other ADAM proteins, which interact with integrin receptors via their disintegrin domains, there is currently no evidence for an ADAM-17-integrin association. By analyzing the adhesion of a series of cell lines with recombinant fragments of the extracellular domain of ADAM-17, we now demonstrate a functional interaction between ADAM-17 and ␣ 5  1 integrin in a trans orientation. Because ADAM-17-mediated adhesion was sensitive to RGD peptides and EDTA, and the integrin-binding site within ADAM-17 was narrowed down to the disintegrin/ cysteine-rich region, the two molecules appear to have a ligand-receptor relationship mediated by the ␣ 5  1 ligand binding pocket. Intriguingly, ADAM-17 and ␣ 5  1 were found to co-localize in both membrane ruffles and focal adhesions in HeLa cells. When confluent HeLa cell monolayers were wounded, ADAM-17 and ␣ 5  1 redistributed to the leading edge and co-localized, which is suggestive of a cis orientation. We postulate that the interaction of ADAM-17 with ␣ 5  1 may target or modulate its metalloproteolytic activity.
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