Tetraspanins are localized at motility-related structures and involved in normal human keratinocyte wound healing migration

We have described previously that beta 1 integrins, which mediate keratinoc yte cell adhesion and migration, are in ligand-occupied conformation at the basal surface but not at the lateral and apical surfaces of keratinocytes. This led us to study the cellular localization and function of tetraspanin molecules, which have been postulated to modulate integrin activity. We fo und that CD9 and CD81 are highly expressed by keratinocytes clearly delinea ting filopodia at lateral and apical surfaces. CD63 and CD151 are largely e xpressed in the intracellular compartment, although some membrane expressio n is observed. We found accumulation of CD9, CD81, and CD151 together with alpha 3 and beta 1 integrins at intercellular junctions. In low calcium med ium, this intercellular space is crossed by a zipper of filopodia enriched in alpha 3 beta 1 and tetraspanin proteins. Interestingly, the expression o f CD9, CD81, and beta 1 and alpha 3 integrins was detected in the footprint s and rippings of motile keratinocytes, suggesting their role in both adhes ion to extracellular matrix and keratinocyte motility. beta 1 integrins wer e only partially activated in the rips, whereas cytoskeleton-linking protei ns such as talin were completely absent. On the other hand, antitetraspanin antibodies did not stain focal adhesions, which contain talin. The involve ment of tetraspanins in keratinocyte motility was assessed in a wound heali ng migration assay. Inhibition of cell migration was observed with antibodi es to CD9, CD81, beta 1, and alpha 3, and, to a lesser extent, to CD151. To gether these results indicate that tetraspanin-integrin complexes might be involved in transient adhesion and integrin recycling during keratinocyte m igration, as well as in intercellular recognition.