Dynamics of the α6β4 Integrin in Keratinocytes

Abstract

The integrin α6β4 has been implicated in two apparently contrasting processes, i.e., the formation of stable adhesions, and cell migration and invasion. To study the dynamic properties of α6β4 in live cells two different β4-chimeras were stably expressed in β4-deficient PA-JEB keratinocytes. One chimera consisted of full-length β4 fused to EGFP at its carboxy terminus (β4-EGFP). In a second chimera the extracellular part of β4 was replaced by EGFP (EGFP-β4), thereby rendering it incapable of associating with α6 and thus of binding to laminin-5. Both chimeras induce the formation of hemidesmosome-like structures, which contain plectin and often also BP180 and BP230. During cell migration and division, the β4-EGFP and EGFP-β4 hemidesmosomes disappear, and a proportion of the β4-EGFP, but not of the EGFP-β4 molecules, become part of retraction fibers, which are occasionally ripped from the cell membrane, thereby leaving “footprints” of the migrating cell. PA-JEB cells expressing β4-EGFP migrate considerably more slowly than those that express EGFP-β4. Studies with a β4-EGFP mutant that is unable to interact with plectin and thus with the cytoskeleton (β4R1281W-EGFP) suggest that the stabilization of the interaction between α6β4 and LN-5, rather than the increased adhesion to LN-5, is responsible for the inhibition of migration. Consistent with this, photobleaching and recovery experiments revealed that the interaction of β4 with plectin renders the bond between α6β4 and laminin-5 more stable, i.e., β4-EGFP is less dynamic than β4R1281W-EGFP. On the other hand, when α6β4 is bound to laminin-5, the binding dynamics of β4 to plectin are increased, i.e., β4-EGFP is more dynamic than EGFP-β4. We suggest that the stability of the interaction between α6β4 and laminin-5 is influenced by the clustering of α6β4 through the deposition of laminin-5 underneath the cells. This clustering ultimately determines whether α6β4 will inhibit cell migration or not.