The integrin α6β4 functions in carcinoma cell migration on laminin-1 by mediating the formation and stabilization of actin-containing motility structures

Functional studies on the alpha 6 beta 4 integrin have focused primarily on its role in the organization of hemidesmosomes, stable adhesive structures that associate with the intermediate filament cytoskeleton. In this study, we examined the function of the alpha 6 beta 4 integrin in clone A cells, a colon carcinoma cell line that expresses alpha 6 beta 4 but no alpha 6 beta 1 integrin and exhibits dynamic adhesion and motility on laminin-1. Time-lapse videomicroscopy of clone A cells on laminin-1 revealed that their migration is characterized by filopodial extension and stabilization followed by lamellae that extend in the direction of stabilized filopodia. A function-blocking mAb specific for the alpha 6 beta 4 integrin inhibited clone A migration on laminin-1. This mAb also inhibited filopodial formation and stabilization and lamella formation. Indirect immunofluorescence microscopy revealed that the alpha 6 beta 4 integrin is localized as discrete clusters in filopodia, lamellae, and retraction fibers. Although beta 1 integrins were also localized in the same structures, a spatial separation of these two integrin populations was evident. In filopodia and lamellae, a striking colocalization of the alpha 6 beta 4 integrin and F-actin was seen. An association between alpha 6 beta 4 and F-actin is supported by the fact that alpha 6 beta 4 integrin and actin were released from clone A cells by treatment with the F-actin-severing protein gelsolin and that alpha 6 beta 4 immunostaining at the marginal edges of clone A cells on laminin-1 was resistant to solubilization with Triton X-100. Cytokeratins were not observed in filopodia and lamellipodia. Moreover, alpha 6 beta 4 was extracted from these marginal edges with a Tween-40/deoxycholate buffer that solubilizes the actin cytoskeleton but not cytokeratins. Three other carcinoma cell lines (MIP-101, CCL-228, and MDA-MB-231) exhibited alpha 6 beta 4 colocalized with actin in filopodia and lamellae. Formation of lamellae in these cells was inhibited with an alpha 6-specific antibody. Together, these results indicate that the alpha 6 beta 4 integrin functions in carcinoma migration on laminin-1 through its ability to promote the formation and stabilization of actin-containing motility structures.