The integrin alpha6beta4 functions in carcinoma cell migration on laminin-1 by mediating the formation and stabilization of actin-containing motility structures

Functional studies on the a 6 b 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 a 6 b 4 integrin in clone A cells, a colon carcinoma cell line that expresses a 6 b 4 but no a 6 b 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 functionblocking mAb specific for the a 6 b 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 a 6 b 4 integrin is localized as discrete clusters in filopodia, lamellae, and retraction fibers. Although b 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 a 6 b 4 integrin and F-actin was seen. An association between a 6 b 4 and F-actin is supported by the fact that a 6 b 4 integrin and actin were released from clone A cells by treatment with the F-actin– severing protein gelsolin and that a 6 b 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, a 6 b 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 a 6 b 4 colocalized with actin in filopodia and lamellae. Formation of lamellae in these cells was inhibited with an a 6-specific antibody. Together, these results indicate that the a 6 b 4 integrin functions in carcinoma migration on laminin-1 through its ability to promote the formation and stabilization of actin-containing motility structures. T he integrin a 6 b 4, a receptor for the laminins, is essential for the organization and maintenance of epithelial structure (11, 57). In many epithelia, this integrin mediates the formation of stable adhesive structures termed hemidesmosomes that link the intermediate filament cytoskeleton with the extracellular matrix (3, 16). Indeed, the ability of a 6 b 4 to associate with intermediate filaments distinguishes it from other integrins that interact primarily with the actin cytoskeleton (21). The importance of this integrin in epithelial structure has been reinforced by the recent generation of b 4-knockout mice that exhibit gross alterations in epithelial morphology and loss of anchorage to the basement membrane (11, 57). Although the a 6 b 4 integrin is also expressed in many carcinomas, its biological functions in these epithelialderived tumors have not been well studied (43). We and others have argued that a 6 b 4 may be associated with the process of carcinoma invasion (6, 12, 26, 43, 45). Initially, this argument was based on immunohistochemical data that correlated a 6 b 4 expression and localization with invasive carcinoma (12, 45, 56). More recently, we demonstrated that ectopic expression of a 6 b 4 in b 4-deficient colon carcinoma cells significantly increased the rate at which these cells invaded laminin matrices (6). Such data that associate a 6 b 4 with carcinoma invasion, however, are not consistent with the established role for this integrin in the formation of stable and rigid adhesive structures and maintenance of cell polarity in normal epithelial cells because invasive carcinoma cells are characterized by their dynamic interactions with extracellular matrices and their rapid rate of migration, as well as a loss of polarity (24). A priori, these dynamic functions of carcinoma cells would be impeded by the presence of a 6 b 4-containing hemidesAddress all correspondence to Arthur M. Mercurio, Beth Israel Deaconess Medical Center-Dana 601, 330 Brookline Ave., Boston, MA 02215. Tel.: (617) 667-7714. Fax: (617) 975-5071. E-mail: [email protected] on N ovem er 0, 2010 jcb.rress.org D ow nladed fom Published December 29, 1997