Dynamics of the α6β4 Integrin in Keratinocytes

Abstract

The integrin ␣6␤4 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 ␣6␤4 in live cells two different ␤4-chimeras were stably expressed in ␤4-deficient PA-JEB keratinocytes. One chimera consisted of full-length ␤4 fused to EGFP at its carboxy terminus (␤4-EGFP). In a second chimera the extracellular part of ␤4 was replaced by EGFP (EGFP-␤4), thereby rendering it incapable of associating with ␣6 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 ␤4-EGFP and EGFP-␤4 hemidesmosomes disappear, and a proportion of the ␤4-EGFP, but not of the EGFP-␤4 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 ␤4-EGFP migrate considerably more slowly than those that express EGFP-␤4. Studies with a ␤4-EGFP mutant that is unable to interact with plectin and thus with the cytoskeleton (␤4 R1281W -EGFP) suggest that the stabilization of the interaction between ␣6␤4 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 ␤4 with plectin renders the bond between ␣6␤4 and laminin-5 more stable, i.e., ␤4-EGFP is less dynamic than ␤4 R1281W-EGFP. On the other hand, when ␣6␤4 is bound to laminin-5, the binding dynamics of ␤4 to plectin are increased, i.e., ␤4-EGFP is more dynamic than EGFP-␤4. We suggest that the stability of the interaction between ␣6␤4 and laminin-5 is influenced by the clustering of ␣6␤4 through the deposition of laminin-5 underneath the cells. This clustering ultimately determines whether ␣6␤4 will inhibit cell migration or not. INTRODUCTIONKeratinocytes adhere to the basement membrane by hemidesmosomes that serve as anchoring sites for the intermediate filament system and play a critical role in stabilizing the association of the dermis with the epidermis. The transmembrane components of hemidesmosomes comprise the laminin-5 (LN-5) binding integrin ␣6␤4 and the bullous pemphigoid antigen (BP)180. These proteins are connected via the hemidesmosomal proteins plectin and BP230 to the keratin intermediate filament system (reviewed by Borradori and Sonnenberg, 1999).Based on their structural constituents, two subtypes of hemidesmosomes are distinguished. Type I hemidesmosomes contain ␣6␤4, plectin, BP180, and BP230 (Green and Jones, 1996), whereas type II hemidesmosomes contain only ␣6␤4 and plectin (Uematsu et al., 1994). Recently, the tetraspanin CD151 was identified as another component of both type I and II hemidesmosomes (Sterk et al., 2000). Type I or classical hemidesmosomes are present in basal keratinocytes of squamous and complex epithelia . Type II hemidesmosomes are found in intestinal epi...

Integrin α6β4-erbB2 Complex Inhibits Haptotaxis by Up-regulating E-cadherin Cell-Cell Junctions in Keratinocytes

“…Later on, cells may start to form ␣3␤1-controlled AJs, but they remain mobile, because sheet migration may now dominate. In contrast to ␣3␤1, ␣6␤4 is involved in blocking rather than promoting migration, as previously shown in primary human keratinocytes (10,39), HaCaT cells (6) and in ␤4-deficient PA-JEB keratinocytes, which were rescued by introducing a ␤4 construct (40). Importantly, Geuijen and Sonnenberg (40) showed that ␣6␤4-triggered motility inhibition was not simply due to increased adhesion to Ln-5 but was a result of increased stability of the interaction between ␣6␤4 and Ln-5.…”

“…In contrast to ␣3␤1, ␣6␤4 is involved in blocking rather than promoting migration, as previously shown in primary human keratinocytes (10,39), HaCaT cells (6) and in ␤4-deficient PA-JEB keratinocytes, which were rescued by introducing a ␤4 construct (40). Importantly, Geuijen and Sonnenberg (40) showed that ␣6␤4-triggered motility inhibition was not simply due to increased adhesion to Ln-5 but was a result of increased stability of the interaction between ␣6␤4 and Ln-5. Thus, ␣6␤4-triggered simultaneous up-regulation of both cell-ECM and cell-cell adhesion may result in an adhesive, non-migratory phenotype and may explain the observation that Ln-5 can guide keratinocytes to switch from a migratory-to a dormant, integrated epithelial phenotype (11,41).…”

“…After eight hours at 37°C, the number of single cells and doublets was assessed as described in of ␣6␤4 in thyroid cells or suprabasal keratinocytes promoted tumorigenesis (47)(48)(49)(50), indicating a role of ␣6␤4 in cancer formation and dissemination. In contrast, overexpression of ␣6␤4 in ␤4 null keratinocytes, breast, and urinary bladder cancer cells led to down-regulation of cell motility (40,51,52). Furthermore, cancer specimen analyses showed loss or down-regulation of ␣6␤4 and/or Ln-5 in breast and prostate cancer tissue (51,(53)(54)(55)(56)(57)(58) and an inverse correlation between ␤4 levels and dissemination potential in gastric cancer (59).…”

The making of hemidesmosome structures in vivo

“…In vitro cell culture studies have suggested that hemidesmosomes undergo active disassembly and reassembly in response to environmental cues. Time-lapse analysis of the hemidesmosome markers BP180 and b4-integrin has revealed the dynamic properties of these proteins, especially during keratinocyte migration and wound healing in scratch assays (Geuijen and Sonnenberg, 2002;Tsuruta et al, 2003). Moreover, it has been observed that hemidesmosomes get rapidly disassembled during carcinoma invasion (Rabinovitz and Mercurio, 1997;Santoro et al, 2003;Rabinovitz et al, 2004).…”

The Calcium/Calcineurin Pathway Promotes Hemidesmosome Stability through Inhibition of β4 Integrin Phosphorylation

“…Although HD provides a stable anchor, its components undergo a rapid turnover, indicating a dynamic equilibrium (12,13). This rapid turnover might allow epithelial cells to efficiently switch between a stationary and a migratory state by quickly changing the balance toward disassembly.…”