Integrin α6β4E variant is associated with actin and CD9 structures and modifies the biophysical properties of cell-cell and cell-ECM interactions.

Integrin α6β4 is an essential, dynamic adhesion receptor for laminin 332 found on epithelial cells, required for formation of strong cell-ECM adhesion and induced migration, and coordinated by regions of the β4C cytoplasmic domain. β4E, a unique splice variant of β4 expressed in normal tissue, contains a cytoplasmic domain of 231 amino acids with a unique 114 amino acids sequence instead of β4C's canonical 1089 amino acids. We determined the distribution of α6β4E within normal human glandular epithelium and its regulation and effect on cellular biophysical properties. Canonical α6β4C expressed in all basal cells, as expected, while α6β4E expressed within a subset of luminal cells. α6β4E expression was induced by 3D culture conditions, activated Src, was reversible, and stabilized by bortezomib, a proteasome inhibitor. α6β4C expressed in all cells during induced migration whereas α6β4E was restricted to a subset of cells with increased kinetics of cell-cell and cell-ECM resistance properties. Interestingly, α6β4E presented in ring-like patterns measuring ∼ 1.75 × 0.72 microns, containing actin and CD9 at cell-ECM locations. In contrast, α6β4C expressed only within hemidesmosome-like structures containing BP180. Integrin α6β4E is an inducible adhesion isoform in normal epithelial cells that can alter biophysical properties of cell-cell and cell-ECM interactions. Movie S1 Movie S1 Live-imaging of induced migration of RWPE-1 cells without doxycycline treatment. RWPE-1 cells were cultured for 4 days and were induced to migrate by a scratch assay procedure. Movie was taken 1 hour after induction for 48 hours. Frames were captured every 10 minutes. Scale bar = 100 microns. Movie S2 Movie S2 Live-imaging of induced migration of RWPE-1 cells treated with doxycycline to induce β4E-tGFP. RWPE-1 cells were cultured for 4 days with doxycycline (500 ng/ml) and were induced to migration by a scratch assay procedure. Movie was taken 1 hour after induction for 48 hours. Frames were captured every 10 minutes. Scale bar = 100 microns. Movie S3 Movie S3 Integrin β4E co-localized with actin in "ring-like" patterns at the cell-ECM region. A representative Z-stack image showing β4E-tGFP (green) and actin (red) localized at "ring-like" patterns in RWPE-1 cells treated with doxycycline for 4 days. Z-stacks were captured with 0.2 μm step size. Scale bar = 10 microns.