α-Actinin-4 is required for vinculin recruitment, permeability barrier formation, and cell–cell adhesion strengthening during junction development. (A) Wide-field immunofluorescence images showing accumulation of α-actinin-4 and actin at the cell junction from young 1 dpc to mature 5 dpc monolayers. (B) Wide-field immunofluorescence images showing α-actinin-4 accumulation preceding that of vinculin at the maturing junction in 2 dpc monolayers. (C) BSA flux assays showing maturation of barrier function over several days, which is compromised in α-actinin-4 knockdown cells. Error bars are standard errors. n = 3. (D) Wide-field immunofluorescence images showing lack of junctional vinculin in α-actinin-4 knockdown cells. (E) Wide-field fluorescence images showing holes in the α-actinin-4 knockdown cell monolayer after mechanical stress. (F) Wide-field immunofluorescence images showing mechanical stress to cell–cell adhesion results in disruption of α-actinin-4 knockdown in the 2 dpc cell monolayer. (G) Western blots showing normal levels of vinculin in α-actinin-4 knockdown cells. Bars, 10 µm.