Figure 4.

The contractile force of actomyosin is necessary to maintain the central sealing elements in proximity. (A) WT EpH4 cells were treated with DMSO (control) or blebbistatin (50 μM) for 2 h, fixed, and then stained with anti-tricellulin mAb (green) and anti–claudin-3 pAb (magenta). Boxed regions are magnified below. Scale bar: 20 μm. (B) Schematic showing LSR-PP1C chimeric construct. (C) Whole-cell lysates of EpH4 WT cells, LSR KO cells, or LSR KO cells expressing either WT LSR (LSR wt) or LSR-PP1C were immunoblotted with the indicated antibodies. Molecular weight measurements are in kD. (D–F) LSR KO EpH4 cells expressing either LSR wt or LSR-PP1C were fixed and stained with anti-LSR pAb (green), anti-PP1γ mAb (magenta), and DAPI (blue; D); anti-2P-MRLC mAb (E); or anti–claudin-3 pAb (green) and anti-tricellulin mAb (magenta; F). (F) Boxed regions are magnified below. Scale bar: 20 μm. (G) The rate of tTJ disruption was quantified as in Fig. 1 D. Student’s t test; ****, P < 0.001. (H) Schematic showing changes in tTJs following myosin inhibition. Loss of actomyosin contractility at tTJs results in increased distance between converging central sealing elements. Source data are available for this figure: SourceData F4.