Fig. S4.

Tension-dependent recruitment of LGN to E-cadherin adhesions. (A) Immunostaining of endogenous LGN at cell–cell contacts in MDCK cells expressing T151 E-cadherin that were cultured at low density, high density, or at high density with 10% biaxial stretch. The quantification shows the mean ratio ± SD of LGN at cell–cell contacts versus cytosol. Similar to wild-type MDCK cells (Fig. 4A), LGN localized to cell–cell contacts in low cell density MDCK T151 cells, but LGN levels at cell–cell contacts were decreased at high cell density. This is explained by the fact that the actomyosin cytoskeleton exerts force on E-cadherin at the plasma membrane regardless of whether or not E-cadherin is involved in cell–cell contacts (31), and thus regardless of whether it forms homotypic interactions with E-cadherin on neighboring cells (which are absent in MDCK T151 cells). Stretch only increases tension on E-cadherin involved in cell–cell contacts (31), and biaxial stretch did not result in the enrichment of LGN at cell–cell contacts in MDCK cells expressing T151 E-cadherin. This finding indicates that LGN recruitment to cell–cell contacts upon stretch requires the transmission of force through the extracellular domain of E-cadherin. (Scale bars, 10 μm.) (B) Schematic overview of E-cadherin:Fc–coated sidewalls in which a vertical silicone sidewall is coated with E-cadherin:Fc (E-cad:Fc). (C and D) Localization of E-cadherin:DsRed and endogenous LGN at the sidewall–cell junction in MDCK cells, incubated for 5 min with 25 µM ML-7 or vehicle control (C), with the mean ratio ± SD of LGN at the cell–sidewall E-cadherin junction versus cytosol from three independent experiments (16 control cells and 13 ML-7–treated cells) (D). Arrowheads indicate enrichment of E-cadherin or LGN at the sidewall–cell junction. (Scale bars, 10 µm.) **P < 0.006, n.s., not significant.