Figure 6.

Lateral lumen polarity requires the presence of E-cadherin but not E-cadherin–mediated cell–cell adhesion or α-catenin. ΔEcadherin expression was either repressed (+dox) or induced (−dox) in ΔEcad-MDCK cells (black and blue bars in E and a and b in A–C). In B and C, ΔEcad-MDCK cells were transduced with a Par1b-adenovirus. Par1b-MDCK cells were either mock depleted (red bars in E and c in A–C, first panel in D), depleted of E-cadherin (green bars in E and d in A–C, second panel in D) or depleted of α-catenin (e in A–C, third panel in D) and cultured in the presence (A) or absence (B–D) of dox. (A) Merged confocal x-y and x-z views of monolayers 24 h after Ca²⁺ switch; gp135 (green), ZO-1 (blue), and phalloidin (single x-z section in b, red). Arrowheads indicate the basal surface as determined by phalloidin staining (data not shown). Note that ΔEcad (b) induces lateral lumina at endogenous Par1b levels, whereas Ecadherin depletion (d) allows apical surface formation. α-Catenin–depleted cells (e) fail to elongate a lateral domain, resulting in gp135 accumulation just above the cell base where it localizes along a circumferential actin belt (data not shown; also see Capaldo and Macara, 2007). (B) Merged confocal x-y views and single x-z sections of recombinant Par1b-expressing monolayers 24 h after Ca²⁺ switch; gp135 (green), ZO-1 (blue), and phalloidin (x-z views, red). Note that E-cadherin depletion (d) but not ΔEcadherin expression (b) inhibits lateral lumen formation. (C) Merged confocal x-y views and single x-z sections of recombinant Par1b-expressing monolayers cultured in low Ca²⁺ medium in the presence of blebbistatin; gp135 (green), ZO-1 (blue), and phalloidin (x-z view; a, red). Note that E-cadherin depletion (d) but not ΔEcadherin expression (b) or α-catenin depletion (e) leads to apical rather than lateral lumen. (D) Merged confocal x-y views of recombinant Par1b-expressing monolayers 24 h after Ca²⁺ switch; gp135 (green) and phalloidin (red). Note the presence of gp135- and phalloidin-positive lateral lumina in the control and α-catenin shRNA panels (white arrows), the gp135-positive endosomes in the α-catenin shRNA panel (white asterisk), and gp135- and phalloidin-positive grape-like vacuoles in the E-cadherin shRNA panel (white arrowhead). Graph, percentage of cells with gp135 forming either apical or lateral lumen or in intracellular VACs/endosomes was quantified from three random images of ∼350 cells in each of three independent experiments. SEs are presented. (E) Percentage of cells with lateral lumina 3, 5, and 8 h after Ca²⁺ switch in the presence of blebbistatin; data from three independent experiments are presented with SE. Asterisk (*) indicates significant difference with p > 0.001. Bars, 10 μm.