Figure 6.

Crb3 is required for the formation of the first apical lumen and recruitment of aPKCζ. Crb3 shRNA knockdowns were prepared as previously described. Isolated cell clones were embedded in Geltrex, fixed after 24 h, and stained with the indicated antibodies. (A, collapsed Z-stacks) In contrast to Luciferase shRNA treated control cells (subpanel a) that formed regular solitary lumina (arrow) at the two-cell stage (lumen size >5 μm), Crb3-depleted cells formed two-cell stages with missing intercellular lumina, because E-cadherin staining is continuous (subpanel b) or strongly decreased lumen size (subpanel c). Knocked-down cells often formed intracytoplasmic accumulations of the apical marker GP135 (subpanel b), with a simultaneous accumulation of aPKCζ. Some cells with impaired lumen formation showed minimal accumulation of aPKCζ at attaching membranes between daughter cells (subpanel b). GP135 internalization was still functional in Crb3 knockdown cells (subpanel d). (B) Quantification of the phenotypes of three different clones. Lumina were quantified according to their size (lumina ≤5 μm were categorized as small). Results were acquired in three independent experiments.