Figure 7.

Expression of GFP-Crb3a rescues the knockdown of Crb3. Crb3 shRNA clone 3 was transduced with empty pRevTRE vector, pRevTRE GFP-Crb3a full-length (FL), or missing its PDZ-binding motif (ΔERLI). (A) Western blot of MDCK wild-type cells versus empty vector–infected cells shows a persisting knockdown of Crb3a. Probing with anti-GFP antibody reveals similar expression levels for FL and ΔERLI constructs. (B, collapsed Z-stacks) Empty vector–transduced cells presented primarily a no-lumen phenotype with minimal recruitment of aPKCζ to the site of cell adhesion similar to the original knockdown (subpanel a). Expression of GFP-Crb3a FL rescues lumen formation and aPKCζ recruitment (subpanel b). GFP-Crb3a ΔERLI expression partially rescues MDCK lumen formation with a smaller average lumen diameter, but not aPKCζ recruitment, which was minimal in all observed two-cell stages (subpanel c). (C) Quantification of phenotypes. Cells with a lumen size >5 μm were categorized as large lumen, and smaller sizes were counted as small lumen. GFP-Crb3a FL expression rescues lumen formation with predominantly large lumina. GFP-Crb3a ΔERLI-expression Crb3a shRNAi cells display a partial rescue with a greater number of small lumina. (D) The average lumen diameter is significantly smaller in ΔERLI-rescued cells (3.2 μm) compared with FL-rescued cells (5.2 μm).