Figure 1.

GFP-Crb3a expression in MDCKII cysts. (A) GFP-Crb3 construct. GFP-Crb3a was cloned into the tetracycline-inducible pRevTRE and virally transduced into wild-type MDCKII cells. (B) Western blot of GFP-Crb3a–expressing clones. MDCKII cells were infected with pRevTRE GFP-Crb3 and selected for 2 wk to generate a stable cell line. Clones were isolated, grown for 3 d, lysed in RIPA buffer, and analyzed by Western blot. The basal GFP-Crb3a expression in the absence of induction was sufficient to yield acceptable protein levels. (C) GFP-Crb3a localizes to the apical membrane, TJs, and cilia. MDCKII cells were grown on transwell filters for 4 or 7 d to obtain cilia, fixed, and costained with GP135, ZO-1, E-cadherin, or polyglutamylated tubulin. (D) Overexpression of GFP-Crb3a increases the size of the apical membrane domain. GFP-Crb3a–expressing MDCKII clones were embedded in collagen as previously described (Straight et al., 2004), grown for 7 d, fixed, and stained for the indicated markers. Clone 6 is a low-level expresser and only shows a moderate expansion of the apical membrane (arrowhead), whereas clone 7 expresses GFP-Crb3a at a higher level and displays folds of surplus apical membrane on the luminal side (arrowheads). (E) MDCKII wild-type cells grown in collagen for 7 d possess a flat apical membrane positive for endogenous Crb3 and GP135.