Figure 4.

Crumbs3 rescues migration, polarity and tight junction formation. (A) iBMK parental, TDCLs or derivatives infected with the retroviral vector (5D/png) or the Crb3-expressing retroviral vector (5D/Crb3) were cultured to a confluent monolayer. Scratching a line through the monolayer with a pipette tip then disrupted a small area. The open gap was then monitored as the cells migrated in and filled the void using computerized video multifield time-lapse microscopy for three days. Epithelial fusion sheet resulting from tight junction formation is indicated below the red line. (B) Restoration of Crb3 expression establishes single layer cell growth, apicobasal polarity and lumen formation in TDCL 5D spheroids. TDCL 5D uninfected or infected with the empty retroviral vector png, or the wild type Crbs3 expressing vector were cultured in Matrigel and monitored by time-lapse microscopy. (C) Spheroids were co-stained with for β-catenin (green) and active caspase-3 (red) and imaged by confocal microscopy at 14 days. Crb3 expression confers single layer cell growth and apoptotic lumen formation in three-dimensional culture. 20–30 spheroids/microscopic field were counted in five different fields for each cell line and the experiment was repeated three times (p<0.05). Greater than 90% of TDCL 5D cells expressing Crbs3 formed spheroids with hollow lumens compared to less than 10% of TDCL 5D infected with the png vector.