Depletion of apical transport proteins perturbs epithelial cyst formation and ciliogenesis

Abstract

Epithelial cells are vital for maintaining the complex architecture and functions of organs in the body. Directed by cues from the extracellular matrix, cells polarize their surface into apical and basolateral domains, and connect by extensive cell-cell junctions to form tightly vowen epithelial layers. In fully polarized cells, primary cilia project from the apical surface. Madin-Darby canine kidney (MDCK) cells provide a model to study organization of cells as monolayers and also in 3D in cysts. In this study retrovirus-mediated RNA interference (RNAi) was used to generate a series of knockdowns (KDs) for proteins implicated in apical transport: annexin-13, caveolin-1, galectin-3, syntaxin-3, syntaxin-2 and VIP17 and/or MAL. Cyst cultures were then employed to study the effects of these KDs on epithelial morphogenesis. Depletion of these proteins by RNAi stalled the development of the apical lumen in cysts and resulted in impaired ciliogenesis. The most severe ciliary defects were observed in annexin-13 and syntaxin-3 KD cysts. Although the phenotypes demonstrate the robustness of the formation of the polarized membrane domains, they indicate the important role of apical membrane biogenesis in epithelial organization.

Regulation of epithelial cell polarity by PAR-3 depends on Girdin transcription and Girdin–Gαi3 signaling

“…In Girdin-depleted cysts, E-cadherin was excluded from luminal surfaces containing apical gp135 (supplementary material Fig. S4G), indicating that the apical domain of each cell was not correctly integrated, similar to reports for PAR-3 and apical transport proteins (Bryant et al, 2010;Horikoshi et al, 2009;Torkko et al, 2008). Gαi3 colocalized with F-actin in the apical membrane of MDCK cysts (Fig.…”

Different responses in transformation of MDCK cells in 2D and 3D culture by v-Src as revealed by microarray techniques, RT-PCR and functional assays

“…Collagen I (BD Biosciences, rat tail collagen I, high concentration) was prepared as described by Torkko et al 19 Ice cold Matrigel and Collagen I were mixed, the cell suspension was added and the mixture pipetted into flexiPERM reusable cell culture chambers (Greiner Bio-One, Frickenhausen, Germany) sealed on a microslide. After polymerization of Matrigel new medium supplement (200 ml D-MEM with 5% FBS) was given.…”

The galectin lattice at a glance

“…Similarly, in the absence of the basolateral sorting adaptor AP-1B, Gal4 promotes transcytosis and apical sorting of the transferrin receptor through apical recycling endosomes (Perez Bay et al, 2014). Gal3, Gal4, Gal7 and Gal9 have all been shown to be determinants of apical sorting, and are required for epithelial morphogenesis and polarity, the development of the apical lumen and ciliogenesis (Delacour et al, 2006(Delacour et al, , 2008Mishra et al, 2010;Mo et al, 2012;Rondanino et al, 2011;Stechly et al, 2009;Torkko et al, 2008). Gal3 trafficking, therefore, involves non-classical secretion from the cytoplasm and glycan-dependent raft endocytosis to recycling endosomes, where it and other galectins play roles in post-Golgi sorting (see poster).…”