The COPII component Sar1 has been shown to be required for luminal matrix assembly and tube expansion of Drosophila trachea ( Tsarouhas et al., 2007). The haunted and ghost genes encode the coat complex II (COPII) proteins Sec23 and Sec24, respectively ( Norum et al., 2010). For example, the Drosophila mutants haunted and ghost show defects in epithelial polarity as well as in secretion into the luminal matrix of the trachea and cuticle deposition. Membrane trafficking through the early secretory pathway has been implicated in tube or lumen formation in several systems in vivo ( Tsarouhas et al., 2007 Grieder et al., 2008 Jayaram et al., 2008 Norum et al., 2010). Increasing evidence shows that directed secretion of ECM is a key requirement in establishment of polarity. Membrane traffic is essential to establish and maintain cell polarity through apical and basal targeting of proteins as well as through directed recycling of components between these membrane domains ( Mellman and Nelson, 2008).

Elucidating the molecular mechanisms underlying the interactions between epithelial cells and basal lamina, culminating in formation of an epithelial sheet or a tube of cells, is crucial for our understanding of development, organ function, and the onset and progression of disease (reviewed by Bryant and Mostov, 2008). The ECM is generally secreted by epithelial cells. Cell–substrate contacts are defined as interactions between cells and a specialized extracellular matrix (ECM), the basal lamina, which consists mainly of collagen and laminin. Our results provide insight into the role of COPII in epithelial morphogenesis and have implications for the interpretation of epithelial polarity and organization assays in cell culture.Įpithelial morphogenesis requires formation of cell–substrate contacts to enable polarization of cells. We conclude that efficient COPII-dependent secretion, notably assembly of Sec13–Sec31, is required to drive epithelial morphogenesis in both two- and three-dimensional cultures in vitro, as well as in vivo. Incorporation of primary fibroblasts within the three-dimensional culture substantially restores normal morphogenesis. When embedded in a three-dimensional matrix, Sec13-depleted Caco-2 cells form cysts but, unlike controls, are defective in lumen expansion. Defects are seen in the ability of cells to adhere to the substrate, form a monolayer and form intercellular junctions. In human intestinal epithelial cells (Caco-2), Sec13 depletion causes defective epithelial polarity and organization on permeable supports. Here, we show that suppression of the outer layer COPII component, Sec13, in zebrafish embryos results in a disorganized gut epithelium. Secretion of collagen and other matrix components requires efficient coat complex II (COPII) vesicle formation at the endoplasmic reticulum. Neither buffer capacity nor counterion permeabilities were key determinants of pH(G), pH(ER) or ER/Golgi H(+) leak rates.Epithelial morphogenesis is directed by interactions with the underlying extracellular matrix. The Golgi is more acidic than the ER because it has an active H(+) pump and fewer or smaller H(+) leaks. Removal of either K(+) or Cl(-) did not affect ER and Golgi H(+) leak rates, or steady-state pH(G) and pH(ER). ER membranes had an apparent H(+) permeability three times greater than that of Golgi membranes. Buffer capacities of the cytosol, ER and Golgi were all similar (6-10 mM/pH). In live, intact cells, ER pH (pH(ER)) was 7.2 +/- 0.2 and Golgi pH (pH(G)) was 6.4 +/- 0.3 and was dissipated by bafilomycin. A pH-sensitive, membrane-permeable fluorescein-biotin was targeted to endoplasmic-reticulum- and Golgi-localized avidin-chimera proteins in HeLa cells. Using a novel method, we tested whether differences in H(+) 'leak' and/or counterion conductances contributed to the pH difference between two secretory pathway organelles. The mechanisms that regulate pH in these organelles are still unknown. The pH values form a decreasing gradient: the endoplasmic reticulum (ER) is nearly neutral, the Golgi is mildly acidic and the secretory granules are more acidic still ( approximately pH 5). Mammalian organelles of the secretory pathway are of differing pH.

Cystol had higher ph than er lumen