28 research outputs found
Cell Surface Biotinylation and Other Techniques for Determination of Surface Polarity of Epithelial Monolayers
hINADl/PATJ, a homolog of discs lost, interacts with crumbs and localizes to tight junctions in human epithelial cells
Requirement for galectin-3 in apical protein sorting.
The central aspect of epithelial cells is their polarized structure, characterized by two distinct domains of the plasma membrane, the apical and the basolateral membrane. Apical protein sorting requires various signals and different intracellular routes to the cell surface. The first apical targeting motif identified is the membrane anchoring of a polypeptide by glycosyl-phosphatidyl-inositol (GPI). A second group of apical signals involves N- and O-glycans, which are exposed to the luminal side of the sorting organelle. Sucrase-isomaltase (SI) and lactase-phlorizin hydrolase (LPH), which use separate transport platforms for trafficking, are two model proteins for the study of apical protein sorting. In contrast to LPH, SI associates with sphingolipid/cholesterol-enriched membrane microdomains or "lipid rafts". After exit form the trans-Golgi network (TGN), the two proteins travel in distinct vesicle populations, SAVs (SI-associated vesicles) and LAVs (LPH-associated vesicles) . Here, we report the identification of the lectin galectin-3 delivering non-raft-dependent glycoproteins in the lumen of LAVs in a carbohydrate-dependent manner. Depletion of galectin-3 from MDCK cells results in missorting of non-raft-dependent apical membrane proteins to the basolateral cell pole. This suggests a direct role of galectin-3 in apical sorting as a sorting receptor
Receptors for and bioresponses to 1,25-dihydroxyvitamin D in a human colon carcinoma cell line (HT-29)
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Methods to estimate the polarized distribution of surface antigens in cultured epithelial cells
Pals1/Mpp5 is required for correct localization of Crb1 at the subapical region in polarized Muller glia cells
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Chapter 2 Methods to Estimate the Polarized Distribution of Surface Antigens in Cultured Epithelial Cells
This chapter reviews the methods employed to transfect and express foreign genes into epithelial cells. It also determines the polarized surface expression of both endogenous and exogenous plasma membrane proteins. The polarized distribution of surface proteins between the apical and the basolateral surfaces of epithelial cells is the basis of their vectorial function. Retroviral promoters, specifically Moloney murine leukemia virus (MuLV) and Rous sarcoma virus long terminal repeats (LTR) have resulted in high levels of expression of exogenous plasma membrane proteins. The neomycin-resistance marker coupled with selection with G418 has been very useful for the selection of stable transfectants. Polarity can be expressed as a ratio between the amounts of a given molecule in apical and basolateral plasma membrane domains. An important consideration in the analysis of this ratio is the fact that the area of the apical domain is usually a fraction of the area of the basolateral domain. It is important to express polarity as a density ratio—that is, a ratio between the amounts of the molecule per unit surface area in each surface domain. Various methods to estimate the surface polarity of a molecule in expressed epithelial cells include isotopic uptake or binding, electrophysiologic procedures, postsection colloidal gold immunocytochernistry, and EM immunocytochemistry