scholarly journals Endocytic pathways in polarized Caco-2 cells: identification of an endosomal compartment accessible from both apical and basolateral surfaces.

1990 ◽  
Vol 110 (2) ◽  
pp. 337-348 ◽  
Author(s):  
E J Hughson ◽  
C R Hopkins
Keyword(s):  
Cell Line ◽  
Concanavalin A ◽  
Transferrin Receptor ◽  
Apical Surface ◽  
Double Labeling ◽  
Endocytic Compartment ◽  
Endosomal Compartment ◽  
Apical Cytoplasm ◽  
Peroxidase Conjugate ◽  
Endocytic Pathways

The enterocyte-like cell line Caco-2 forms a polarized epithelium when grown on filters. We have investigated the interaction of endocytic pathways from the apical and basolateral surfaces. The transferrin receptor was an appropriate marker for the basolateral route; uptake of radiolabeled transferrin was highly polarized, and recycling of this ligand back to the basolateral surface occurred with an efficiency of 95%, even after prolonged incubations with transferrin. Using a transferrin-peroxidase conjugate to delineate the morphological pathway, we have identified an early endocytic compartment in the basolateral cytoplasm of the cells. Longer incubations revealed a deeper endocytic compartment in the apical cytoplasm. Concanavalin A complexed to gold was used to simultaneously label the apical endocytic route. After 60 min, extensive mixing of the two labels was seen in endocytic elements throughout the apical cytoplasm, including in the Golgi area, but never in the basal cytoplasm. Using a second double labeling procedure in which antitransferrin receptor antibody complexed to gold was applied to the basolateral surface for up to 2 h and free peroxidase applied to the apical surface for shorter periods, we demonstrated that this apical marker rapidly (within 5 min) reached endosomes containing antibody-gold. Our results indicate that, in Caco-2 cells, the endocytic pathways from the apical and basolateral surfaces meet in an endosomal compartment from which transferrin can still be recycled.

scholarly journals Biogenetic pathways of plasma membrane proteins in Caco-2, a human intestinal epithelial cell line.

1990 ◽  
Vol 111 (4) ◽  
pp. 1351-1361 ◽  
Author(s):  
A Le Bivic ◽  
A Quaroni ◽  
B Nichols ◽  
E Rodriguez-Boulan
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Transferrin Receptor ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Epithelial Cell Line ◽  
Apical Surface ◽  
Intestinal Epithelial ◽  
Human Intestinal Epithelial Cell

We studied the sorting and surface delivery of three apical and three basolateral proteins in the polarized epithelial cell line Caco-2, using pulse-chase radiolabeling and surface domain-selective biotinylation (Le Bivic, A., F. X. Real, and E. Rodriguez-Boulan. 1989. Proc. Natl. Acad. Sci. USA. 86:9313-9317). While the basolateral proteins (antigen 525, HLA-I, and transferrin receptor) were targeted directly and efficiently to the basolateral membrane, the apical markers (sucrase-isomaltase [SI], aminopeptidase N [APN], and alkaline phosphatase [ALP]) reached the apical membrane by different routes. The large majority (80%) of newly synthesized ALP was directly targeted to the apical surface and the missorted basolateral pool was very inefficiently transcytosed. SI was more efficiently targeted to the apical membrane (greater than 90%) but, in contrast to ALP, the missorted basolateral pool was rapidly transcytosed. Surprisingly, a distinct peak of APN was detected on the basolateral domain before its accumulation in the apical membrane; this transient basolateral pool (at least 60-70% of the enzyme reaching the apical surface, as measured by continuous basal addition of antibodies) was efficiently transcytosed. In contrast with their transient basolateral expression, apical proteins were more stably localized on the apical surface, apparently because of their low endocytic capability in this membrane. Thus, compared with two other well-characterized epithelial models, MDCK cells and the hepatocyte, Caco-2 cells have an intermediate sorting phenotype, with apical proteins using both direct and indirect pathways, and basolateral proteins using only direct pathways, during biogenesis.

scholarly journals Endocytosis of Wingless via a dynamin-independent pathway is necessary for signaling in Drosophila wing discs

2016 ◽  
Vol 113 (45) ◽  
pp. E6993-E7002 ◽  
Author(s):  
Anupama Hemalatha ◽  
Chaitra Prabhakara ◽  
Satyajit Mayor
Keyword(s):  
Signal Transduction ◽  
Wing Disc ◽  
Low Ph ◽  
Endocytic Pathway ◽  
Apical Surface ◽  
Receptor Complexes ◽  
Drosophila Wing ◽  
Wing Discs ◽  
Ph Dependent ◽  
Endocytic Pathways

Endocytosis of ligand-receptor complexes regulates signal transduction during development. In particular, clathrin and dynamin-dependent endocytosis has been well studied in the context of patterning of the Drosophila wing disc, wherein apically secreted Wingless (Wg) encounters its receptor, DFrizzled2 (DFz2), resulting in a distinctive dorso-ventral pattern of signaling outputs. Here, we directly track the endocytosis of Wg and DFz2 in the wing disc and demonstrate that Wg is endocytosed from the apical surface devoid of DFz2 via a dynamin-independent CLIC/GEEC pathway, regulated by Arf1, Garz, and class I PI3K. Subsequently, Wg containing CLIC/GEEC endosomes fuse with DFz2-containing vesicles derived from the clathrin and dynamin-dependent endocytic pathway, which results in a low pH-dependent transfer of Wg to DFz2 within the merged and acidified endosome to initiate Wg signaling. The employment of two distinct endocytic pathways exemplifies a mechanism wherein cells in tissues leverage multiple endocytic pathways to spatially regulate signaling.

Differential localization of villin and fimbrin during development of the mouse visceral endoderm and intestinal epithelium

Development ◽  
1989 ◽  
Vol 106 (2) ◽  
pp. 407-419 ◽  
Author(s):  
R.M. Ezzell ◽  
M.M. Chafel ◽  
P.T. Matsudaira
Keyword(s):  
Intestinal Epithelium ◽  
Actin Filaments ◽  
Early Embryo ◽  
Actin Binding ◽  
Common Mechanism ◽  
Apical Surface ◽  
Visceral Endoderm ◽  
Apical Cytoplasm ◽  
Gut Epithelium ◽  
Microfilament Bundles

The apical surface of transporting epithelia is specially modified to absorb nutrients efficiently by amplifying its surface area as microvilli. Each microvillus is supported by an underlying core of bundled actin filaments. Villin and fimbrin are two actin-binding proteins that bundle actin filaments in the intestine and kidney brush border epithelium. To better understand their function in the assembly of the cytoskeleton during epithelial differentiation, we examined the pattern of villin and fimbrin expression in the developing mouse using immunofluorescence and immunoelectron microscopy. Villin is first detected at day 5 in the primitive endoderm of the postimplantation embryo and is later restricted to the visceral endoderm. By day 8.5, villin becomes redistributed to the apical surface in the visceral endoderm, appearing in the gut at day 10 and concentrating in the apical cytoplasm of the differentiating intestinal epithelium 2–3 days later. In contrast, fimbrin is found in the oocyte and in all tissues of the early embryo. In both the visceral endoderm and gut epithelium, fimbrin concentrates at the apical surface 2–3 days after villin; this redistribution occurs when the visceral endoderm microvilli first contain organized microfilament bundles and when microvilli first begin to appear in the gut. These results suggest a common mechanism of assembly of the absorptive surface of two different tissues in the embryo and identify villin as a useful marker for the visceral endoderm.

Clonal sublines that are morphologically and functionally distinct from parental OK cells

1989 ◽  
Vol 256 (4) ◽  
pp. F672-F679 ◽  
Author(s):  
J. A. Cole ◽  
L. R. Forte ◽  
W. J. Krause ◽  
P. K. Thorne
Keyword(s):  
Cell Line ◽  
Phosphate Transport ◽  
Parental Line ◽  
Apical Surface ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Sodium Dependent ◽  
Clonal Lines ◽  
P Cells ◽  
Camp Formation

Three clonal subpopulations of opossum kidney (OK) cells were derived from the parental line. The distribution of apical microvilli suggested that the OK cell line was heterogeneous. The clonal OK sublines appeared homogeneous as reflected by microvilli, which were uniformly distributed on the apical surface. Parathyroid hormone (PTH), forskolin (FSK), and prostaglandin E1 (PGE1) increased adenosine 3',5'-cyclic monophosphate (cAMP) formation in OK cells and all of the clones. PTH inhibited sodium-dependent phosphate transport in parental cells and in OK/B and OK/P clones with maximal effects appearing at 4, 2, and 1 h, respectively. PTH had no effect on phosphate transport in OK/H cells. FSK inhibited phosphate transport in parental cells and OK/B and OK/P clones but was relatively ineffective in OK/H cells. PGE1 decreased phosphate transport in OK/B and OK/P cells but was ineffective in the parental line and in OK/H cells. Phorbol 12-myristate 13-acetate, a potent inhibitor of phosphate transport in the parental OK cell line, had little effect in the clonal sublines. These clonal lines have remained phenotypically stable for 10 passages and should prove useful in studying the regulation of phosphate transport by PTH as well as addressing the question of whether PTH receptor subclasses exist which couple to cAMP and/or calcium effector systems in kidney cells.

Inhibition of H+ secretion by lectins in turtle bladder: role of a cell type on acidification

1985 ◽  
Vol 248 (5) ◽  
pp. R584-R594
Author(s):  
D. M. Potter ◽  
J. A. Arruda
Keyword(s):  
Concanavalin A ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Granular Cell ◽  
Cell Fraction ◽  
Dependent Manner ◽  
O2 Consumption ◽  
Cell Type ◽  
Double Labeling ◽  
Morphological Studies

Because certain lectins have been shown to bind to the intercalated cell of the cortical collecting tubule, we investigated the effect of concanavalin A and wheat germ agglutinin on urinary acidification in isolated turtle bladders. After addition to the mucosal but not serosal fluid, concanavalin A and wheat germ agglutinin decreased H+ secretion in a dose-dependent manner, and these effects were specifically inhibited by the competitive antagonists of concanavalin A (alpha-methyl-D-mannoside) and of wheat germ agglutinin (N-acetylglucosamine). Concanavalin A decreased H+ secretion by decreasing both the proton motive force and the active conductance of protons. Although electroneutral HCO3 secretion was not inhibited by either lectin, Na transport was decreased by 18 and 25%, respectively, after concanavalin A and wheat germ agglutinin. Concanavalin A failed to inhibit O2 consumption by the granular cell fraction but significantly inhibited O2 consumption by the carbonic anhydrase rich cell fraction. Morphological studies utilizing peroxidase or fluorescein-labeled concanavalin A showed that concanavalin A stained one cell type and that this staining was specific since it could be blocked by the competitive antagonist alpha-methyl-D-mannoside. Studies utilizing double labeling with fluorescein concanavalin A and acridine orange suggested that both probes stain the same cell type. The data strongly suggest that concanavalin A interacts specifically with the cell responsible for H+ secretion.

scholarly journals Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor μ2 kinase

2003 ◽  
Vol 163 (2) ◽  
pp. 231-236 ◽  
Author(s):  
Antony P. Jackson ◽  
Alexander Flett ◽  
Carl Smythe ◽  
Lindsay Hufton ◽  
Frank R. Wettey ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Heavy Chain ◽  
Transferrin Receptor ◽  
Coated Pits ◽  
Permeabilized Cells ◽  
Clathrin Heavy Chain

Endocytic cargo such as the transferrin receptor is incorporated into clathrin-coated pits by associating, via tyrosine-based motifs, with the AP2 complex. Cargo–AP2 interactions occur via the μ2 subunit of AP2, which needs to be phosphorylated for endocytosis to occur. The most likely role for μ2 phosphorylation is in cargo recruitment because μ2 phosphorylation enhances its binding to internalization motifs. Here, we investigate the control of μ2 phosphorylation. We identify clathrin as a specific activator of the μ2 kinase and, in permeabilized cells, we show that ligand sequestration, driven by exogenous clathrin, results in elevated levels of μ2 phosphorylation. Furthermore, we show that AP2 containing phospho-μ2 is mainly associated with assembled clathrin in vivo, and that the level of phospho-μ2 is strongly reduced in a chicken B cell line depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via the modulation of phospho-μ2 levels.

The Effect of Apotransferrin on Iron Release From Caco-2 Cells, an Intestinal Epithelial Cell Line

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3974-3979 ◽  
Author(s):  
Xavier Alvarez-Hernandez ◽  
Margaret Smith ◽  
Jonathan Glass
Keyword(s):  
Cell Line ◽  
Laser Scanning ◽  
Nitrilotriacetic Acid ◽  
Transepithelial Transport ◽  
Epithelial Cell Line ◽  
Apical Surface ◽  
Intestinal Epithelial ◽  
Iron Release ◽  
Transport Analysis ◽  
Texas Red

Abstract The Caco-2 cell line grown in bicameral chambers was used to study the effect of transferrin in the basal chamber on the transepithelial transport of iron. We have shown that when iron was offered as59Fe on the apical surface of the Caco-2 cells, transport of 59Fe into the basal chamber was stimulated by 50 μmol/L apotransferrin. Here, we examined the effect on59Fe transport of lower concentrations of apotransferrin, as well as the effects on transport of ovo-, cobalt-, and ferri-transferrin and of iron chelators with an affinity for iron greater than that of transferrin. The stimulation of 59Fe transport was more sensitive to the presence of apotransferrin with a Km of 0.078 ± 0.008 μmol/L compared with ferri-transferrin with a Km of 1.24 ± 0.39 μmol/L (P < .006). 59Fe transport was less sensitive to diethylenetriaminopenta-acetic acid (DTPA) than apotransferrin with Kms of 1.52 ± 0.70. The chelator nitrilotriacetic acid (NTA) exhibited no stimulation of59Fe transport. Analysis of laser scanning confocal micrographs showed that apotransferrin labeled with Texas Red is internalized by Caco-2 cells from the basal side and localizes in distinct vesicles above the nucleus. The sensitivity of apotransferrin in stimulating Fe transport suggests a unique interaction of apotransferrin with the basal surface of the intestinal epithelium.

scholarly journals Rapid endocytosis of the transferrin receptor in the absence of bound transferrin.

1985 ◽  
Vol 100 (2) ◽  
pp. 633-637 ◽  
Author(s):  
C Watts
Keyword(s):  
Cell Surface ◽  
Ligand Binding ◽  
Cell Line ◽  
Transferrin Receptor ◽  
Protein A ◽  
Transferrin Receptors ◽  
Coated Pits ◽  
Prolonged Incubation ◽  
Surface Receptors ◽  
Recycling Pathway

The rate of endocytosis of transferrin receptors, occupied or unoccupied with transferrin, was measured on the cell line K562. At 37 degrees C, receptors, radioiodinated on the cell surface at 4 degrees C, were internalized equally rapidly in the presence or absence of transferrin. In both cases, 50% of the labeled receptors became resistant to externally added trypsin in 5 min. An antitransferrin antibody was used to show directly that the receptors had entered the cells without bound transferrin. The distribution of the receptors on the cell surface was revealed by antibody and protein A-gold staining after prolonged incubation in the presence or absence of transferrin. The receptors were concentrated in coated pits under both conditions. The data suggest that endocytosis of transferrin receptors is not "triggered" by ligand binding and raise the possibility that ligand-induced down-regulation of surface receptors may not occur by this mechanism. Instead receptors may be recognized as being ligand-occupied, not at the cell surface, but at some other site in the recycling pathway such as the endosome.

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