scholarly journals Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes.

1994 ◽  
Vol 125 (1) ◽  
pp. 67-86 ◽  
Author(s):  
G Apodaca ◽  
L A Katz ◽  
K E Mostov
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Cell ◽  
Fluid Phase ◽  
Secretory Component ◽  
Apical Plasma Membrane ◽  
Recycling Endosome ◽  
Endosomal Compartment ◽  
Early Endosomes

Classically, the polymeric immunoglobulin receptor and its ligand, IgA, are thought to be sorted from basolateral early endosomes into transcytotic vesicles that directly fuse with the apical plasma membrane. In contrast, we have found that in MDCK cells IgA is delivered from basolateral endosomes to apical endosomes and only then to the apical cell surface. When internalized from the basolateral surface of MDCK cells IgA is found to accumulate under the apical plasma membrane in a compartment that is accessible to two apically added membrane markers: anti-secretory component Fab fragments, and avidin internalized from the biotinylated apical pole of the cell. This accumulation occurs in the presence of apical trypsin, which prevents internalization of the ligand from the apical cell surface. Using a modification of the diaminobenzidine density-shift assay, we estimate that approximately 80% of basolaterally internalized IgA resides in the apical endosomal compartment. In addition, approximately 50% of basolaterally internalized transferrin, a basolateral recycling protein, has access to this apical endosomal compartment and is efficiently recycled back to the basolateral surface. Microtubules are required for the organization of the apical endosomal compartment and it is dispersed in nocodazole-treated cells. Moreover, this compartment is largely inaccessible to fluid-phase markers added to either pole of the cell, and therefore seems analogous to the recycling endosome described in nonpolarized cells. We propose a model in which transcytosis is not a specialized pathway that uses unique transcytotic vesicles, but rather combines portions of pathways used by non-transcytosing molecules.

scholarly journals Annexin XIIIb: a novel epithelial specific annexin is implicated in vesicular traffic to the apical plasma membrane.

1995 ◽  
Vol 128 (6) ◽  
pp. 1043-1053 ◽  
Author(s):  
K Fiedler ◽  
F Lafont ◽  
R G Parton ◽  
K Simons
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Cell ◽  
Protein Composition ◽  
Apical Plasma Membrane ◽  
Vesicular Traffic ◽  
Transport Vesicles ◽  
Apical Vesicles

The sorting of apical and basolateral proteins into vesicular carriers takes place in the trans-Golgi network (TGN) in MDCK cells. We have previously analyzed the protein composition of immunoisolated apical and basolateral transport vesicles and have now identified a component that is highly enriched in apical vesicles. Isolation of the encoding cDNA revealed that this protein, annexin XIIIb, is a new isoform of the epithelial specific annexin XIII sub-family which includes the previously described intestine-specific annexin (annexin XIIIa; Wice, B. M., and J. I. Gordon. 1992. J. Cell Biol. 116:405-422). Annexin XIIIb differs from annexin XIIIa in that it contains a unique insert of 41 amino acids in the NH2 terminus and is exclusively expressed in dog intestine and kidney. Immunofluorescence microscopy demonstrated that annexin XIIIb was localized to the apical plasma membrane and underlying punctate structures. Since annexins have been suggested to play a role in membrane-membrane interactions in exocytosis and endocytosis, we investigated whether annexin XIIIb is involved in delivery to the apical cell surface. To this aim we used permeabilized MDCK cells and a cytosol-dependent in vitro transport assay. Antibodies specific for annexin XIIIb significantly inhibited the transport of influenza virus hemagglutinin from the TGN to the apical plasma membrane while the transport of vesicular stomatitis virus glycoprotein to the basolateral cell surface was unaffected. We propose that annexin XIIIb plays a role in vesicular transport to the apical plasma membrane in MDCK cells.

Brefeldin A rapidly disrupts plasma membrane polarity by blocking polar sorting in common endosomes of MDCK cells

2001 ◽  
Vol 114 (18) ◽  
pp. 3309-3321 ◽  
Author(s):  
Exing Wang ◽  
Janice G. Pennington ◽  
James R. Goldenring ◽  
Walter Hunziker ◽  
Kenneth W. Dunn
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Brefeldin A ◽  
Apical Plasma Membrane ◽  
Transferrin Receptors ◽  
Recycling Endosome ◽  
Polarized Cells ◽  
Endocytic Sorting ◽  
On Line

Recent studies showing thorough intermixing of apical and basolateral endosomes have demonstrated that endocytic sorting is critical to maintaining the plasma membrane polarity of epithelial cells. Our studies of living, polarized cells show that disrupting endocytosis with brefeldin-A rapidly destroys the polarity of transferrin receptors in MDCK cells while having no effect on tight junctions. Brefeldin-A treatment induces tubulation of endosomes, but the sequential compartments and transport steps of the transcytotic pathway remain intact. Transferrin is sorted from LDL, but is then missorted from common endosomes to the apical recycling endosome, as identified by its nearly neutral pH, and association with GFP chimeras of Rabs 11a and 25. From the apical recycling endosome, transferrin is then directed to the apical plasma membrane. These data are consistent with a model in which polarized sorting of basolateral membrane proteins occurs via a brefeldin-A-sensitive process of segregation into basolateral recycling vesicles. Although disruption of polar sorting correlates with dissociation of γ-adaptin from endosomes, γ-adaptin does not appear to be specifically involved in sorting into recycling vesicles, as we find it associated with the transcytotic pathway, and particularly to the post-sorting transcytotic apical recycling endosome. Movies available on-line

scholarly journals Apical and basolateral transferrin receptors in polarized BeWo cells recycle through separate endosomes.

1991 ◽  
Vol 114 (6) ◽  
pp. 1149-1158 ◽  
Author(s):  
D P Cerneus ◽  
A van der Ende
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Cell Line ◽  
Apical Cell ◽  
Bewo Cell ◽  
Transferrin Receptors ◽  
Membrane Domains ◽  
Bewo Cells ◽  
Early Endosomes ◽  
Plasma Membrane Domains

Contrary to most other epithelia, trophoblasts in the human placenta, which form the physical barrier between the fetal and the maternal blood circulation, express high numbers of transferrin receptors on their apical cell surface. This study describes the establishment of a polarized trophoblast-like cell line BeWo, which exhibit a high expression of transferrin receptors on the apex of the cells. Cultured on permeable filter supports, BeWo cells formed a polarized monolayer with microvilli on their apical cell surface. Across the monolayer a transepithelial resistance developed of approximately 600 omega.cm2 within 4 d. Depletion of Ca2+ from the medium decreased the resistance to background levels, showing its dependence on the integrity of tight junctions. Within the same period of time the secretion of proteins became polarized. In addition, the compositions of integral membrane proteins at the apical and basolateral plasma membrane domains were distinct as determined by domain-selective iodination. Similar to placental trophoblasts, binding of 125I-labeled transferrin to BeWo monolayers revealed that the transferrin receptor was expressed at both plasma membrane domains. Apical and basolateral transferrin receptors were found in a 1:2 surface ratio and exhibited identical dissociation constants and molecular weights. After uptake, transferrin recycled predominantly to the domain of administration, indicating separate recycling pathways from the apical and basolateral domain. This was confirmed by using diaminobenzidine cytochemistry, a technique by which colocalization of endocytosed 125I-labeled and HRP-conjugated transferrin can be monitored. No mixing of the two types of ligands was observed, when both ligands were simultaneously internalized for 10 or 60 min from opposite domains, demonstrating that BeWo cells possess separate populations of apical and basolateral early endosomes. In conclusion, the trophoblast-like BeWo cell line can serve as a unique model to compare the apical and basolateral endocytic pathways of a single ligand, transferrin, in polarized epithelial cells.

scholarly journals Rab8 Regulates Basolateral Secretory, But Not Recycling, Traffic at the Recycling Endosome

2008 ◽  
Vol 19 (5) ◽  
pp. 2059-2068 ◽  
Author(s):  
Lauren Henry ◽  
David R. Sheff
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Surface ◽  
Recycling Endosome ◽  
Recycling Pathway ◽  
Polarized Epithelial Cells ◽  
Pass Through

Rab8 is a monomeric GTPase that regulates the delivery of newly synthesized proteins to the basolateral surface in polarized epithelial cells. Recent publications have demonstrated that basolateral proteins interacting with the μ1-B clathrin adapter subunit pass through the recycling endosome (RE) en route from the TGN to the plasma membrane. Because Rab8 interacts with these basolateral proteins, these findings raise the question of whether Rab8 acts before, at, or after the RE. We find that Rab8 overexpression during the formation of polarity in MDCK cells, disrupts polarization of the cell, explaining how Rab8 mutants can disrupt basolateral endocytic and secretory traffic. However, once cells are polarized, Rab8 mutants cause mis-sorting of newly synthesized basolateral proteins such as VSV-G to the apical surface, but do not cause mis-sorting of membrane proteins already at the cell surface or in the endocytic recycling pathway. Enzymatic ablation of the RE also prevents traffic from the TGN from reaching the RE and similarly results in mis-sorting of newly synthesized VSV-G. We conclude that Rab8 regulates biosynthetic traffic through REs to the plasma membrane, but not trafficking of endocytic cargo through the RE. The data are consistent with a model in which Rab8 functions in regulating the delivery of TGN-derived cargo to REs.

Polarized GP2 secretion in MDCK cells via GPI targeting and apical membrane-restricted proteolysis

1996 ◽  
Vol 270 (1) ◽  
pp. G176-G183 ◽  
Author(s):  
B. A. Fritz ◽  
A. W. Lowe
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Apical Cell ◽  
Secretory Protein ◽  
Apical Plasma Membrane ◽  
Zymogen Granule ◽  
Gpi Anchor ◽  
Pancreatic Acini

The major zymogen granule membrane protein in the exocrine pancreas is glycoprotein 2 (GP2), a glycosyl phosphatidylinositol (GPI)-linked membrane protein. Despite its GPI anchor, GP2 is secreted into the pancreatic duct. We examined the mechanism underlying the secretion of GP2 in isolated pancreatic acini and transfected Madin-Darby canine kidney (MDCK) cells (MDCK-GP2). MDCK-GP2 cells release GP2 almost exclusively (> 95%) from the apical membrane. Using GP2 as a model, we defined a novel mechanism of polarized protein secretion in which a secretory protein is targeted via a GPI anchor to the apical plasma membrane, whereupon the mature form is released by proteolysis. Furthermore, we described two features of MDCK cells that enhance the polarized release of GP2: an apical plasma membrane-restricted distribution of the protease responsible for GP2 membrane cleavage, and a transcytotic pathway to reroute basolateral plasma membrane GP2 to the apical cell surface.

scholarly journals Sorting of an apical plasma membrane glycoprotein occurs before it reaches the cell surface in cultured epithelial cells.

1984 ◽  
Vol 99 (6) ◽  
pp. 2131-2139 ◽  
Author(s):  
K S Matlin ◽  
K Simons
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Plasma Membrane ◽  
Epithelial Cell Line ◽  
Membrane Glycoprotein ◽  
Apical Surface ◽  
Infected Cells ◽  
Surface Domains ◽  
Darby Canine Kidney

In Madin-Darby canine kidney (MDCK) cells (a polarized epithelial cell line) infected with influenza virus, the hemagglutinin behaves as an apical plasma membrane glycoprotein. To determine biochemically the domain on the plasma membrane, apical or basolateral, where newly synthesized hemagglutinin first appears, cells were cultured on Millipore filters to make both cell surface domains independently accessible. Hemagglutinin in virus-infected cells was pulse-labeled, chased, and detected on the plasma membrane with a sensitive trypsin assay. Under all conditions tested, newly made hemagglutinin appeared simultaneously on both domains, with the bulk found in the apical membrane. When trypsin was continuously present on the basolateral surface during the chase, little hemagglutinin was cleaved relative to the amount transported apically. In addition, specific antibodies against the hemagglutinin placed basolaterally had no effect on transport to the apical domain. These observations suggested that most newly synthesized hemagglutinin does not transiently appear on the basolateral surface but rather is delivered directly to the apical surface in amounts that account for its final polarized distribution.

Vasopressin-induced changes in the three-dimensional structure of toad bladder apical surface

1987 ◽  
Vol 253 (5) ◽  
pp. C707-C720 ◽  
Author(s):  
J. H. Hartwig ◽  
D. A. Ausiello ◽  
D. Brown
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Apical Cell ◽  
Three Dimensional ◽  
Granular Cell ◽  
Cytochalasin D ◽  
Toad Bladder ◽  
Dimensional Structure ◽  
Apical Plasma Membrane ◽  
Apical Surface

The apical plasma membrane of toad bladder granular cells undergoes a rapid and dramatic increase in water permeability in response to vasopressin stimulation. Previous studies have shown that this permeability increase is accompanied by characteristic changes in the morphology of this membrane and that these changes may be involved in the hormonal response. In this report, we have used the technique of rapid freezing and freeze drying to obtain high resolution stereo images of the surface of the granular cell apical plasma membrane before and during vasopressin stimulation. Using this approach, we confirmed that vasopressin induces a ridge-to-villus transformation of the cell surface even in the absence of osmotic water flow, but now show that this transformation occurs at least in part via a retraction of segments of preexisting ridges, rather than by the growth of new microvilli from the apical cell surface. This is also demonstrated by the finding that vasopressin induces the ridge-to-villus transformation of the cell surface even in the presence of cytochalasin D. In addition, the rapid-freeze, freeze-dry technique reveals that the surface glycocalyx of the epithelial cells consists of a complex, three-dimensional network of filaments that is heterogeneous among different cells. Finally, vasopressin-induced tubular invaginations of the apical plasma membrane were visualized in stereomicrographs, and the number and size of such invaginations were altered in the presence of cytochalasin D. These may represent surface images of vasopressin-induced exo- and endocytotic events that are related to membrane permeability changes.

Selective localization of the polytopic membrane protein prominin in microvilli of epithelial cells - a combination of apical sorting and retention in plasma membrane protrusions

1999 ◽  
Vol 112 (7) ◽  
pp. 1023-1033 ◽  
Author(s):  
D. Corbeil ◽  
K. Roper ◽  
M.J. Hannah ◽  
A. Hellwig ◽  
W.B. Huttner
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Plasma Membrane ◽  
Membrane Domain ◽  
Membrane Protrusions ◽  
Selective Retention ◽  
Polytopic Membrane Protein ◽  
Plasma Membrane Domain

Prominin is a recently identified polytopic membrane protein expressed in various epithelial cells, where it is selectively associated with microvilli. When expressed in non-epithelial cells, prominin is enriched in plasma membrane protrusions. This raises the question of whether the selective association of prominin with microvilli in epithelial cells is solely due to its preference for, and stabilization in, plasma membrane protrusions, or is due to both sorting to the apical plasma membrane domain and subsequent enrichment in plasma membrane protrusions. To investigate this question, we have generated stably transfected MDCK cells expressing either full-length or C-terminally truncated forms of mouse prominin. Confocal immunofluorescence and domain-selective cell surface biotinylation experiments on transfected MDCK cells grown on permeable supports demonstrated the virtually exclusive apical localization of prominin at steady state. Pulse-chase experiments in combination with domain-selective cell surface biotinylation showed that newly synthesized prominin was directly targeted to the apical plasma membrane domain. Immunoelectron microscopy revealed that prominin was confined to microvilli rather than the planar region of the apical plasma membrane. Truncation of the cytoplasmic C-terminal tail of prominin impaired neither its apical cell surface expression nor its selective retention in microvilli. Both the apical-specific localization of prominin and its selective retention in microvilli were maintained when MDCK cells were cultured in low-calcium medium, i.e. in the absence of tight junctions. Taken together, our results show that: (i) prominin contains dual targeting information, for direct delivery to the apical plasma membrane domain and for the enrichment in the microvillar subdomain; and (ii) this dual targeting does not require the cytoplasmic C-terminal tail of prominin and still occurs in the absence of tight junctions. The latter observation suggests that entry into, and retention in, plasma membrane protrusions may play an important role in the establishment and maintenance of the apical-basal polarity of epithelial cells.

scholarly journals Sorting and endocytosis of viral glycoproteins in transfected polarized epithelial cells.

1986 ◽  
Vol 102 (4) ◽  
pp. 1242-1255 ◽  
Author(s):  
T A Gottlieb ◽  
A Gonzalez ◽  
L Rizzolo ◽  
M J Rindler ◽  
M Adesnik ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
G Protein ◽  
Apical Cell ◽  
Viral Envelope ◽  
Apical Plasma Membrane ◽  
Protein Synthesis Inhibitor ◽  
Transfected Cells ◽  
Cell Biol ◽  
Viral Glycoproteins

Previous studies (Rindler, M. J., I. E., Ivanov, H. Plesken, and D. D. Sabatini, 1985, J. Cell Biol., 100: 136-151; Rindler, M. J., I. E. Ivanov, H. Plesken, E. J. Rodriguez-Boulan, and D. D. Sabatini, 1984, J. Cell Biol., 98: 1304-1319) have demonstrated that in polarized Madin-Darby canine kidney cells infected with vesicular stomatitis virus (VSV) or influenza virus the viral envelope glycoproteins G and HA are segregated to the basolateral and apical plasma membrane domains, respectively, where budding of the corresponding viruses takes place. Furthermore, it has been shown that this segregation of the glycoproteins reflects the polarized delivery of the newly synthesized polypeptides to each surface domain. In transfection experiments using eukaryotic expression plasmids that contain cDNAs encoding the viral glycoproteins, it is now shown that even in the absence of other viral components, both proteins are effectively segregated to the appropriate cell surface domain. In transfected cells, the HA glycoprotein was almost exclusively localized in the apical cell surface, whereas the G protein, although preferentially localized in the basolateral domains, was also present in lower amounts, in the apical surfaces of many cells. Using transfected and infected cells, it was demonstrated that, after reaching the cell surface, the G protein, but not the HA protein, undergoes interiorization by endocytosis. Thus, in the presence of chloroquine, a drug that blocks return of interiorized plasma membrane proteins to the cell surface, the G protein was quantitatively trapped in endosome- or lysosome-like vesicles. The sequestration of G was a rapid process that was completed in many cells by 1-2 h after chloroquine treatment. The fact that in transfected cells the surface content of G protein was not noticeably reduced during a 5-h incubation with cycloheximide, a protein synthesis inhibitor that did not prevent the effect of chloroquine, implies that normally, G protein molecules are not only interiorized but are also recycled to the cell surface.

scholarly journals Transcytotic Efflux from Early Endosomes Is Dependent on Cholesterol and Glycosphingolipids in Polarized Hepatic Cells

2003 ◽  
Vol 14 (7) ◽  
pp. 2689-2705 ◽  
Author(s):  
Lydia K. Nyasae ◽  
Ann L. Hubbard ◽  
Pamela L. Tuma
Keyword(s):  
Plasma Membrane ◽  
Protein Trafficking ◽  
Protein Solubility ◽  
Fluid Phase ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Pharmacological Agents ◽  
Hepatic Cells ◽  
Early Endosomes ◽  
Golgi Network

We examined the role that lipid rafts play in regulating apical protein trafficking in polarized hepatic cells. Rafts are postulated to form in the trans-Golgi network where they recruit newly synthesized apical residents and mediate their direct transport to the apical plasma membrane. In hepatocytes, single transmembrane and glycolipid-anchored apical proteins take the “indirect” route. They are transported from the trans-Golgi to the basolateral plasma membrane where they are endocytosed and transcytosed to the apical surface. Do rafts sort hepatic apical proteins along this circuitous pathway? We took two approaches to answer this question. First, we determined the detergent solubility of selected apical proteins and where in the biosynthetic pathway insolubility was acquired. Second, we used pharmacological agents to deplete raft components and assessed their effects on basolateral-to-apical transcytosis. We found that cholesterol and glycosphingolipids are required for delivery from basolateral early endosomes to the subapical compartment. In contrast, fluid phase uptake and clathrin-mediated internalization of recycling receptors were only mildly impaired. Apical protein solubility did not correlate with raft depletion or impaired transcytosis, suggesting other factors contribute to apical protein insolubility. Examination of apical proteins in Fao cells also revealed that raft-dependent sorting does not require the polarized cell context.

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