scholarly journals Effect of nocodazole on vesicular traffic to the apical and basolateral surfaces of polarized MDCK cells.

1990 ◽  
Vol 111 (6) ◽  
pp. 2365-2373 ◽  
Author(s):  
P P Breitfeld ◽  
W C McKinnon ◽  
K E Mostov
Keyword(s):  
Apical Membrane ◽  
Mdck Cells ◽  
Polymeric Immunoglobulin Receptor ◽  
Apical Surface ◽  
Membrane Domains ◽  
Immunoglobulin Receptor ◽  
Vesicular Traffic ◽  
Protein Traffic ◽  
Microtubule Disruption ◽  
Recycling Pathway

A polarized cell, to maintain distinct basolateral and apical membrane domains, must tightly regulate vesicular traffic terminating at either membrane domain. In this study we have examined the extent to which microtubules regulate such traffic in polarized cells. Using the polymeric immunoglobulin receptor expressed in polarized MDCK cells, we have examined the effects of nocodazole, a microtubule-disrupting agent, on three pathways that deliver proteins to the apical surface and two pathways that deliver proteins to the basolateral surface. The biosynthetic and transcytotic pathways to the apical surface are dramatically altered by nocodazole in that a portion of the protein traffic on each of these two pathways is misdirected to the basolateral surface. The apical recycling pathway is slowed in the presence of nocodazole but targeting is not disrupted. In contrast, the biosynthetic and recycling pathways to the basolateral surface are less affected by nocodazole and therefore appear to be more resistant to microtubule disruption.

Opposite sorting and transcytosis of the polymeric immunoglobulin receptor in transfected endothelial and epithelial cells

1998 ◽  
Vol 111 (9) ◽  
pp. 1197-1206
Author(s):  
T. Su ◽  
K.K. Stanley
Keyword(s):  
Endothelial Cells ◽  
Steady State ◽  
Epithelial Cells ◽  
Cell Line ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Secretory Component ◽  
Polymeric Immunoglobulin Receptor ◽  
Apical Surface ◽  
Immunoglobulin Receptor

We have transfected a polarised endothelial cell line, ECV 304, and an epithelial cell line, MDCK, with a well characterised epithelial protein, the rat polymeric immunoglobulin receptor (pIgR), in order to study the protein sorting and transcytosis in endothelial cells. The expressed protein was normally processed and the steady state distribution between apical and basolateral surfaces was similar in both cell types. MDCK cells, however, showed a marked polarity in the delivery of newly synthesised pIgR to the cell surface, and in the release of secretory component. 88% of newly synthesised pIgR in MDCK cells was first delivered to the basolateral surface and 99% of secretory component was released from the apical surface. In contrast the basolateral targeting signal of pIgR was only partially recognised in endothelial cells, with 63% of the newly synthesised pIgR being first delivered to the basolateral surface. At steady state only 43% of the pIgR was found on the basolateral membrane. The direction of dimeric IgA transcytosis in endothelial cells was from apical to basolateral surfaces, opposite to that in MDCK cells. These data suggest that endothelial cells poorly recognise the targeting signals of proteins from epithelial cells, and that the direction of transcytosis is linked to the biological role of the cells.

The basolateral sorting signal of the polymeric immunoglobulin receptor contains two functional domains

1996 ◽  
Vol 109 (8) ◽  
pp. 2133-2139
Author(s):  
V. Reich ◽  
K. Mostov ◽  
B. Aroeti
Keyword(s):  
Mdck Cells ◽  
Minor Effect ◽  
Polymeric Immunoglobulin Receptor ◽  
Type I ◽  
Apical Surface ◽  
Alanine Scanning Mutagenesis ◽  
Beta Turn ◽  
Immunoglobulin Receptor ◽  
Sorting Signal ◽  
Basolateral Targeting

Basolateral sorting of the polymeric immunoglobulin receptor (pIgR) expressed in Madin-Darby canine kidney (MDCK) cells is mediated by a 17-residue sorting signal that resides in the cytoplasmic domain. We have recently analyzed the sequence requirements of the signal by alanine scanning mutagenesis. We found that basolateral sorting is mediated primarily by three amino acids: H656, R657 and V660. Individual mutations of each of these residues to Ala caused a substantial decrease in basolateral sorting and a corresponding increase in targeting to the apical surface. Structural analysis of 17-residue peptides corresponding to the signal revealed that V660 is in a beta-turn (probably type I) secondary structure, and its mutation to Ala destabilized the turn. H656 and R657 were not part of the turn and substitution of Arg657 to Ala had no effect on the turn stability. These results suggested that the signal is comprised of two structurally distinct domains: a critical V660 in the context of the beta-turn and an additional two residues (H656 and R657) that are not in the turn and probably are unimportant for its stability. Here we provide evidence suggesting that the two domains are distinguishable not only by their structure but also by their function. Basolateral targeting of pIgR mutants bearing Ala mutations at either 656 or 657 was not affected by treatment with brefeldin A (BFA), while basolateral targeting of pIgR containing an Ala substitution at position 660 was markedly and uniquely stimulated by BFA. Compared to single Ala substitutions, simultaneous mutations of H656 and R657 to Ala caused an additional minor effect on basolateral and apical sorting, whereas double mutations of V660 and either H656 or R657 resulted in a maximal decrease in basolateral targeting and corresponding increase in apical targeting. These results suggest the existence of two domains in the signal. When both domains are destroyed, basolateral targeting is maximally inhibited. The results also imply that V660 mediates basolateral sorting by a different mechanism from H656 and R657. We suggest that V660 and perhaps more generally the beta-turn may interact with BFA-sensitive adaptor complexes.

scholarly journals Postendocytotic sorting of the ligand for the polymeric immunoglobulin receptor in Madin-Darby canine kidney cells.

1989 ◽  
Vol 109 (2) ◽  
pp. 475-486 ◽  
Author(s):  
P P Breitfeld ◽  
J M Harris ◽  
K E Mostov
Keyword(s):  
Mdck Cells ◽  
Polymeric Immunoglobulin Receptor ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Immunoglobulin Receptor ◽  
Intracellular Sorting ◽  
Polymeric Immunoglobulins ◽  
Darby Canine Kidney ◽  
Canine Kidney

The polymeric immunoglobulin receptor (pIg-R) is responsible for the receptor-mediated transcytosis of polymeric immunoglobulins (IgA and IgM) across various epithelia. We have expressed the cDNA for the pIg-R in Madin-Darby canine kidney (MDCK) cells and found that this system mimics that found in vivo (Mostov, K. E., and D. L. Deitcher. 1986. Cell. 46:613-621). We have now investigated the postendocytotic pathway of the ligand for the pIg-R. After a 5-min internalization at the basolateral surface, approximately 45% of internalized ligand recycles to the basolateral medium and 30% is transcytosed to the apical medium. We have also examined why transcytosis of ligand is unidirectional, going only from basolateral to apical, but not from apical to basolateral. Several factors could explain this, such as proteolytic cleavage of the pIg-R at the apical surface, decreased apical endocytosis of ligand, or an intracellular sorting event. In this report, we show that the protease inhibitor, leupeptin, inhibits the cleavage of the pIg-R but does not alter the unidirectionality of transcytosis. In addition, we demonstrate that there is a significant amount of apical endocytosis of ligand (70% of that observed basolaterally). Finally, we demonstrate that apically endocytosed ligand can return only to the apical surface. Thus, once ligand reaches the apical surface, it is "trapped" and cannot return to the basolateral surface. We propose that the unidirectionality of transcytosis is the result of intracellular sorting, and that this results from a signal(s) present on the pIg-R.

scholarly journals MAL2, a novel raft protein of the MAL family, is an essential component of the machinery for transcytosis in hepatoma HepG2 cells

2002 ◽  
Vol 159 (1) ◽  
pp. 37-44 ◽  
Author(s):  
María C. de Marco ◽  
Fernando Martín-Belmonte ◽  
Leonor Kremer ◽  
Juan P. Albar ◽  
Isabel Correas ◽  
...  
Keyword(s):  
Hepg2 Cells ◽  
Mdck Cells ◽  
Functional Characterization ◽  
Essential Element ◽  
Polymeric Immunoglobulin Receptor ◽  
Apical Surface ◽  
Immunoglobulin Receptor ◽  
Indirect Route ◽  
Hepatoma Hepg2

Transcytosis is used alone (e.g., hepatoma HepG2 cells) or in combination with a direct pathway from the Golgi (e.g., epithelial MDCK cells) as an indirect route for targeting proteins to the apical surface. The raft-associated MAL protein is an essential element of the machinery for the direct route in MDCK cells. Herein, we present the functional characterization of MAL2, a member of the MAL protein family, in polarized HepG2 cells. MAL2 resided selectively in rafts and is predominantly distributed in a compartment localized beneath the subapical F-actin cytoskeleton. MAL2 greatly colocalized in subapical endosome structures with transcytosing molecules en route to the apical surface. Depletion of endogenous MAL2 drastically blocked transcytotic transport of exogenous polymeric immunoglobulin receptor and endogenous glycosylphosphatidylinositol-anchored protein CD59 to the apical membrane. MAL2 depletion did not affect the internalization of these molecules but produced their accumulation in perinuclear endosome elements that were accessible to transferrin. Normal transcytosis persisted in cells that expressed exogenous MAL2 designed to resist the depletion treatment. MAL2 is therefore essential for transcytosis in HepG2 cells.

scholarly journals An anchor-minus form of the polymeric immunoglobulin receptor is secreted predominantly apically in Madin-Darby canine kidney cells.

1987 ◽  
Vol 105 (5) ◽  
pp. 2031-2036 ◽  
Author(s):  
K E Mostov ◽  
P Breitfeld ◽  
J M Harris
Keyword(s):  
Mdck Cells ◽  
Secretory Protein ◽  
Site Directed Mutagenesis ◽  
Polymeric Immunoglobulin Receptor ◽  
Apical Surface ◽  
Basal Secretion ◽  
Madin Darby Canine Kidney ◽  
Immunoglobulin Receptor ◽  
Darby Canine Kidney ◽  
Canine Kidney

The polymeric immunoglobulin receptor is expressed in a variety of polarized epithelial cells. Newly made receptor travels first to the basolateral surface. The receptor is then endocytosed, transported across the cell in vesicles, and exocytosed at the apical surface. We have now deleted the membrane spanning and cytoplasmic portions of the receptor by site-directed mutagenesis, thus converting the receptor to a secretory protein. When expressed in polarized Madin-Darby canine kidney (MDCK) cells the truncated protein is secreted at both surfaces, with a ratio of apical-to-basal secretion of 3.4. In contrast, when the exogenous secretory protein chicken lysozyme is expressed in these cells, it is released at both sides with a ratio of apical-to-basal secretion of 0.43. (Koder-Koch, C., R. Bravo, S. Fuller, D. Cutler, and H. Garoff, 1985, J. Cell Biol., 43:297-306). Lysozyme is thought to lack a signal that targets it to one surface or the other, and so its secretion may represent a default, bulk flow pathway to both surfaces. When compared with lysozyme, the truncated polymeric immunoglobulin receptor is preferentially secreted apically by a factor of 3.4:0.43 or 7.8. We suggest that the lumenal portion of the polymeric immunoglobulin receptor contains a signal that targets it to the apical surface.

scholarly journals Apical and basolateral coated pits of MDCK cells differ in their rates of maturation into coated vesicles, but not in the ability to distinguish between mutant hemagglutinin proteins with different internalization signals.

1995 ◽  
Vol 129 (5) ◽  
pp. 1241-1250 ◽  
Author(s):  
H Y Naim ◽  
D T Dodds ◽  
C B Brewer ◽  
M G Roth
Keyword(s):  
Binding Sites ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Apical Cell ◽  
Single Amino Acid ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Membrane Domains ◽  
Coated Pits ◽  
Influenza Virus Hemagglutinin

In polarized epithelial MDCK cells, all known endogenous endocytic receptors are found on the basolateral domain. The influenza virus hemagglutinin (HA) which is normally sorted to the apical plasma membrane, can be converted to a basolateral protein by specific mutations in its short cytoplasmic domain that also create internalization signals. For some of these mutations, sorting to the basolateral surface is incomplete, allowing internalization of two proteins that differ by a single amino acid of the internalization signal to be compared at both the apical and basolateral surfaces of MDCK cells. The rates of internalization of HA-Y543 and HA-Y543,R546 from the basolateral surface of polarized MDCK cells resembled those in nonpolarized cells, whereas their rates of internalization from the apical cell surface were fivefold slower. However, HA-Y543,R546 was internalized approximately threefold faster than HA-Y543 at both membrane domains, indicating that apical endocytic pits in polarized MDCK cells retained the ability to discriminate between different internalization signals. Slower internalization from the apical surface could not be explained by a limiting number of coated pits: apical membrane contained 0.7 as many coated pits per cell cross-section as did basolateral membranes. 10-14% of HA-Y543 at the apical surface of polarized MDCK cells was found in coated pits, a percentage not significantly different from that observed in apical coated pits of nonpolarized MDCK cells, where internalization was fivefold faster. Thus, there was no lack of binding sites for HA-Y543 in apical coated pits of polarized cells. However, at the apical surface many more shallow pits, and fewer deep, mature pits, were observed than were seen at the basolateral. These results suggest that the slower internalization at the apical surface is due to slower maturation of coated pits, and not to a difference in recognition of internalization signals.

Polymeric immunoglobulin receptor expressed in MDCK cells transcytoses IgA

Cell ◽  
1986 ◽  
Vol 46 (4) ◽  
pp. 613-621 ◽  
Author(s):  
Keith E. Mostov ◽  
David L. Deitcher
Keyword(s):  
Mdck Cells ◽  
Polymeric Immunoglobulin Receptor ◽  
Immunoglobulin Receptor

The transcytotic pathway of an apical plasma membrane protein (B10) in hepatocytes is similar to that of IgA and occurs via a tubular pericentriolar compartment

1996 ◽  
Vol 109 (6) ◽  
pp. 1215-1227 ◽  
Author(s):  
I. Hemery ◽  
A.M. Durand-Schneider ◽  
G. Feldmann ◽  
J.P. Vaerman ◽  
M. Maurice
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Apical Membrane ◽  
Rat Hepatocytes ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Plasma Membrane Protein ◽  
Microtubule Disruption

In hepatocytes, newly synthesized apical plasma membrane proteins are first delivered to the basolateral surface and are supposed to reach the apical surface by transcytosis. The transcytotic pathway of apical membrane proteins and its relationship with other endosomal pathways has not been demonstrated morphologically. We compared the intracellular route of an apical plasma membrane protein, B10, with that of polymeric IgA (pIgA), which is transcytosed, transferrin (Tf) which is recycled, and asialoorosomucoid (ASOR) which is delivered to lysosomes. Ligands and anti-B10 monoclonal IgG were linked to fluorochromes or with peroxidase. The fate of each ligand was followed by confocal and electron microscopy in polarized primary monolayers of rat hepatocytes. When fluorescent anti-B10 IgG and fluorescent pIgA were simultaneously endocytosed for 15–30 minutes, they both uniformly labelled a juxtanuclear compartment. By 30–60 minutes, they reached the bile canaliculi. Tf and ASOR were also routed to the juxtanuclear area, but their fluorescence patterns were more punctate. Microtubule disruption prevented all ligands from reaching the juxtanuclear area. This area corresponded, at least partially, to the localization of the mannose 6-phosphate receptor, an endosomal marker. By electron microscopy, the juxtanuclear compartment was made up of anastomosing tubules connected to vacuoles, and was organized around the centrioles. B10 and pIgA were mainly found in the tubules, whereas ASOR was segregated inside the vacuolar elements and Tf within thinner, recycling tubules. In conclusion, transcytosis of the apical membrane protein B10 occurs inside tubules similar to those carrying pIgA, and involves passage via the pericentriolar area. In the pericentriolar area, the transcytotic tubules appear to maintain connections with other endosomal elements where sorting between recycled and degraded ligands occurs.

scholarly journals Antimicrotubule drugs inhibit the polarized insertion of an intracellular glycoprotein pool into the apical membrane of Madin-Darby canine kidney (MDCK) cells

1992 ◽  
Vol 103 (3) ◽  
pp. 677-687 ◽  
Author(s):  
G.K. Ojakian ◽  
R. Schwimmer
Keyword(s):  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Immunogold Electron Microscopy ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Transport Pathways ◽  
Intracellular Targeting ◽  
Polarized Delivery ◽  
Membrane Recognition

Previous experiments on MDCK cells have demonstrated that the polarized appearance of a 135 kDa glycoprotein (gp135) on the apical plasma membrane can occur through the insertion of both newly synthesized gp135 as well as a pre-existing gp135 intracellular pool. In this study, anticytoskeletal drugs were utilized to determine the role of the cytoskeleton in the polarized delivery of gp135. Colchicine and nocodazole produced a 15–20% inhibition in the apical surface accumulation of newly synthesized gp135 and inhibited the appearance of the gp135 pool by approximately 33%, while cytochalasin D had no affect on the apical accumulation of either newly synthesized gp135 or the gp135 pool. These results indicate that microtubules, but not microfilaments, are involved in the intracellular targeting of gp135. Quantitative immunogold electron microscopy of nocodazole-treated cells demonstrated that gp135 was not mistargeted to the basolateral membrane, suggesting the possibility that some vesicles containing gp135 did not fuse with the apical membrane and remained in the cells. These experiments demonstrate that microtubules are an important component of gp135 insertion into the apical membrane. They also suggest that gp135 resides within vesicles which have an apical membrane recognition signal and cannot fuse with the basolateral membrane. The possibility that these data, and those of others, could support a hypothesis for the presence of two constitutive apical transport pathways is discussed.

scholarly journals Gp135/podocalyxin and NHERF-2 participate in the formation of a preapical domain during polarization of MDCK cells

2005 ◽  
Vol 168 (2) ◽  
pp. 303-313 ◽  
Author(s):  
Doris Meder ◽  
Anna Shevchenko ◽  
Kai Simons ◽  
Joachim Füllekrug
Keyword(s):  
Free Surface ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Single Cells ◽  
Basolateral Membrane ◽  
Pdz Domain ◽  
Binding Motif ◽  
Membrane Domains ◽  
Regulatory Factor ◽  
Pdz Protein

Epithelial polarization involves the segregation of apical and basolateral membrane domains, which are stabilized and maintained by tight junctions and membrane traffic. We report that unlike most apical and basolateral proteins in MDCK cells, which separate only after junctions have formed, the apical marker gp135 signifies an early level of polarized membrane organization established already in single cells. We identified gp135 as the dog orthologue of podocalyxin. With a series of domain mutants we show that the COOH-terminal PSD-95/Dlg/ZO-1 (PDZ)–binding motif is targeting podocalyxin to the free surface of single cells as well as to a subdomain of the terminally polarized apical membrane. This special localization of podocalyxin is shared by the cytoplasmic PDZ-protein Na+/H+ exchanger regulatory factor (NHERF)-2. Depleting podocalyxin by RNA interference caused defects in epithelial polarization. Together, our data suggest that podocalyxin and NHERF-2 function in epithelial polarization by contributing to an early apical scaffold based on PDZ domain-mediated interactions.

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