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

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.

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Apical Membrane Targeting of Nedd4 Is Mediated by an Association of Its C2 Domain with Annexin Xiiib

The Journal of Cell Biology ◽

10.1083/jcb.149.7.1473 ◽

2000 ◽

Vol 149 (7) ◽

pp. 1473-1484 ◽

Cited By ~ 104

Author(s):

Pamela J. Plant ◽

Frank Lafont ◽

Sandra Lecat ◽

Paul Verkade ◽

Kai Simons ◽

...

Keyword(s):

Plasma Membrane ◽

Apical Membrane ◽

Mdck Cells ◽

Immunogold Electron Microscopy ◽

Apical Plasma Membrane ◽

Apical Region ◽

Apical Surface ◽

Dependent Manner ◽

Interacting Proteins ◽

C2 Domain

Nedd4 is a ubiquitin protein ligase (E3) containing a C2 domain, three or four WW domains, and a ubiquitin ligase HECT domain. We have shown previously that the C2 domain of Nedd4 is responsible for its Ca2+-dependent targeting to the plasma membrane, particularly the apical region of epithelial MDCK cells. To investigate this apical preference, we searched for Nedd4-C2 domain-interacting proteins that might be involved in targeting Nedd4 to the apical surface. Using immobilized Nedd4-C2 domain to trap interacting proteins from MDCK cell lysate, we isolated, in the presence of Ca2+, a ∼35–40-kD protein that we identified as annexin XIII using mass spectrometry. Annexin XIII has two known isoforms, a and b, that are apically localized, although XIIIa is also found in the basolateral compartment. In vitro binding and coprecipitation experiments showed that the Nedd4-C2 domain interacts with both annexin XIIIa and b in the presence of Ca2+, and the interaction is direct and optimal at 1 μM Ca2+. Immunofluorescence and immunogold electron microscopy revealed colocalization of Nedd4 and annexin XIIIb in apical carriers and at the apical plasma membrane. Moreover, we show that Nedd4 associates with raft lipid microdomains in a Ca2+-dependent manner, as determined by detergent extraction and floatation assays. These results suggest that the apical membrane localization of Nedd4 is mediated by an association of its C2 domain with the apically targeted annexin XIIIb.

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The Cytoplasmic Tail of Rhodopsin Acts as a Novel Apical Sorting Signal in Polarized MDCK Cells

The Journal of Cell Biology ◽

10.1083/jcb.142.5.1245 ◽

1998 ◽

Vol 142 (5) ◽

pp. 1245-1256 ◽

Cited By ~ 98

Author(s):

Jen-Zen Chuang ◽

Ching-Hwa Sung

Keyword(s):

Secretory Pathway ◽

Apical Membrane ◽

Mdck Cells ◽

Basolateral Membrane ◽

Brefeldin A ◽

Cytoplasmic Tail ◽

Apical Plasma Membrane ◽

Glutathione S Transferase ◽

Sorting Signals ◽

Apical Sorting

All basolateral sorting signals described to date reside in the cytoplasmic domain of proteins, whereas apical targeting motifs have been found to be lumenal. In this report, we demonstrate that wild-type rhodopsin is targeted to the apical plasma membrane via the TGN upon expression in polarized epithelial MDCK cells. Truncated rhodopsin with a deletion of 32 COOH-terminal residues shows a nonpolar steady-state distribution. Addition of the COOH-terminal 39 residues of rhodopsin redirects the basolateral membrane protein CD7 to the apical membrane. Fusion of rhodopsin's cytoplasmic tail to a cytosolic protein glutathione S-transferase (GST) also targets this fusion protein (GST–Rho39Tr) to the apical membrane. The targeting of GST–Rho39Tr requires both the terminal 39 amino acids and the palmitoylation membrane anchor signal provided by the rhodopsin sequence. The apical transport of GST–Rho39Tr can be reversibly blocked at the Golgi complex by low temperature and can be altered by brefeldin A treatment. This indicates that the membrane-associated GST–Rho39Tr protein may be sorted along a yet unidentified pathway that is similar to the secretory pathway in polarized MDCK cells. We conclude that the COOH-terminal tail of rhodopsin contains a novel cytoplasmic apical sorting determinant. This finding further indicates that cytoplasmic sorting machinery may exist in MDCK cells for some apically targeted proteins, analogous to that described for basolaterally targeted proteins.

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Aminopeptidase N is directly sorted to the apical domain in MDCK cells.

The Journal of Cell Biology ◽

10.1083/jcb.111.6.2923 ◽

1990 ◽

Vol 111 (6) ◽

pp. 2923-2930 ◽

Cited By ~ 23

Author(s):

H P Wessels ◽

G H Hansen ◽

C Fuhrer ◽

A T Look ◽

H Sjöström ◽

...

Keyword(s):

Apical Membrane ◽

Mdck Cells ◽

Basolateral Membrane ◽

Cell Types ◽

Aminopeptidase N ◽

Apical Surface ◽

Apical Domain ◽

Single Protein ◽

Cell Type Specific ◽

Golgi Network

In different epithelial cell types, integral membrane proteins appear to follow different sorting pathways to the apical surface. In hepatocytes, several apical proteins were shown to be transported there indirectly via the basolateral membrane, whereas in MDCK cells a direct sorting pathway from the trans-Golgi-network to the apical membrane has been demonstrated. However, different proteins had been studied in these cells. To compare the sorting of a single protein in both systems, we have expressed aminopeptidase N, which already had been shown to be sorted indirectly in hepatocytes, in transfected MDCK cells. As expected, it was predominantly localized to the apical domain of the plasma membrane. By monitoring the appearance of newly synthesized aminopeptidase N at the apical and basolateral surface, it was found to be directly sorted to the apical domain in MDCK cells, indicating that the sorting pathways are indeed cell type-specific.

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

The Journal of Cell Biology ◽

10.1083/jcb.111.4.1351 ◽

1990 ◽

Vol 111 (4) ◽

pp. 1351-1361 ◽

Cited By ~ 139

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.

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Selective Alterations in Biosynthetic and Endocytic Protein Traffic in Madin-Darby Canine Kidney Epithelial Cells Expressing Mutants of the Small GTPase Rac1

Molecular Biology of the Cell ◽

10.1091/mbc.11.1.287 ◽

2000 ◽

Vol 11 (1) ◽

pp. 287-304 ◽

Cited By ~ 51

Author(s):

Tzuu-Shuh Jou ◽

Som-Ming Leung ◽

Linette M. Fung ◽

Wily G. Ruiz ◽

W. James Nelson ◽

...

Keyword(s):

Apical Membrane ◽

Mdck Cells ◽

Basolateral Membrane ◽

Membrane Traffic ◽

Small Gtpase ◽

Apical Plasma Membrane ◽

Membrane Domains ◽

Madin Darby Canine Kidney ◽

Darby Canine Kidney ◽

Canine Kidney

Madin-Darby canine kidney (MDCK) cells expressing constitutively active Rac1 (Rac1V12) accumulate a large central aggregate of membranes beneath the apical membrane that contains filamentous actin, Rac1V12, rab11, and the resident apical membrane protein GP-135. To examine the roles of Rac1 in membrane traffic and the formation of this aggregate, we analyzed endocytic and biosynthetic trafficking pathways in MDCK cells expressing Rac1V12 and dominant inactive Rac1 (Rac1N17). Rac1V12 expression decreased the rates of apical and basolateral endocytosis, whereas Rac1N17 expression increased those rates from both membrane domains. Basolateral-to-apical transcytosis of immunoglobulin A (IgA) (a ligand for the polymeric immunoglobulin receptor [pIgR]), apical recycling of pIgR-IgA, and accumulation of newly synthesized GP-135 at the apical plasma membrane were all decreased in cells expressing Rac1V12. These effects of Rac1V12 on trafficking pathways to the apical membrane were the result of the delivery and trapping of these proteins in the central aggregate. In contrast to abnormalities in apical trafficking events, basolateral recycling of transferrin, degradation of EGF internalized from the basolateral membrane, and delivery of newly synthesized pIgR from the Golgi to the basolateral membrane were all relatively unaffected by Rac1V12 expression. Rac1N17 expression had little or no effect on these postendocytic or biosynthetic trafficking pathways. These results show that in polarized MDCK cells activated Rac1 may regulate the rate of endocytosis from both membrane domains and that expression of dominant active Rac1V12 specifically alters postendocytic and biosynthetic membrane traffic directed to the apical, but not the basolateral, membrane.

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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.

The Journal of Cell Biology ◽

10.1083/jcb.129.5.1241 ◽

1995 ◽

Vol 129 (5) ◽

pp. 1241-1250 ◽

Cited By ~ 31

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.

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PBA increases CFTR expression but at high doses inhibits Cl−secretion in Calu-3 airway epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1999.277.4.l700 ◽

1999 ◽

Vol 277 (4) ◽

pp. L700-L708 ◽

Cited By ~ 11

Author(s):

Johannes Loffing ◽

Bryan D. Moyer ◽

Donna Reynolds ◽

Bruce A. Stanton

Keyword(s):

Protein Expression ◽

Apical Membrane ◽

Basolateral Membrane ◽

Airway Epithelial Cells ◽

Cell Phenotype ◽

Apical Plasma Membrane ◽

Airway Epithelial ◽

High Concentration ◽

Cycle Enzyme ◽

Cf Transmembrane Conductance Regulator

Sodium 4-phenylbutyrate (PBA), a short-chain fatty acid, has been approved to treat patients with urea cycle enzyme deficiencies and is being evaluated in the management of sickle cell disease, thalassemia, cancer, and cystic fibrosis (CF). Because relatively little is known about the effects of PBA on the expression and function of the wild-type CF transmembrane conductance regulator (wt CFTR), the goal of this study was to examine the effects of PBA and related compounds on wt CFTR-mediated Cl−secretion. To this end, we studied Calu-3 cells, a human airway cell line that expresses endogenous wt CFTR and has a serous cell phenotype. We report that chronic treatment of Calu-3 cells with a high concentration (5 mM) of PBA, sodium butyrate, or sodium valproate but not of sodium acetate reduced basal and 8-(4-chlorophenylthio)-cAMP-stimulated Cl−secretion. Paradoxically, PBA enhanced CFTR protein expression 6- to 10-fold and increased the intensity of CFTR staining in the apical plasma membrane. PBA also increased protein expression of Na+-K+-ATPase. PBA reduced CFTR Cl−currents across the apical membrane but had no effect on Na+-K+-ATPase activity in the basolateral membrane. Thus a high concentration of PBA (5 mM) reduces Cl−secretion by inhibiting CFTR Cl−currents across the apical membrane. In contrast, lower therapeutic concentrations of PBA (0.05–2 mM) had no effect on cAMP-stimulated Cl−secretion across Calu-3 cells. We conclude that PBA concentrations in the therapeutic range are unlikely to have a negative effect on Cl−secretion. However, concentrations >5 mM might reduce transepithelial Cl−secretion by serous cells in submucosal glands in individuals expressing wt CFTR.

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Intracellular traffic of the ecto-nucleotide pyrophosphatase/phosphodiesterase NPP3 to the apical plasma membrane of MDCK and Caco-2 cells: apical targeting occurs in the absence of N-glycosylation

Journal of Cell Science ◽

10.1242/jcs.113.23.4193 ◽

2000 ◽

Vol 113 (23) ◽

pp. 4193-4202 ◽

Cited By ~ 2

Author(s):

N.R. Meerson ◽

V. Bello ◽

J.L. Delaunay ◽

T.A. Slimane ◽

D. Delautier ◽

...

Keyword(s):

Cell Surface ◽

Mdck Cells ◽

Transmembrane Proteins ◽

Cell Types ◽

Apical Plasma Membrane ◽

Apical Surface ◽

Intracellular Traffic ◽

Transmembrane Glycoprotein ◽

Direct Demonstration ◽

Targeting Signal

Glycosylation was considered the major signal candidate for apical targeting of transmembrane proteins in polarized epithelial cells. However, direct demonstration of the role of glycosylation has proved difficult because non-glycosylated apical transmembrane proteins usually do not reach the cell surface. Here we were able to follow the targeting of the apical transmembrane glycoprotein NPP3 both when glycosylated and non-glycosylated. Transfected in polarized MDCK and Caco-2 cells, NPP3 was exclusively expressed at the apical membrane. The transport kinetics of the protein to the cell surface were studied after metabolic (35)S-labeling and surface immunoprecipitation. The newly synthesized protein was mainly targeted directly to the apical surface in MDCK cells, whereas 50% transited through the basolateral surface in Caco-2 cells. In both cell types, the basolaterally targeted pool was effectively transcytosed to the apical surface. In the presence of tunicamycin, NPP3 was not N-glycosylated. The non-glycosylated protein was partially retained intracellularly but the fraction that reached the cell surface was nevertheless predominantly targeted apically. However, transcytosis of the non-glycosylated protein was partially impaired in MDCK cells. These results provide direct evidence that glycosylation cannot be considered an apical targeting signal for NPP3, although glycosylation is necessary for correct trafficking of the protein to the cell surface.

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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

Journal of Cell Science ◽

10.1242/jcs.109.6.1215 ◽

1996 ◽

Vol 109 (6) ◽

pp. 1215-1227 ◽

Cited By ~ 2

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.

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The MAL Proteolipid Is Necessary for Normal Apical Transport and Accurate Sorting of the Influenza Virus Hemagglutinin in Madin-Darby Canine Kidney Cells

The Journal of Cell Biology ◽

10.1083/jcb.145.1.141 ◽

1999 ◽

Vol 145 (1) ◽

pp. 141-151 ◽

Cited By ~ 128

Author(s):

Rosa Puertollano ◽

Fernando Martín-Belmonte ◽

Jaime Millán ◽

María del Carmen de Marco ◽

Juan P. Albar ◽

...

Keyword(s):

Influenza Virus ◽

Mdck Cells ◽

Basolateral Membrane ◽

Ectopic Expression ◽

Apical Surface ◽

Madin Darby Canine Kidney ◽

Transport Pathway ◽

Influenza Virus Hemagglutinin ◽

Darby Canine Kidney ◽

Canine Kidney

The MAL (MAL/VIP17) proteolipid is a nonglycosylated integral membrane protein expressed in a restricted pattern of cell types, including T lymphocytes, myelin-forming cells, and polarized epithelial cells. Transport of the influenza virus hemagglutinin (HA) to the apical surface of epithelial Madin-Darby canine kidney (MDCK) cells appears to be mediated by a pathway involving glycolipid- and cholesterol- enriched membranes (GEMs). In MDCK cells, MAL has been proposed previously as being an element of the protein machinery for the GEM-dependent apical transport pathway. Using an antisense oligonucleotide-based strategy and a newly generated monoclonal antibody to canine MAL, herein we have approached the effect of MAL depletion on HA transport in MDCK cells. We have found that MAL depletion diminishes the presence of HA in GEMs, reduces the rate of HA transport to the cell surface, inhibits the delivery of HA to the apical surface, and produces partial missorting of HA to the basolateral membrane. These effects were corrected by ectopic expression of MAL in MDCK cells whose endogenous MAL protein was depleted. Our results indicate that MAL is necessary for both normal apical transport and accurate sorting of HA.

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