Faculty Opinions recommendation of Delivery of raft-associated, GPI-anchored proteins to the apical surface of polarized MDCK cells by a transcytotic pathway.

Membrane microdomains (“lipid rafts”) enriched in glycosylphosphatidylinositol (GPI)-anchored proteins, glycosphingolipids, and cholesterol have been implicated in events ranging from membrane trafficking to signal transduction. Although there is biochemical evidence for such membrane microdomains, they have not been visualized by light or electron microscopy. To probe for microdomains enriched in GPI- anchored proteins in intact cell membranes, we used a novel form of digital microscopy, imaging fluorescence resonance energy transfer (FRET), which extends the resolution of fluorescence microscopy to the molecular level (<100 Å). We detected significant energy transfer between donor- and acceptor-labeled antibodies against the GPI-anchored protein 5′ nucleotidase (5′ NT) at the apical membrane of MDCK cells. The efficiency of energy transfer correlated strongly with the surface density of the acceptor-labeled antibody. The FRET data conformed to theoretical predictions for two-dimensional FRET between randomly distributed molecules and were inconsistent with a model in which 5′ NT is constitutively clustered. Though we cannot completely exclude the possibility that some 5′ NT is in clusters, the data imply that most 5′ NT molecules are randomly distributed across the apical surface of MDCK cells. These findings constrain current models for lipid rafts and the membrane organization of GPI-anchored proteins.

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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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Bovine colostrum supports the serum-free proliferation of epithelial cells but not of fibroblasts in long-term culture

The Journal of Cell Biology ◽

10.1083/jcb.84.3.808 ◽

1980 ◽

Vol 84 (3) ◽

pp. 808-814 ◽

Cited By ~ 44

Author(s):

M Klagsbrun

Keyword(s):

Epithelial Cells ◽

Mdck Cells ◽

Calf Serum ◽

Morphological Characteristics ◽

Bovine Colostrum ◽

Apical Surface ◽

Early Passage ◽

Rat Fibroblasts ◽

Canine Kidney

Medium lacking serum but supplemented with milk will support the growth of sparse cells in culture. Milk obtained within 8 h after the birth of a calf (day 1 colostrum) is the most effective in supporting proliferation. In mixed cultures of early-passage bovine embryonic kidney (BEK) or early-passage calf kidney (CK) cells, both epithelial cells and fibroblasts grow in Dulbecco's modified eagle's medium (DMEM) supplemented with serum. However, only cells that appear to be epithelial-like grow in DMEM supplemented with colostrum. Sparse cultures of early-passage human and rat fibroblasts that grow readily in DMEM supplemented with serum do not grow in DMEM supplemented with colostrum. Canine kidney epithelial cells (MDCK), when plated sparsely, grow exponentially in DMEM supplemented with day 1 bovine colostrum. The generation time is 26 h, the same growth rate as in DMEM supplemented with calf serum. The MDCK cells can be subcultured and regrown to confluence repeatedly in colostrum-supplemented DMEM. Growth in DMEM supplemented with colostrum does not alter the morphological characteristics of the MDCK cells, which are polygonal, contain microvilli at the apical surface, and are connected by tight junctions and desmosomes. MDCK cells do not proliferate in DMEM supplemented with milk obtained 1 wk after the birth of a calf.

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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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Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells.

The Journal of Cell Biology ◽

10.1083/jcb.105.4.1623 ◽

1987 ◽

Vol 105 (4) ◽

pp. 1623-1635 ◽

Cited By ~ 299

Author(s):

G van Meer ◽

E H Stelzer ◽

R W Wijnaendts-van-Resandt ◽

K Simons

Keyword(s):

Plasma Membrane ◽

Golgi Complex ◽

Mdck Cells ◽

Laser Fluorescence ◽

Apical Plasma Membrane ◽

Apical Surface ◽

Madin Darby Canine Kidney ◽

Basolateral Side ◽

Darby Canine Kidney ◽

Canine Kidney

To study the intracellular transport of newly synthesized sphingolipids in epithelial cells we have used a fluorescent ceramide analog, N-6[7-nitro-2,1,3-benzoxadiazol-4-yl] aminocaproyl sphingosine (C6-NBD-ceramide; Lipsky, N. G., and R. E. Pagano, 1983, Proc. Natl. Acad. Sci. USA, 80:2608-2612) as a probe. This ceramide was readily taken up by filter-grown Madin-Darby canine kidney (MDCK) cells from liposomes at 0 degrees C. After penetration into the cell, the fluorescent probe accumulated in the Golgi area at temperatures between 0 and 20 degrees C. Chemical analysis showed that C6-NBD-ceramide was being converted into C6-NBD-sphingomyelin and C6-NBD-glucosyl-ceramide. An analysis of the fluorescence pattern after 1 h at 20 degrees C by means of a confocal scanning laser fluorescence microscope revealed that the fluorescent marker most likely concentrated in the Golgi complex itself. Little fluorescence was observed at the plasma membrane. Raising the temperature to 37 degrees C for 1 h resulted in intense plasma membrane staining and a loss of fluorescence from the Golgi complex. Addition of BSA to the apical medium cleared the fluorescence from the apical but not from the basolateral plasma membrane domain. The basolateral fluorescence could be depleted only by adding BSA to the basal side of a monolayer of MDCK cells grown on polycarbonate filters. We conclude that the fluorescent sphingomyelin and glucosylceramide were delivered from the Golgi complex to the plasma membrane where they accumulated in the external leaflet of the membrane bilayer. The results also demonstrated that the fatty acyl labeled lipids were unable to pass the tight junctions in either direction. Quantitation of the amount of NBD-lipids delivered to the apical and the basolateral plasma membranes during incubation for 1 h at 37 degrees C showed that the C6-NBD-glucosylceramide was two- to fourfold enriched on the apical as compared to the basolateral side, while C6-NBD-sphingomyelin was about equally distributed. Since the surface area of the apical plasma membrane is much smaller than that of the basolateral membrane, both lipids achieved a higher concentration on the apical surface. Altogether, our results suggest that the NBD-lipids are sorted in MDCK cells in a way similar to their natural counterparts.

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

Journal of Cell Science ◽

10.1242/jcs.103.3.677 ◽

1992 ◽

Vol 103 (3) ◽

pp. 677-687 ◽

Cited By ~ 4

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.

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GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells

The Journal of Cell Biology ◽

10.1083/jcb.200507116 ◽

2006 ◽

Vol 172 (7) ◽

pp. 1023-1034 ◽

Cited By ~ 75

Author(s):

Simona Paladino ◽

Thomas Pocard ◽

Maria Agata Catino ◽

Chiara Zurzolo

Keyword(s):

Plasma Membrane ◽

Mdck Cells ◽

Basolateral Membrane ◽

Apical Surface ◽

Madin Darby Canine Kidney ◽

Biochemical Assays ◽

Intracellular Sorting ◽

Apical Sorting ◽

Golgi Network ◽

Darby Canine Kidney

The polarity of epithelial cells is dependent on their ability to target proteins and lipids in a directional fashion. The trans-Golgi network, the endosomal compartment, and the plasma membrane act as sorting stations for proteins and lipids. The site of intracellular sorting and pathways used for the apical delivery of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are largely unclear. Using biochemical assays and confocal and video microscopy in living cells, we show that newly synthesized GPI-APs are directly delivered to the apical surface of fully polarized Madin–Darby canine kidney cells. Impairment of basolateral membrane fusion by treatment with tannic acid does not affect the direct apical delivery of GPI-APs, but it does affect the organization of tight junctions and the integrity of the monolayer. Our data clearly demonstrate that GPI-APs are directly sorted to the apical surface without passing through the basolateral membrane. They also reinforce the hypothesis that apical sorting of GPI-APs occurs intracellularly before arrival at the plasma membrane.

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Epithelial sorting of a glycosylphosphatidylinositol-anchored bacterial protein expressed in polarized renal MDCK and intestinal Caco-2 cells

Journal of Cell Science ◽

10.1242/jcs.108.1.369 ◽

1995 ◽

Vol 108 (1) ◽

pp. 369-377 ◽

Cited By ~ 1

Author(s):

K.L. Soole ◽

M.A. Jepson ◽

G.P. Hazlewood ◽

H.J. Gilbert ◽

B.H. Hirst

Keyword(s):

Epithelial Cells ◽

Cell Surface ◽

Intestinal Epithelial Cells ◽

Mdck Cells ◽

Basolateral Membrane ◽

Apical Cell ◽

Apical Surface ◽

Intestinal Epithelial ◽

Gpi Anchor ◽

Sorting Signal

To evaluate whether a glycosylphosphatidylinositol (GPI) anchor can function as a protein sorting signal in polarized intestinal epithelial cells, the GPI-attachment sequence from Thy-1 was fused to bacterial endoglucanase E' (EGE') from Clostridium thermocellum and polarity of secretion of the chimeric EGE'-GPI protein was evaluated. The chimeric EGE'-GPI protein was shown to be associated with a GPI anchor by TX-114 phase-partitioning and susceptibility to phosphoinositol-specific phospholipase C. In polarized MDCK cells, EGE' was localized almost exclusively to the apical cell surface, while in polarized intestinal Caco-2 cells, although 80% of the extracellular form of the enzyme was routed through the apical membrane over a 24 hour period, EGE' was also detected at the basolateral membrane. Rates of delivery of EGE'-GPI to the two membrane domains in Caco-2 cells, as determined with a biotinylation protocol, revealed apical delivery was approximately 2.5 times that of basolateral. EGE' delivered to the basolateral cell surface was transcytosed to the apical surface. These data indicate that a GPI anchor does represent a dominant apical sorting signal in intestinal epithelial cells. However, the mis-sorting of a proportion of EGE'GPI to the basolateral surface of Caco-2 cells provides an explanation for additional sorting signals in the ectodomain of some endogenous GPI-anchored proteins.

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Sorting of rat liver and ileal sodium-dependent bile acid transporters in polarized epithelial cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1998.275.5.g1045 ◽

1998 ◽

Vol 275 (5) ◽

pp. G1045-G1055 ◽

Cited By ~ 8

Author(s):

An-Qiang Sun ◽

Meenakshisundaram Ananthanarayanan ◽

Carol J. Soroka ◽

Sundararajah Thevananther ◽

Benjamin L. Shneider ◽

...

Keyword(s):

Bile Acid ◽

Amino Acid ◽

Mdck Cells ◽

Cytoplasmic Tail ◽

Apical Surface ◽

Acid Transport ◽

Transport Activity ◽

Wild Type ◽

Bile Acid Transport ◽

Apical Domain

The rat ileal apical Na+-dependent bile acid transporter (ASBT) and the liver Na+-taurocholate cotransporting polypeptide (Ntcp) are members of a new family of anion transporters. These transport proteins share limited sequence homology and almost identical predicted secondary structures but are localized to the apical surface of ileal enterocytes and the sinusoidal surface of hepatocytes, respectively. Stably transfected Madin-Darby canine kidney (MDCK) cells appropriately localized wild-type ASBT and Ntcp apically and basolaterally as assessed by functional activity and immunocytochemical localization studies. Truncated and chimeric transporters were used to determine the functional importance of the cytoplasmic tail in bile acid transport activity and membrane localization. Two cDNAs were created encoding a truncated transporter in which the 56-amino-acid COOH-terminal tail of Ntcp was removed or substituted with an eight-amino-acid epitope FLAG. For both mutants there was some loss of fidelity in basolateral sorting in that ∼75% of each protein was delivered to the basolateral surface compared with ∼90% of the wild-type Ntcp protein. In contrast, deletion of the cytoplasmic tail of ASBT led to complete loss of transport activity and sorting to the apical membrane. An Ntcp chimera in which the 56-amino-acid COOH-terminal tail of Ntcp was replaced with the 40-amino-acid cytoplasmic tail of ASBT was largely redirected (82.4 ± 3.9%) to the apical domain of stably transfected MDCK cells, based on polarity of bile acid transport activity and localization by confocal immunofluorescence microscopy. These results indicate that a predominant signal for sorting of the Ntcp protein to the basolateral domain is located in a region outside of the cytoplasmic tail. These studies have further shown that a novel apical sorting signal is localized to the cytoplasmic tail of ASBT and that it is transferable and capable of redirecting a protein normally sorted to the basolateral surface to the apical domain of MDCK cells.

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