scholarly journals Selective inhibition of protein targeting to the apical domain of MDCK cells by brefeldin A.

1992 ◽  
Vol 118 (1) ◽  
pp. 51-62 ◽  
Author(s):  
S H Low ◽  
B L Tang ◽  
S H Wong ◽  
W Hong
Keyword(s):  
Protein Targeting ◽  
Mdck Cells ◽  
Brefeldin A ◽  
Selective Inhibition ◽  
Surface Expression ◽  
Apical Surface ◽  
Golgi Transport ◽  
Apical Domain ◽  
Golgi Network ◽  
Basolateral Targeting

Dipeptidyl peptidase IV (DPPIV) is mainly vectorially targeted to the apical surface in MDCK cells. BFA was found to abolish the apical targeting of DPPIV. This BFA effect could be achieved under conditions where the ER to Golgi transport and the total surface expression of DPPIV were essentially unaffected. BFA executed its effect during the transport from the trans-Golgi network (TGN) to the surface. The inhibition of apical targeting resulted in enhanced mis-targeting to the basolateral surface. The mistargeted DPPIV was transcytosed back to the apical domain only after BFA withdrawal. In contrast, the basolateral targeting of uvomorulin was unaffected by BFA. These results established that the apical targeting of DPPIV was selectively abolished by BFA.

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

1990 ◽  
Vol 111 (6) ◽  
pp. 2923-2930 ◽  
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.

scholarly journals Cell-specific basolateral membrane sorting of the human liver Na+-dependent bile acid cotransporter

2001 ◽  
Vol 280 (6) ◽  
pp. G1305-G1313 ◽  
Author(s):  
An-Qiang Sun ◽  
I'Kyori Swaby ◽  
Shuhua Xu ◽  
Frederick J. Suchy
Keyword(s):  
Low Temperature ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Brefeldin A ◽  
Temperature Shift ◽  
Surface Expression ◽  
Apical Surface ◽  
Uptake Studies ◽  
Green Fluorescent

The human Na+-taurocholate cotransporting polypeptide (Ntcp) is located exclusively on the basolateral membrane of hepatocyte, but the mechanisms underlying its membrane sorting domain have not been fully elucidated. In the present study, a green fluorescent protein-fused human NTCP (NTCP-GFP) was constructed using the polymerase chain reaction and was stably transfected into Madin-Darby canine kidney (MDCK) and Caco-2 cells. Taurocholate uptake studies and confocal microscopy demonstrated that the polarity of basolateral surface expression of NTCP-GFP was maintained in MDCK cells but was lost in Caco-2 cells. Nocodazole (33 μM), an agent that causes microtubular depolymerization, partially disrupted the basolateral localization of NTCP-GFP by increasing apical surface expression to 33.5% compared with untreated cells ( P < 0.05). Brefeldin A (BFA; 1–2 μM) disrupted the polarized basolateral localization of NTCP, but monensin (1.4 μM) had no affect on NTCP-GFP localization. In addition, low-temperature shift (20°C) did not affect the polarized basolateral surface sorting of NTCP-GFP and repolarization of this protein after BFA interruption. In summary, these data suggest that the polarized basolateral localization of human NTCP is cell specific and is mediated by a novel sorting pathway that is BFA sensitive and monensin and low-temperature shift insensitive. The process may also involve microtubule motors.

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

2006 ◽  
Vol 172 (7) ◽  
pp. 1023-1034 ◽  
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.

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

1998 ◽  
Vol 275 (5) ◽  
pp. G1045-G1055 ◽  
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.

Apical membrane and intracellular distribution of endogenous alpha 2A-adrenergic receptors in MDCK cells

1994 ◽  
Vol 267 (3) ◽  
pp. F347-F353 ◽  
Author(s):  
M. D. Okusa ◽  
K. R. Lynch ◽  
D. L. Rosin ◽  
L. Huang ◽  
Y. Y. Wei
Keyword(s):  
Cell Surface ◽  
De Novo ◽  
Mdck Cells ◽  
Specific Binding ◽  
Surface Expression ◽  
Surface Proteins ◽  
Cell Surface Expression ◽  
Surface Binding ◽  
Binding Studies ◽  
Apical Domain

The purpose of the current studies was to characterize the endogenous alpha 2-adrenergic receptor (AR) subtypes present in Madin-Darby canine kidney (MDCK) cells and to determine their level of expression and pattern of distribution. By saturation binding analysis with [3H]MK-912, MDCK cells expressed high levels of alpha 2-ARs with a maximum receptor density (Bmax) of 798 +/- 55 fmol/mg protein and an equilibrium dissociation constant (Kd) of 0.98 +/- 0.32 nM. Competitive binding studies using prazosin, oxymetazoline, phentolamine, and epinephrine to displace [3H]MK-912 demonstrated inhibition constant (Ki) values of 1,270 +/- 250, 5.0 +/- 0.4, 5.5 +/- 0.3, and 392 +/- 150 nM (n = 3), respectively. In Northern blot analysis we found that MDCK cells expressed transcripts encoding alpha 2A-AR and not alpha 2B-AR or alpha 2C-AR. Surface binding experiments suggested that approximately 60% of alpha 2A-ARs are distributed at the cell surface domain. Specific binding of [3H]MK-912 to soluble apical and basolateral surface proteins isolated by surface biotinylation indicated the expression of surface alpha 2A-ARs was limited to the apical domain of MDCK cells. No alpha 2A-ARs were detected on the basolateral surface. We conclude that endogenous alpha 2A-ARs are targeted to the apical domain of MDCK cells and that the intracellular compartment may contain ARs as a reservoir for de novo cell surface expression or, alternatively, may represent internalized receptors.

Brefeldin A causes structural and functional alterations of the trans-Golgi network of MDCK cells

1994 ◽  
Vol 107 (4) ◽  
pp. 933-943 ◽  
Author(s):  
M. Wagner ◽  
A.K. Rajasekaran ◽  
D.K. Hanzel ◽  
S. Mayor ◽  
E. Rodriguez-Boulan
Keyword(s):  
Mdck Cells ◽  
Brefeldin A ◽  
Adaptor Proteins ◽  
Fungal Metabolite ◽  
Surface Transport ◽  
Structural Alterations ◽  
Golgi Stack ◽  
Trans Golgi Network ◽  
Influenza Hemagglutinin ◽  
Golgi Network

The trans-Golgi network (TGN) of MDCK cells is exquisitely sensitive to the fungal metabolite brefeldin A (BFA), in contrast to the refractory Golgi stack of these cells. At a concentration of 1 microgram/ml, BFA promoted extensive tubulation of the TGN while the medical Golgi marker alpha-mannosidase II was not affected. Tubules emerging minutes after addition of the drug contained both the apical marker influenza hemagglutinin (HA), previously accumulated at 20 degrees C, and the fusion protein interleukin receptor/TGN38 (TGG), a TGN marker that recycles basolaterally, indicating that, in contrast to TGN vesicles, TGN-derived tubules cannot sort apical and basolateral proteins. After 60 minutes treatment with BFA, HA and TGG tubules formed extensive networks widely spread throughout the cell, different from the focused centrosomal localization previously described in non-polarized cells. The TGG network partially codistributed with an early endosomal tubular network loaded with transferrin, suggesting that the TGG and endosomal networks had fused or that TGG had entered the endosomal network via surface recycling and endocytosis. The extensive structural alterations of the TGN were accompanied by functional disruptions, such as the extensive mis-sorting of influenza HA, and by the release of the TGN marker gamma-adaptin. Our results suggest the involvement of BFA-sensitive adaptor proteins in TGN--&gt;surface transport.

scholarly journals Sorting of newly synthesized galactosphingolipids to the two surface domains of epithelial cells.

1996 ◽  
Vol 132 (5) ◽  
pp. 813-821 ◽  
Author(s):  
P van der Bijl ◽  
M Lopes-Cardozo ◽  
G van Meer
Keyword(s):  
Fatty Acid ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Surface ◽  
High Concentration ◽  
Selective Transport ◽  
Endogenous Lipid ◽  
Surface Domains ◽  
Golgi Network

The high concentration of glycosphingolipids on the apical surface of epithelial cells may be generated by selective transport from their site of synthesis to the cell surface. Previously, we showed that canine kidney MDCK and human intestinal Caco-2 cells converted a ceramide carrying the short fluorescent fatty acid C6-NBD to glucosylceramide (GlcCer) and sphingomyelin (SM), and that GlcCer was preferentially transported to the apical surface as compared to SM. Here, we address the point that not all glycosphingolipid classes are apically enriched in epithelia. We show that a ceramide containing the 2-hydroxy fatty acid C6OH was preferentially converted by MDCK and Caco-2 cells to galactosylceramide (GalCer) and its derivatives galabiosylceramide (Ga2Cer) and sulfatide (SGalCer) as compared to SM and GlcCer--all endogenous lipid classes of these cells. Transport to the apical and basolateral cell surface was monitored by a BSA-depletion assay. In MDCK cells, GalCer reached the cell surface with two- to sixfold lower apical/basolateral polarity than GlcCer. Remarkably, in Caco-2 cells GalCer and GlcCer displayed the same apical/basolateral polarity, but it was sixfold lower for lipids with a C6OH chain than for C6-NBD lipids. Therefore, the sorting of a sphingolipid appears to depend on lipid structure and cell type. We propose that the different ratios of gluco- and galactosphingolipid synthesis in the various epithelial tissues govern lipid sorting in the membrane of the trans Golgi network by dictating the composition of the domains from where vesicles bud to the apical and basolateral cell surface.

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.

Effect of brefeldin A on heparan sulphate biosynthesis in Madin–Darby canine kidney cells

2002 ◽  
Vol 362 (2) ◽  
pp. 359-366 ◽  
Author(s):  
Svein Olav KOLSET ◽  
Kristian PRYDZ ◽  
Katja FJELDSTAD ◽  
Fariba SAFAIYAN ◽  
Tram Thu VUONG ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Heparan Sulphate ◽  
Brefeldin A ◽  
Sodium Chlorate ◽  
Cell Types ◽  
Madin Darby Canine Kidney ◽  
Golgi Stack ◽  
Golgi Network ◽  
Darby Canine Kidney ◽  
Canine Kidney

Brefeldin A (BFA) perturbs the organization of the Golgi apparatus, such that Golgi stack components are fused with the endoplasmic reticulum (ER) and separated from the trans-Golgi network. In many cell types, BFA blocks the secretion of macromolecules but still allows the action of Golgi enzymes in the ER. Treatment of cells with BFA has been reported to inhibit the secretion of heparan sulphate (HS) proteoglycans and alter the structure of their HS components, but the nature of such structural alterations has not been characterized in detail. We analysed the effect of BFA on HS biosynthesis in Madin—Darby canine kidney (MDCK) cells, in which the Golgi complex is more resistant towards BFA than in most other cell types. We found that MDCK cells were able to secrete HS proteoglycans in spite of BFA treatment. However, the secretion of HS was reduced and the secreted HS differed from that produced by untreated cells. In BFA-treated cells, two structurally distinct pools of HS were generated. One pool was similar to HS from control cells, with the exception that the 6-O-sulphation of glucosamine (GlcN) residues was reduced. In contrast, the other pool consisted of largely unmodified N-acetylheparosan polymers with a low (<20%) proportion of N-sulphated GlcN residues but a substantial proportion of N-unsubstituted GlcN units, indicating that it had been acted upon by N-deacetylases and partly by the N-sulphotransferases, but not by O-sulphotransferases. Together, these findings represent a previously unrecognized alteration in HS biosynthesis caused by BFA, and differ dramatically from our previous findings in MDCK cells pertaining to the undersulphation of HS caused by sodium chlorate treatment.

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