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.

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

1999 ◽  
Vol 145 (1) ◽  
pp. 141-151 ◽  
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.

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

1987 ◽  
Vol 105 (4) ◽  
pp. 1623-1635 ◽  
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.

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 Serine phosphorylation site of the 46-kDa mannose 6-phosphate receptor is required for transport to the plasma membrane in Madin-Darby canine kidney and mouse fibroblast cells.

1997 ◽  
Vol 8 (4) ◽  
pp. 567-576 ◽  
Author(s):  
P Breuer ◽  
C Körner ◽  
C Böker ◽  
A Herzog ◽  
R Pohlmann ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Mdck Cells ◽  
Phosphorylation Site ◽  
Serine Phosphorylation ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Mannose 6 Phosphate ◽  
Darby Canine Kidney ◽  
Canine Kidney

Up to 4% of the human 46-kDa mannose 6-phosphate receptor (MPR46) expressed in Madin-Darby canine kidney (MDCK) cells are localized at the cell surface. At steady state, the expression of MPR46 on the apical surface of filter-grown MDCK cells is about sixfold lower than on the basolateral surface. The cytoplasmic domain of the MPR46 is phosphorylated on serine 56 at low stoichiometry. By expressing mutant MPR46 we have shown that the MPR46 phosphorylation site is required for delivery to the plasma membrane. In addition, mutant MPR46 expressed in MPR-deficient mouse embryonic fibroblasts were not detected at the cell surface and their ability to sort newly synthesized cathepsin D was not altered. Since the loss of MPR46 phosphorylation correlates with the lack of cell surface expression, phosphorylation of serine 56 may either function as a direct plasma membrane targeting signal or inhibit MPR46 recycling from endosomes to Golgi, resulting in trafficking to the cell surface.

scholarly journals Transport of influenza HA from the trans-Golgi network to the apical surface of MDCK cells permeabilized in their basolateral plasma membranes: energy dependence and involvement of GTP-binding proteins.

1990 ◽  
Vol 111 (6) ◽  
pp. 2893-2908 ◽  
Author(s):  
D Gravotta ◽  
M Adesnik ◽  
D D Sabatini
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Binding Proteins ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Surface ◽  
Gtp Binding Proteins ◽  
Trans Golgi Network ◽  
Gtp Binding ◽  
Golgi Network

A procedure employing streptolysin O to effect the selective permeabilization of either the apical or basolateral plasma membrane domains of MDCK cell monolayers grown on a filter support was developed which permeabilizes the entire monolayer, leaves the opposite cell surface domain intact, and does not abolish the integrity of the tight junctions. This procedure renders the cell interior accessible to exogenous macromolecules and impermeant reagents, permitting the examination of their effects on membrane protein transport to the intact surface. The last stages of the transport of the influenza virus hemagglutinin (HA) to the apical surface were studied in pulse-labeled, virus-infected MDCK cells that were incubated at 19.5 degrees C for 90 min to accumulate newly synthesized HA in the trans-Golgi network (TGN), before raising the temperature to 35 degrees C to allow synchronized transport to the plasma membrane. In cells permeabilized immediately after the cold block, 50% of the intracellular HA molecules were subsequently delivered to the apical surface. This transport was dependent on the presence of an exogenous ATP supply and was markedly inhibited by the addition of GTP-gamma-S at the time of permeabilization. On the other hand, the GTP analogue had no effect when it was added to cells that, after the cold block, were incubated for 15 min at 35 degrees C before permeabilization, even though at this time most HA molecules were still intracellular and their appearance at the cell surface was largely dependent on exogenous ATP. These findings indicate that GTP-binding proteins are involved in the constitutive process that effects vesicular transport from the TGN to the plasma membrane and that they are charged early in this process. Transport of HA to the cell surface could be made dependent on the addition of exogenous cytosol when, after permeabilization, cells were washed to remove endogenous cytosolic components. This opens the way towards the identification of cell components that mediate the sorting of apical and basolateral membrane components in the TGN and their polarized delivery to the cell surface.

scholarly journals MAL regulates clathrin-mediated endocytosis at the apical surface of Madin–Darby canine kidney cells

2003 ◽  
Vol 163 (1) ◽  
pp. 155-164 ◽  
Author(s):  
Fernando Martín-Belmonte ◽  
José A. Martínez-Menárguez ◽  
Juan F. Aranda ◽  
José Ballesta ◽  
María C. de Marco ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Microscopic Analysis ◽  
Time Lapse ◽  
Kidney Cells ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Apical Vesicles ◽  
Darby Canine Kidney ◽  
Canine Kidney

MAL is an integral protein component of the machinery for apical transport in epithelial Madin–Darby canine kidney (MDCK) cells. To maintain its distribution, MAL cycles continuously between the plasma membrane and the Golgi complex. The clathrin-mediated route for apical internalization is known to differ from that at the basolateral surface. Herein, we report that MAL depends on the clathrin pathway for apical internalization. Apically internalized polymeric Ig receptor (pIgR), which uses clathrin for endocytosis, colocalized with internalized MAL in the same apical vesicles. Time-lapse confocal microscopic analysis revealed cotransport of pIgR and MAL in the same endocytic structures. Immunoelectron microscopic analysis evidenced colabeling of MAL with apically labeled pIgR in pits and clathrin-coated vesicles. Apical internalization of pIgR was abrogated in cells with reduced levels of MAL, whereas this did not occur either with its basolateral entry or the apical internalization of glycosylphosphatidylinositol-anchored proteins, which does not involve clathrin. Therefore, MAL is critical for efficient clathrin-mediated endocytosis at the apical surface in MDCK cells.

scholarly journals TGN38 recycles basolaterally in polarized Madin-Darby canine kidney cells.

1994 ◽  
Vol 5 (10) ◽  
pp. 1093-1103 ◽  
Author(s):  
A K Rajasekaran ◽  
J S Humphrey ◽  
M Wagner ◽  
G Miesenböck ◽  
A Le Bivic ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Protein Localization ◽  
Evolutionary Relationship ◽  
Cytoplasmic Domain ◽  
Chimeric Proteins ◽  
Madin Darby Canine Kidney ◽  
Surface Domains ◽  
Darby Canine Kidney ◽  
Canine Kidney

Sorting of newly synthesized plasma membrane proteins to the apical or basolateral surface domains of polarized cells is currently thought to take place within the trans-Golgi network (TGN). To explore the relationship between protein localization to the TGN and sorting to the plasma membrane in polarized epithelial cells, we have expressed constructs encoding the TGN marker, TGN38, in Madin-Darby canine kidney (MDCK) cells. We report that TGN38 is predominantly localized to the TGN of these cells and recycles via the basolateral membrane. Analyses of the distribution of Tac-TGN38 chimeric proteins in MDCK cells suggest that the cytoplasmic domain of TGN38 has information leading to both TGN localization and cycling through the basolateral surface. Mutations of the cytoplasmic domain that disrupt TGN localization also lead to nonpolarized delivery of the chimeric proteins to both surface domains. These results demonstrate an apparent equivalence of basolateral and TGN localization determinants and support an evolutionary relationship between TGN and plasma membrane sorting processes.

scholarly journals Actin Is Required for Endocytosis at the Apical Surface of Madin-Darby Canine Kidney Cells where ARF6 and Clathrin Regulate the Actin Cytoskeleton

2006 ◽  
Vol 17 (1) ◽  
pp. 427-437 ◽  
Author(s):  
Tehila Hyman ◽  
Miri Shmuel ◽  
Yoram Altschuler
Keyword(s):  
Bound State ◽  
Cell Cortex ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Epithelial Surface ◽  
Biochemical Assays ◽  
Epithelial Cell Lines ◽  
Darby Canine Kidney ◽  
Canine Kidney

In epithelial cell lines, apical but not basolateral clathrin-mediated endocytosis has been shown to be affected by actin-disrupting drugs. Using electron and fluorescence microscopy, as well as biochemical assays, we show that the amount of actin dedicated to endocytosis is limiting at the apical surface of epithelia. In part, this contributes to the low basal rate of clathrin-dependent endocytosis observed at this epithelial surface. ARF6 in its GTP-bound state triggers the recruitment of actin from the cell cortex to the clathrin-coated pit to enable dynamin-dependent endocytosis. In addition, we show that perturbation of the apical endocytic system by expression of a clathrin heavy-chain mutant results in the collapse of microvilli. This phenotype was completely reversed by the expression of an ARF6-GTP-locked mutant. These observations indicate that concomitant to actin recruitment, the apical clathrin endocytic system is deeply involved in the morphology of the apical plasma membrane.

scholarly journals Microtubule perturbation inhibits intracellular transport of an apical membrane glycoprotein in a substrate-dependent manner in polarized Madin-Darby canine kidney epithelial cells.

1990 ◽  
Vol 1 (12) ◽  
pp. 921-936 ◽  
Author(s):  
M J van Zeijl ◽  
K S Matlin
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Intracellular Transport ◽  
Mdck Cells ◽  
Sodium Dodecyl ◽  
Apical Plasma Membrane ◽  
Dependent Manner ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

The effects of microtubule perturbation on the transport of two different viral glycoproteins were examined in infected Madin-Darby canine kidney (MDCK) cells grown on both permeable and solid substrata. Quantitative biochemical analysis showed that the microtubule-depolymerizing drug nocodazole inhibited arrival of influenza hemagglutinin on the apical plasma membrane in MDCK cells grown on both substrata. In contrast, the microtubule-stabilizing drug taxol inhibited apical appearance of hemagglutinin only when MDCK cells were grown on permeable substrata. On the basis of hemagglutinin mobility on sodium dodecyl sulfate gels and its sensitivity to endo H, it was evident that nocodazole and taxol arrested hemagglutinin at different intracellular sites. Neither drug caused a significant increase in the amount of hemagglutinin detected on the basolateral plasma membrane domain. In addition, neither drug had any noticeable effect on the transport of the vesicular stomatitis virus (VSV)-G protein to the basolateral surface. These results shed light on previous conflicting reports using this model system and support the hypothesis that microtubules play a role in the delivery of membrane glycoproteins to the apical, but not the basolateral, domain of epithelial cells.

Expression of 5-HT1C receptors in transfected MDCK cells is functionally and anatomically asymmetric

1993 ◽  
Vol 265 (1) ◽  
pp. C193-C200 ◽  
Author(s):  
H. Luo ◽  
A. Tesfaye ◽  
I. Schieren ◽  
H. S. Chase
Keyword(s):  
Binding Sites ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Lysergic Acid ◽  
Madin Darby Canine Kidney ◽  
High Affinity ◽  
G418 Selection ◽  
Selection Medium ◽  
Darby Canine Kidney ◽  
Canine Kidney

Madin-Darby canine kidney (MDCK) cells were transfected with the cDNA for the rat 5-HT1C receptor (pMV7-SR1c) using electroporation. Cells that survived G418 selection medium were loaded with indo-1 and run through a fluorescence-activated cell sorter (FACS); 10% responded to serotonin (5-HT) with an increase in intracellular Ca2+ concentration ([Ca2+]i). Responding cells were separated with the FACS, grown to confluence, and resorted two more times until a clone of 100% respondents was obtained (SR-MDCK). In SR-MDCK cells grown on porous filters, [Ca2+]i increased only when 5-HT was applied to the basolateral membrane (change in [Ca2+]i = 190 +/- 43 nM); there was no response of [Ca2+]i to apical application of 5-HT. The asymmetric response to 5-HT was likely due to targeting of 5-HT1C receptors exclusively to the basolateral membrane of SR-MDCK cells; 125I-labeled lysergic acid diethylamide binding sites, a marker of high-affinity 5-HT receptors, were located only in the basolateral membrane. These experiments demonstrate that epithelial cells can be stably transfected to express G protein-linked, calcium-mobilizing receptors and that the receptors may be targeted asymmetrically to specific domains of the plasma membrane.

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