scholarly journals Microtubule-acting drugs lead to the nonpolarized delivery of the influenza hemagglutinin to the cell surface of polarized Madin-Darby canine kidney cells.

1987 ◽  
Vol 104 (2) ◽  
pp. 231-241 ◽  
Author(s):  
M J Rindler ◽  
I E Ivanov ◽  
D D Sabatini
Keyword(s):  
Golgi Apparatus ◽  
Cell Surface ◽  
G Protein ◽  
Mdck Cells ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Vesicular Stomatitis ◽  
Ha Protein ◽  
Darby Canine Kidney ◽  
Canine Kidney

The synchronized directed transfer of the envelope glycoproteins of the influenza and vesicular stomatitis viruses from the Golgi apparatus to the apical and basolateral surfaces, respectively, of polarized Madin-Darby canine kidney (MDCK) cells can be achieved using temperature-sensitive mutant viruses and appropriate temperature shift protocols (Rindler, M. J., I. E. Ivanov, H. Plesken, and D. D. Sabatini, 1985, J. Cell Biol., 100:136-151). The microtubule-depolymerizing agents colchicine and nocodazole, as well as the microtubule assembly-promoting drug taxol, were found to interfere with the normal polarized delivery and exclusive segregation of hemagglutinin (HA) to the apical surface but not with the delivery and initial accumulation of G on the basolateral surface. Immunofluorescence analysis of permeabilized monolayers of influenza-infected MDCK cells treated with the microtubule-acting drugs demonstrated the presence of substantial amounts of HA protein on both the apical and basolateral surfaces. Moreover, in cells infected with the wild-type influenza virus, particles budded from both surfaces. Viral counts in electron micrographs showed that approximately 40% of the released viral particles accumulated in the intercellular spaces or were trapped between the cell and monolayer and the collagen support as compared to less than 1% on the basolateral surface of untreated infected cells. The effect of the microtubule inhibitors was not a result of a rapid redistribution of glycoprotein molecules initially delivered to the apical surface since a redistribution was not observed when the inhibitors were added to the cells after the HA was permitted to reach the apical surface at the permissive temperature and the synthesis of new HA was inhibited with cycloheximide. The altered segregation of the HA protein that occurs may result from the dispersal of the Golgi apparatus induced by the inhibitors or from the disruption of putative microtubules containing tracks that could direct vesicles from the trans Golgi apparatus to the cell surface. Since the vesicular stomatitis virus G protein is basolaterally segregated even when the Golgi elements are dispersed and hypothetical tracks disrupted, it appears that the two viral envelope glycoproteins are segregated by fundamentally different mechanisms and that the apical surface may be incapable of accepting vesicles carrying the G protein.

scholarly journals Steady-state distribution and biogenesis of endogenous Madin-Darby canine kidney glycoproteins: evidence for intracellular sorting and polarized cell surface delivery.

1989 ◽  
Vol 109 (5) ◽  
pp. 2117-2127 ◽  
Author(s):  
M P Lisanti ◽  
A Le Bivic ◽  
M Sargiacomo ◽  
E Rodriguez-Boulan
Keyword(s):  
Steady State ◽  
Epithelial Cell ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Surface ◽  
Steady State Distribution ◽  
State Distribution ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

We used domain-selective biotinylation/125I-streptavidin blotting (Sargiacomo, M., M. P. Lisanti, L. Graeve, A. Le Bivic, and E. Rodriguez-Boulan. 1989 J. Membr. Biol. 107:277-286), in combination with lectin precipitation, to analyze the apical and basolateral glycoprotein composition of Madin-Darby canine kidney (MDCK) cells and to explore the role of glycosylation in the targeting of membrane glycoproteins. All six lectins used recognized both apical and basolateral glycoproteins, indicating that none of the sugar moieties detected were characteristic of the particular epithelial cell surface. Pulse-chase experiments coupled with domain-selective glycoprotein recovery were designed to detect the initial appearance of newly synthesized glycoproteins at the apical or basolateral cell surface. After a short pulse with a radioactive precursor, glycoproteins reaching each surface were biotinylated, extracted, and recovered via precipitation with immobilized streptavidin. Several basolateral glycoproteins (including two sulfated proteins) and at least two apical glycoproteins (one of them the major sulfated protein of MDCK cells) appeared at the corresponding surface after 20-40 min of chase, but were not detected in the opposite surface, suggesting that they were sorted intracellularly and vectorially delivered to their target membrane. Several "peripheral" apical proteins were detected at maximal levels on the apical surface immediately after the 15-min pulse, suggesting a very fast intracellular transit. Finally, domain-selective labeling of surface carbohydrates with biotin hydrazide (after periodate oxidation) revealed strikingly different integral and peripheral glycoprotein patterns, resembling the Con A pattern, after labeling with sulfo-N-hydroxy-succinimido-biotin. The approaches described here should be useful in characterizing the steady-state distribution and biogenesis of endogenous cell surface components in a variety of epithelial cell lines.

scholarly journals Viral glycoproteins destined for apical or basolateral plasma membrane domains traverse the same Golgi apparatus during their intracellular transport in doubly infected Madin-Darby canine kidney cells.

1984 ◽  
Vol 98 (4) ◽  
pp. 1304-1319 ◽  
Author(s):  
M J Rindler ◽  
I E Ivanov ◽  
H Plesken ◽  
E Rodriguez-Boulan ◽  
D D Sabatini
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
G Protein ◽  
Intracellular Transport ◽  
Mdck Cells ◽  
Simian Virus ◽  
Apical Plasma Membrane ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

Madin-Darby canine kidney (MDCK) cells can sustain double infection with pairs of viruses of opposite budding polarity (simian virus 5 [SV5] and vesicular stomatitis virus [VSV] or influenza and VSV), and we observed that in such cells the envelope glycoproteins of the two viruses are synthesized simultaneously and assembled into virions at their characteristic sites. Influenza and SV5 budded exclusively from the apical plasma membrane of the cells, while VSV emerged only from the basolateral surfaces. Immunoelectron microscopic examination of doubly infected MDCK cells showed that the influenza hemagglutinin (HA) and the VSV G glycoproteins traverse the same Golgi apparatus and even the same Golgi cisternae. This indicates that the pathways of the two proteins towards the plasma membrane do not diverge before passage through the Golgi apparatus and therefore that critical sorting steps must take place during or after passage of the glycoproteins through this organelle. After its passage through the Golgi, the HA accumulated primarily at the apical membrane, where influenza virion assembly occurred. A small fraction of HA did, however, appear on the lateral surface and was incorporated into the envelope of budding VSV virions. Although predominantly found on the basolateral surface, significant amounts of G protein were observed on the apical plasma membrane well before disruption of the tight junctions was detectable. Nevertheless, assembly of VSV virions was restricted to the basolateral domain and in doubly infected cells the G protein was only infrequently incorporated into the envelope of budding influenza virions. These observations indicate that the site of VSV budding is not determined exclusively by the presence of G polypeptides. Therefore, it is likely that, at least for VSV, other cellular or viral components are responsible for the selection of the appropriate budding domain.

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 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 The N-terminal segment of endothelin-converting enzyme (ECE)-1b contains a di-leucine motif that can redirect neprilysin to an intracellular compartment in Madin–Darby canine kidney (MDCK) cells

1999 ◽  
Vol 341 (1) ◽  
pp. 119-126 ◽  
Author(s):  
Françoise CAILLER ◽  
Jacques P. ZAPPULLA ◽  
Guy BOILEAU ◽  
Philippe CRINE
Keyword(s):  
Cell Surface ◽  
Mdck Cells ◽  
Intracellular Localization ◽  
Site Directed Mutagenesis ◽  
Converting Enzyme ◽  
Madin Darby Canine Kidney ◽  
Intracellular Compartment ◽  
Endothelin Converting Enzyme ◽  
Darby Canine Kidney ◽  
Canine Kidney

Endothelin-converting enzyme (ECE)-1 is a membrane-bound metallopeptidase of the neprilysin (NEP) family. ECE-1 is responsible for the conversion of inactive big-endothelins into active endothelins. Three different isoforms of human ECE-1 (ECE-1a, ECE-1b and ECE-1c) have been identified. They differ in their N-terminal cytosolic regions, have distinct tissue distribution and intracellular localization. ECE-1a and ECE-1c are both located at the cell surface whereas ECE-1b is targeted to an intracellular compartment. To better understand the nature of the signal responsible for the targeting of ECE-1b to the intracellular compartment, we have constructed several ECE/NEP chimaeric proteins and expressed them by transfection into Madin-Darby canine kidney (MDCK) cells. This allowed us to identify a nine amino acid segment in the cytosolic tail of ECE-1b that is sufficient to relocate NEP from the cell surface to an intracellular compartment. Site-directed mutagenesis on these chimaeras led to the identification of two leucine residues as part of the intracellular retention signal.

scholarly journals An Intact Dilysine-like Motif in the Carboxyl Terminus of MAL Is Required for Normal Apical Transport of the Influenza Virus Hemagglutinin Cargo Protein in Epithelial Madin-Darby Canine Kidney Cells

2001 ◽  
Vol 12 (6) ◽  
pp. 1869-1883 ◽  
Author(s):  
Rosa Puertollano ◽  
José Angel Martı́nez-Menárguez ◽  
Alicia Batista ◽  
José Ballesta ◽  
Miguel Angel Alonso
Keyword(s):  
Influenza Virus ◽  
Mdck Cells ◽  
Protein Sorting ◽  
Apical Surface ◽  
Carboxy Terminus ◽  
Madin Darby Canine Kidney ◽  
Influenza Virus Hemagglutinin ◽  
Carboxyl Terminal ◽  
Darby Canine Kidney ◽  
Canine Kidney

The MAL proteolipid, a component of the integral protein sorting machinery, has been demonstrated as being necessary for normal apical transport of the influenza virus hemagglutinin (HA) and the overall apical membrane proteins in Madin-Darby canine kidney (MDCK) cells. The MAL carboxy terminus ends with the sequence Arg-Trp-Lys-Ser-Ser (RWKSS), which resembles dilysine-based motifs involved in protein sorting. To investigate whether the RWKSS pentapeptide plays a role in modulating the distribution of MAL and/or its function in apical transport, we have expressed MAL proteins with distinct carboxy terminus in MDCK cells whose apical transport was impaired by depletion of endogenous MAL. Apical transport of HA was restored to normal levels by expression of MAL with an intact but not with modified carboxyl terminal sequences bearing mutations that impair the functioning of dilysine-based sorting signals, although all the MAL proteins analyzed incorporated efficiently into lipid rafts. Ultrastructural analysis indicated that compared with MAL bearing an intact RWKSS sequence, a mutant with lysine −3 substituted by serine showed a twofold increased presence in clathrin-coated cytoplasmic structures and a reduced expression on the plasma membrane. These results indicate that the carboxyl-terminal RWKSS sequence modulates the distribution of MAL in clathrin-coated elements and is necessary for HA transport to the apical surface.

scholarly journals Intracellular sorting and basolateral appearance of the G protein of vesicular stomatitis virus in Madin-Darby canine kidney cells.

1985 ◽  
Vol 101 (2) ◽  
pp. 470-476 ◽  
Author(s):  
S Pfeiffer ◽  
S D Fuller ◽  
K Simons
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Apical Membrane ◽  
Madin Darby Canine Kidney ◽  
Transport Pathway ◽  
Basolateral Side ◽  
Viral Glycoproteins ◽  
Vesicular Stomatitis ◽  
Darby Canine Kidney ◽  
Canine Kidney

The polarity of the surface distribution of viral glycoproteins during virus infection has been studied in the Madin-Darby canine kidney epithelial cell line on nitrocellulose filters. Using a surface radioimmunoassay on Madin-Darby canine kidney strain I cells that had been infected with vesicular stomatitis virus or with avian influenza fowl plague virus, we found that the surface G protein was 97% basolateral, whereas the fowl plague virus hemagglutinin was 88% apical. Newly synthesized, pulse-labeled vesicular stomatitis virus appeared first on the basolateral plasma membrane as measured by an immunoprecipitation assay in which the anti-G protein antibody was applied to the monolayer either from the apical or the basolateral side. Labeled G protein could be accumulated inside the cell at a late stage of transport by decreasing the temperature to 20 degrees C during the chase. Reversal to 37 degrees C led to its rapid and synchronous transport to the basolateral surface at an initial rate 61-fold greater than that of transport to the apical side. These results demonstrate that the newly synthesized G protein is transported directly to the basolateral membrane and does not pass over the apical membrane en route. Since a previous study of the surface appearance of influenza virus hemagglutinins showed that the newly synthesized hemagglutinins were inserted directly from an intracellular site into the apical membrane (Matlin, K., and K. Simons, 1984, J. Cell Biol., 99:2131-2139), we conclude that the divergence of the transport pathway for the apical and basolateral viral glycoproteins has to occur intracellularly, i.e., before reaching the cell surface.

scholarly journals Penetration of Salmonella through a polarized Madin-Darby canine kidney epithelial cell monolayer.

1988 ◽  
Vol 107 (1) ◽  
pp. 221-230 ◽  
Author(s):  
B B Finlay ◽  
B Gumbiner ◽  
S Falkow
Keyword(s):  
Mdck Cell ◽  
Pathogenic Bacteria ◽  
Mdck Cells ◽  
Cell Monolayer ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Intracellular Parasites ◽  
Epithelial Barriers ◽  
Darby Canine Kidney ◽  
Canine Kidney

Many intracellular parasites are capable of penetrating host epithelial barriers. To study this process in more detail we examined the interactions between the pathogenic bacteria Salmonella choleraesuis and polarized epithelial monolayers of Madin-Darby canine kidney (MDCK) cells grown on membrane filters. Association of bacteria with the MDCK cell apical surface was an active event, requiring bacterial RNA and protein synthesis, and was blocked by low temperatures. Salmonella were internalized within a membrane-bound vacuole and exhibited penetration through, but not between MDCK cells. A maximum of 14 Salmonella per MDCK cell crossed the monolayer per hour to the basolateral surface yet the monolayer remained viable and impermeable to Escherichia coli. Apical S. choleraesuis infection resulted in an increase in paracellular permeability but the MDCK intercellular contacts were not significantly disrupted. Basolateral S. choleraesuis infection was inefficient, and only small numbers of S. choleraesuis penetrated to the apical medium.

scholarly journals The MAL Proteolipid Is Necessary for the Overall Apical Delivery of Membrane Proteins in the Polarized Epithelial Madin–Darby Canine Kidney and Fischer Rat Thyroid Cell Lines

2000 ◽  
Vol 11 (6) ◽  
pp. 2033-2045 ◽  
Author(s):  
Fernando Martı́n-Belmonte ◽  
Rosa Puertollano ◽  
Jaime Millán ◽  
Miguel A. Alonso
Keyword(s):  
Membrane Proteins ◽  
Mdck Cells ◽  
Neurotrophin Receptor ◽  
Thyroid Cell ◽  
Apical Surface ◽  
Placental Alkaline Phosphatase ◽  
Madin Darby Canine Kidney ◽  
Rat Thyroid ◽  
Darby Canine Kidney ◽  
Canine Kidney

The MAL proteolipid has been recently demonstrated as being necessary for correct apical sorting of the transmembrane influenza virus hemagglutinin (HA) in Madin–Darby canine kidney (MDCK) cells. The fact that, in contrast to MDCK cells, Fischer rat thyroid (FRT) cells target the majority of glycosylphosphatidylinositol (GPI)-anchored proteins to the basolateral membrane provides us with the opportunity to determine the role of MAL in apical transport of membrane proteins under conditions in which the majority of GPI-anchored proteins are (MDCK cells) or are not (FRT cells) targeted to the apical surface. Using an antisense oligonucleotide-based strategy to deplete endogenous MAL, we have observed that correct transport of apical transmembrane proteins associated (HA) or not (exogenous neurotrophin receptor and endogenous dipeptidyl peptidase IV) with lipid rafts, as well as that of the bulk of endogenous apical membrane, takes place in FRT cells by a pathway that requires normal MAL levels. Even transport of placental alkaline phosphatase, a GPI-anchored protein that is targeted apically in FRT cells, was dependent on normal MAL levels. Similarly, in addition to the reported effect of MAL on HA transport, depletion of MAL in MDCK cells caused a dramatic reduction in the apical delivery of the GPI-anchored gD1-DAF protein, neurotrophin receptor, and the bulk of membrane proteins. These results suggest that MAL is necessary for the overall apical transport of membrane proteins in polarized MDCK and FRT cells.

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