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 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.

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 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 Transcytosis of the G protein of vesicular stomatitis virus after implantation into the apical plasma membrane of Madin-Darby canine kidney cells. I. Involvement of endosomes and lysosomes.

1984 ◽  
Vol 99 (3) ◽  
pp. 796-782 ◽  
Author(s):  
M Pesonen ◽  
W Ansorge ◽  
K Simons
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Vesicular Stomatitis ◽  
Darby Canine Kidney ◽  
Canine Kidney

The G protein of vesicular stomatitis virus, implanted into the apical plasma membrane of Madin-Darby canine kidney cells, is rapidly transcytosed to the basolateral membrane. In this and the accompanying paper (Pesonen, M., R. Bravo, and K. Simons, 1984, J. Cell Biol. 99:803-809.) we have studied the intracellular route by which the G protein traverses during transcytosis. Using Percoll density gradient centrifugation and free flow electrophoresis we could demonstrate that the G protein is endocytosed into a nonlysosomal compartment with a density of approximately 1.05 g/cm3, which has many of the characteristics of endosomes. Transcytosis to the basolateral membrane appeared to occur from this compartment. No direct evidence for the involvement of lysosomes in the transcytotic route could be obtained. No G protein was detected in the lysosomes when transcytosis of G protein was occurring. Moreover, at 21 degrees C when passage of G protein to the lysosomes was shown to be arrested, transcytosis of G protein could still be demonstrated.

scholarly journals Transepithelial transport of a viral membrane glycoprotein implanted into the apical plasma membrane of Madin-Darby canine kidney cells. II. Immunological quantitation.

1983 ◽  
Vol 97 (3) ◽  
pp. 638-643 ◽  
Author(s):  
M Pesonen ◽  
K Simons
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Binding Assay ◽  
Protein A ◽  
Apical Plasma Membrane ◽  
Kidney Cells ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

The envelope of vesicular stomatitis virus was fused with the apical plasma membrane of Madin-Darby canine kidney cells by low pH treatment. The fate of the implanted G protein was then followed using a protein A-binding assay, which was designed to quantitate the amount of G protein in the apical and the basolateral membranes. The implanted G protein was rapidly internalized at 31 degrees C, whereas at 10 degrees C no uptake was observed. Already after 15 min at 31 degrees C, a fraction of the G protein could be detected at the basolateral membrane. After 60 min 25-48% of the G protein was basolateral as measured by the protein A-binding assay. At the same time, 25-33% of the implanted G protein was detected at the apical membrane. Internalization of G protein was not affected by 20 mM ammonium chloride or by 10 microM monensin. However, the endocytosed G protein accumulated in intracellular vacuoles and redistribution back to the plasma membrane was inhibited. We conclude that the implanted G protein was rapidly internalized from the apical surface of Madin-Darby canine kidney cells and a major fraction was routed to the basolateral domain.

scholarly journals Expression of macrophage-lymphocyte Fc receptors in Madin-Darby canine kidney cells: polarity and transcytosis differ for isoforms with or without coated pit localization domains.

1989 ◽  
Vol 109 (6) ◽  
pp. 3291-3302 ◽  
Author(s):  
W Hunziker ◽  
I Mellman
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Apical Plasma Membrane ◽  
Madin Darby Canine Kidney ◽  
Coated Pits ◽  
Receptor Isoforms ◽  
Basolateral Plasma Membrane ◽  
Plasma Membrane Domain ◽  
Darby Canine Kidney ◽  
Canine Kidney

Many cells of the immune system and certain epithelia express receptors for the Fc domain of IgG (FcR). On mouse macrophages and lymphocytes, two distinct receptor isoforms have been identified, designated FcRII-B1 and FcRII-B2. The isoforms are identical except for an in-frame insertion of 47 amino acids in the cytoplasmic tail of FcRII-B1 that blocks its ability to be internalized by clathrin-coated pits. We have recently found that at least one IgG-transporting epithelium, namely placental syncytial trophoblasts, expresses transcripts encoding a receptor similar or identical to macrophage-lymphocyte FcRII. To determine whether FcRII of hematopoietic cells might also function as a transcytotic receptor if expressed in epithelial cells, FcRII-B1 and -B2 were transfected into Madin-Darby canine kidney (MDCK) cells and grown on permeable filter units. The two FcRII isoforms exhibited different patterns of polarized expression: FcRII-B1 was localized mainly to the apical plasma membrane domain, whereas FcRII-B2 was found predominantly on the basolateral surface. As expected for FcR in placenta, FcRII-B2 and to a lesser extent FcRII-B1 mediated transcellular transport of IgG-complexes from the apical to the basolateral plasma membrane. Neither receptor mediated transcytosis in the opposite direction, although FcRII-B2 also delivered ligand to lysosomes when internalized from either the basolateral or apical domains. Furthermore, FcRII-B2 was capable of transporting monovalent antireceptor antibody Fab fragments across the cell, suggesting that transcytosis was not dependent on receptor cross-linking. These findings suggest the possibility that FcRII can mediate transepithelial IgG transport when expressed in placental syncytial trophoblasts in addition to its "classical" endocytic and signaling activities when expressed in macrophages. Because FcRII-B1 and -B2 are expressed with distinct polarities, the results also suggest that interactions with clathrin-coated pits may play a role in generating the polarized distribution of at least some plasma membrane proteins in MDCK cells.

scholarly journals Transepithelial transport of a viral membrane glycoprotein implanted into the apical plasma membrane of Madin-Darby canine kidney cells. I. Morphological evidence.

1983 ◽  
Vol 97 (3) ◽  
pp. 627-637 ◽  
Author(s):  
K Matlin ◽  
D F Bainton ◽  
M Pesonen ◽  
D Louvard ◽  
N Genty ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Basolateral Membrane ◽  
Viral Envelope ◽  
Morphological Evidence ◽  
Apical Plasma Membrane ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

The G protein of vesicular stomatitis virus was implanted in the apical plasma membrane of Madin-Darby canine kidney cells by low pH-dependent fusion of the viral envelope with the cellular membrane. The amount of fusion as determined by removal of unfused virions, either by tryptic digestion or by EDTA treatment at 0 degree C, was 22-24% of the cell-bound virus radioactivity. Upon incubation of cells after implantation, the amount of G protein as detected by immunofluorescence diminished on the apical membrane and appeared within 30 min on the basolateral membrane. At the same time some G protein fluorescence was also seen in intracellular vacuoles. The observations by immunofluorescence were confirmed and extended by electron microscopy. Using immunoperoxidase localization, G protein was seen to move into irregularly shaped vacuoles (endosomes) and multivesicular bodies and to appear on the basolateral plasma membrane. These results suggest that the apical and basolateral domains of Madin-Darby canine kidney cells are connected by an intracellular route.

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 The lateral mobility of the (Na+,K+)-dependent ATPase in Madin-Darby canine kidney cells.

1986 ◽  
Vol 102 (4) ◽  
pp. 1256-1263 ◽  
Author(s):  
A J Jesaitis ◽  
J Yguerabide
Keyword(s):  
Plasma Membrane ◽  
Developmental Stage ◽  
Developmental Stages ◽  
Mdck Cells ◽  
Lateral Mobility ◽  
Madin Darby Canine Kidney ◽  
Confluent Cell ◽  
Restricted Mobility ◽  
Darby Canine Kidney ◽  
Canine Kidney

Fluorescence microphotolysis (recovery after photobleaching) was used to determine the lateral mobility of the (Na+,K+)ATPase and a fluorescent lipid analogue in the plasma membrane of Madin-Darby canine kidney (MDCK) cells at different stages of development. Fluorescein-conjugated Fab' fragments prepared from rabbit anti-dog (Na+,K+)ATPase antibodies (IgG) and 5-(N-hexadecanoyl)aminofluorescein (HEDAF) were used to label the plasma membrane of confluent and subconfluent cultures of MDCK cells. Fractional fluorescence recovery was 50% and 80-90% for the protein and lipid probes, respectively, and was independent of developmental stage. The estimated diffusion constants of the mobile fraction were approximately 5 X 10(-10) cm2/s for the (Na+,K+)ATPase and approximately 2 X 10(-9) cm2/s for HEDAF. Only HEDAF diffusion showed dependency on developmental stage in that D for confluent cells was approximately twice that for subconfluent cells. These results indicate that (Na+,K+)ATPase is 50% immobilized in all developmental stages, whereas lipids in confluent MDCK cells are more mobile than in subconfluent cells. They suggest, furthermore, that the degree of immobilization of the (Na+,K+)ATPase is insufficient to explain its polar distribution, and they support restricted mobility of the ATPase through the tight junctions as the likely mechanism for preventing the diffusion of this protein into the apical domain of the plasma membrane in confluent cell cultures.

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