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 Transcytosis of the G protein of vesicular stomatitis virus after implantation into the apical membrane of Madin-Darby canine kidney cells. II. Involvement of the Golgi complex.

1984 ◽  
Vol 99 (3) ◽  
pp. 803-809 ◽  
Author(s):  
M Pesonen ◽  
R Bravo ◽  
K Simons
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Golgi Complex ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Vesicular Stomatitis ◽  
Darby Canine Kidney ◽  
Canine Kidney

In the preceding paper (Pesonen M., W. Ansorge, and K. Simons, 1984, J. Cell Biol., 99:796-802), we have shown that transcellular transport of the membrane glycoprotein G of vesicular stomatitis virus implanted into the apical membrane of Madin-Darby canine kidney cells is transcytosed through the endosomal compartment to the basolateral plasma membrane. To determine whether the Golgi complex was involved in this process, G protein lacking sialic acid or all of the terminal sugars was implanted into the apical membrane and allowed to move to the basolateral membrane. Using the criteria of endoglycosidase H sensitivity, binding to Ricinus communis agglutinin and two-dimensional gel electrophoresis, the sugars on the transcytosed G protein were found to be the same as in the starting material. The absence of any involvement of the Golgi complex in transcytosis was supported by subcellular fractionation studies in which transcytosing G protein was never found in fractions containing galactosyl transferase.

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 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 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 An efficient method for introducing defined lipids into the plasma membrane of mammalian cells.

1983 ◽  
Vol 97 (5) ◽  
pp. 1365-1374 ◽  
Author(s):  
G van Meer ◽  
K Simons
Keyword(s):  
Plasma Membrane ◽  
Efficient Method ◽  
Mammalian Cells ◽  
Lipid Vesicles ◽  
Apical Plasma Membrane ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Ha Protein ◽  
Darby Canine Kidney ◽  
Canine Kidney

An efficient method has been devised to introduce lipid molecules into the plasma membrane of mammalian cells. This method has been applied to fuse lipid vesicles with the apical plasma membrane of Madin-Darby canine kidney cells. The cells were infected with fowl plague or influenza N virus. 4 h after infection, the hemagglutinin (HA) spike glycoprotein of the virus was present in the apical plasma membrane of the cells. Lipid vesicles containing egg phosphatidylcholine, cholesterol, and an HA receptor (ganglioside) were then bound to the cells at 0 degrees C. More than 85% of the vesicles were released by external neuraminidase at 0 degrees C or by simply warming the cells to 37 degrees C for 10 s, probably because of the action of the viral neuraminidase at the cell surface. However, when the cells were warmed to 37 degrees C in a pH 5.3 medium for 30 s, 50% of the bound vesicles could no longer be released by external neuraminidase. This only occurred when the HA protein had been cleaved into its HA1 and HA2 subunits. When we used influenza N virus, whose HA is not cleaved in Madin-Darby canine kidney cells, cleavage with external trypsin was required. The fact that the HA protein has fusogenic properties at low pH only in its cleaved form suggests that fusion of the vesicles with the plasma membrane had taken place. Further confirmation for fusion was obtained using an assay based on the decrease of energy transfer between two fluorescent phospholipids in a vesicle upon fusion of the vesicle with the plasma membrane (Struck, D. K., D. Hoekstra, and R. E. Pagano. 1981. Biochemistry, 20:4093-4099).

Polarity of endogenous and exogenous glycosyl-phosphatidylinositol-anchored membrane proteins in Madin-Darby canine kidney cells

1990 ◽  
Vol 96 (1) ◽  
pp. 143-149
Author(s):  
J.M. Wilson ◽  
N. Fasel ◽  
J.P. Kraehenbuhl
Keyword(s):  
Plasma Membrane ◽  
Common Mechanism ◽  
Apical Plasma Membrane ◽  
Kidney Cells ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Transfected Cells ◽  
Glycosyl Phosphatidylinositol ◽  
Darby Canine Kidney ◽  
Canine Kidney

Madin-Darby canine kidney cells (MDCK) were transfected with a cDNA encoding the glycosyl-phosphatidylinositol (GPI)-anchored protein mouse Thy-1 in order to study the steady-state surface distribution of exogenous and endogenous GPI-linked proteins. Immunofluorescence of transfected cells grown on collagen-coated coverslips showed that expression of Thy-1 was variable throughout the epithelium, with some cells expressing large amounts of Thy-1 adjacent to very faintly staining cells. Selective surface iodination of cells grown on collagen-coated or uncoated transwell filters followed by immunoprecipitation of Thy-1 demonstrated that all the Thy-1 was present exclusively in the apical plasma membrane. Although cells grown on uncoated filters had much smaller amounts of Thy-1, it was consistently localized on the apical surfaces. Immunofluorescent localization of Thy-1 on 1 micron frozen sections of filter-grown cells demonstrated that all the Thy-1 was on the apical surface and there was no detectable intracellular pool. Phosphatidylinositol-specific phospholipase C digestion of intact iodinated monolayers released Thy-1 only into the apical medium, indicating that Thy-1 was processed normally in transfected cells and was anchored by a GPI-tail. In agreement with previous findings, endogenous GPI-linked proteins were found only on the apical plasma membrane. These results suggest that there is a common mechanism for sorting and targeting of GPI-linked proteins in polarized epithelial cells.

scholarly journals Polarized delivery of viral glycoproteins to the apical and basolateral plasma membranes of Madin-Darby canine kidney cells infected with temperature-sensitive viruses.

1985 ◽  
Vol 100 (1) ◽  
pp. 136-151 ◽  
Author(s):  
M J Rindler ◽  
I E Ivanov ◽  
H Plesken ◽  
D D Sabatini
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Plasma Membranes ◽  
Temperature Sensitive ◽  
Kidney Cells ◽  
Apical Surface ◽  
Nonpermissive Temperature ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

The intracellular route followed by viral envelope glycoproteins in polarized Madin-Darby canine kidney cells was studied by using temperature-sensitive mutants of vesicular stomatitis virus (VSV) and influenza, in which, at the nonpermissive temperature (39.5 degrees C), the newly synthesized glycoproteins (G proteins) and hemagglutinin (HA), respectively, are not transported out of the endoplasmic reticulum. After infection with VSV and incubation at 39.5 degrees C for 4-5 h, synchronous transfer of G protein to the plasma membrane was initiated by shifting to the permissive temperature (32.5 degrees C). Immunoelectron microscopy showed that under these conditions the protein moved to the Golgi apparatus and from there directly to a region of the lateral plasma membrane near this organelle. G protein then seemed to diffuse progressively to basal regions of the cell surface and, only after it had accumulated in the basolateral domain, it began to appear on the apical surface near the intercellular junctions. The results of these experiments indicate that the VSV G protein must be sorted before its arrival at the cell surface, and suggest that passage to the apical domain occurs only late in infection when tight junctions are no longer an effective barrier. In complementary experiments, using the temperature-sensitive mutant of influenza, cultures were first shifted from the nonpermissive temperature (39.5 degrees C) to 18.5 degrees C, to allow entrance of the glycoprotein into the Golgi apparatus (see Matlin, K.S., and K. Simons, 1983, Cell, 34:233-243). Under these conditions HA accumulated in Golgi stacks and vesicles but did not reach the plasma membrane. When the temperature was subsequently shifted to 32.5 degrees C, HA rapidly appeared in discrete regions of the apical surface near, and often directly above, the Golgi elements, and later diffused throughout this surface. To ensure that the anti-HA antibodies had access to lateral domains, monolayers were treated with a hypertonic medium to dilate the intercellular spaces. Some labeling was then observed in the lateral plasma membranes soon after the shift, but this never increased beyond 1.0 gold particle/micron, whereas characteristic densities of labeling in apical surfaces soon became much higher (approximately 10 particles/micron). Our results suggest that the bulk of HA follows a direct pathway leading from the Golgi to regions of the apical surface close to trans-Golgi cisternae.

scholarly journals Molecular characterization of gp40, a mucin-type glycoprotein from the apical plasma membrane of Madin–Darby canine kidney cells (type I)

1997 ◽  
Vol 326 (1) ◽  
pp. 99-108 ◽  
Author(s):  
Gert ZIMMER ◽  
Friedrich LOTTSPEICH ◽  
Andrea MAISNER ◽  
Hans-Dieter KLENK ◽  
Georg HERRLER
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Apical Plasma Membrane ◽  
Type I ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
A Cell ◽  
Polarized Transport ◽  
Darby Canine Kidney ◽  
Canine Kidney

gp40 has been recently identified as a major apical cell-surface sialoglycoprotein of type-I Madin–Darby canine kidney cells, a cell line widely used for the study of polarized transport. The determination of two internal amino acid sequences of the purified glycoprotein by Edman degradation enabled us to isolate the cDNA encoding the 18.6 kDa protein backbone of gp40. Sequence analysis revealed that gp40 is a type-I membrane protein which has several characteristics in common with glycophorin A and other mucin-type glycoproteins. At least 14 serine/threonine residues were found to be used for O-glycosylation. No potential sites for N-glycosylation were detected. gp40 turned out to represent the canine homologue of a cell-surface antigen expressed by various epithelial and non-epithelial cells in rat and mouse. Potential O-glycosylation sites, transmembrane and cytoplasmic domains were found to be highly conserved in the three species. gp40 was detected in canine lung, intestine, kidney, brain and heart but not in liver and spleen. The subline II of Madin–Darby canine kidney cells was found not to express gp40. Stable expression of gp40 in transfected type-II cells revealed that gp40 is predominantly delivered to the apical plasma membrane. N-Glycans and a glycosylphosphatidylinositol anchor, both proposed apical targeting signals, are absent from gp40, indicating that other determinants are responsible for its polarized transport.

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.

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