scholarly journals Microtubules are involved in the secretion of proteins at the apical cell surface of the polarized epithelial cell, Madin-Darby canine kidney

1989 ◽  
Vol 264 (28) ◽  
pp. 16837-16846
Author(s):  
K Parczyk ◽  
W Haase ◽  
C Kondor-Koch
Keyword(s):  
Epithelial Cell ◽  
Cell Surface ◽  
Apical Cell ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

scholarly journals Inhibition by brefeldin A of protein secretion from the apical cell surface of Madin-Darby canine kidney cells.

1991 ◽  
Vol 266 (27) ◽  
pp. 17729-17732 ◽  
Author(s):  
S.H. Low ◽  
S.H. Wong ◽  
B.L. Tang ◽  
P. Tan ◽  
V.N. Subramaniam ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein Secretion ◽  
Apical Cell ◽  
Brefeldin A ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

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 Jaagsiekte Sheep Retrovirus Transformation in Madin-Darby Canine Kidney Epithelial Cell Three-Dimensional Culture

2010 ◽  
Vol 84 (10) ◽  
pp. 5379-5390 ◽  
Author(s):  
Chassidy Johnson ◽  
Kiah Sanders ◽  
Hung Fan
Keyword(s):  
Epithelial Cell ◽  
Mdck Cells ◽  
Mapk Pathway ◽  
Three Dimensional ◽  
Bronchioloalveolar Carcinoma ◽  
Envelope Gene ◽  
Madin Darby Canine Kidney ◽  
Jaagsiekte Sheep Retrovirus ◽  
Darby Canine Kidney ◽  
Canine Kidney

ABSTRACT Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a contagious lung cancer in sheep that shares similarities with human bronchioloalveolar carcinoma (BAC). JSRV is unique because the envelope gene (env) is the oncogene, as it can transform cells in culture and induce tumors in animals. The phosphatidylinositol 3-kinase (PI3K)-Akt-mTOR and H/N-Ras-MEK-mitogen-activated protein kinase (MAPK) pathways have been shown to be critical for Env transformation. However, the question still remains of how disruption of these pathways relates to tumor formation. To address this, JSRV Env transformation was studied in the context of epithelial structure, using the polarized Madin-Darby canine kidney (MDCK) epithelial cell three-dimensional (3-D) culture system. The results indicated that JSRV Env-transformed MDCK cells were larger and had full or multiple lumens, in contrast to the single lumens observed in controls. The altered phenotype was largely mediated by an increase in proliferation, in addition to overcoming the proliferative suppression signal. JSRV Env was not found to disrupt polarity or tight junctions or to inhibit lumen apoptosis. The PI3K-Akt-mTOR pathway was important for Env transformation in MDCK cells, although the mechanisms of action differed in 3-D and monolayer cultures. PI3K-dependent signaling to mTOR occurred in monolayers, while PI3K-independent signaling to mTOR occurred in 3-D culture. In contrast, the H/N-Ras-MEK-MAPK pathway was found to be inhibitory to transformation in both normal and transformed MDCK cells in 3-D culture. However, in monolayer culture, inhibition of MEK reverted the transformed phenotype, suggesting a different mechanism(s) of action in monolayer versus 3-D culture.

Expression of proximal tubular Na-Pi and Na-SO4 cotransporters in MDCK and LLC-PK1 cells by transfection

1996 ◽  
Vol 270 (1) ◽  
pp. F220-F228 ◽  
Author(s):  
E. S. Quabius ◽  
H. Murer ◽  
J. Biber
Keyword(s):  
Mdck Cells ◽  
Apical Cell ◽  
Madin Darby Canine Kidney ◽  
Sodium Dependent ◽  
Proximal Tubular ◽  
Polymerase Chain ◽  
Suitable System ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
Cotransport Systems

Two cD-NAs coding for proximal tubular Na-Pi cotransport (NaPi-2) and Na-SO4 cotransport (NaSi-1) have been transfected by the use of a dexamethasone-inducible vector (pLK-neo) into MDCK and LLC-PK1 cells. By reverse transcription-polymerase chain reaction, expression of corresponding mRNAs was observed after stimulation with dexamethasone only. Similarly, expression of the NaPi-2 protein was detected only after induction with dexamethasone. In transfected Madin-Darby canine kidney (MDCK) cells, dexamethasone induced a large increase of Na-Pi or Na-SO4 cotransport, whereas, in transfected LLC-PK1, cell transport was only minimally expressed. In MDCK cells grown on filter supports, transfected Na-Pi-cotransport activity was equally expressed at both cell surfaces; dual location of expressed NaPi-2 protein was also observed by immunohistochemistry. In contrast, transfected Na-SO4 cotransport activity was predominantly expressed at the apical cell surface of MDCK cells. The results demonstrate that 1), in MDCK cells, the sorting behavior of two proximal tubular cotransport systems seems to be different: apical for Na-SO4 cotransport (NaSi-1) and dual location for Na-Pi cotransport (NaPi-2); and 2) LLC-PK1 cells seem not to be a suitable system to functionally express sodium-dependent cotransport systems for phosphate and sulfate.

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.

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