scholarly journals Mammalian Homolog of Drosophila Tumor Suppressor Lethal (2) Giant Larvae Interacts with Basolateral Exocytic Machinery in Madin-Darby Canine Kidney Cells

2002 ◽  
Vol 13 (1) ◽  
pp. 158-168 ◽  
Author(s):  
Anne Mu∸sch ◽  
David Cohen ◽  
Charles Yeaman ◽  
W. James Nelson ◽  
Enrique Rodriguez-Boulan ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Tumor Suppressor ◽  
Cell Polarity ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Epithelial Cell Line ◽  
Madin Darby Canine Kidney ◽  
Late Step ◽  
Darby Canine Kidney ◽  
Canine Kidney

The Drosophila tumor suppressor protein lethal (2) giant larvae [l(2)gl] is involved in the establishment of epithelial cell polarity during development. Recently, a yeast homolog of the protein has been shown to interact with components of the post-Golgi exocytic machinery and to regulate a late step in protein secretion. Herein, we characterize a mammalian homolog of l(2)gl, called Mlgl, in the epithelial cell line Madin-Darby canine kidney (MDCK). Consistent with a role in cell polarity, Mlgl redistributes from a cytoplasmic localization to the lateral membrane after contact-naive MDCK cells make cell-cell contacts and establish a polarized phenotype. Phosphorylation within a highly conserved region of Mlgl is required to restrict the protein to the lateral domain, because a recombinant phospho-mutant is distributed in a nonpolar manner. Membrane-bound Mlgl from MDCK cell lysates was coimmunoprecipitated with syntaxin 4, a component of the exocytic machinery at the basolateral membrane, but not with other plasma membrane solubleN-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins that are either absent from or not restricted to the basolateral membrane domain. These data suggest that Mlgl contributes to apico-basolateral polarity by regulating basolateral exocytosis.

scholarly journals Association of Rab25 and Rab11a with the Apical Recycling System of Polarized Madin–Darby Canine Kidney Cells

1999 ◽  
Vol 10 (1) ◽  
pp. 47-61 ◽  
Author(s):  
James E. Casanova ◽  
Xiaoye Wang ◽  
Ravindra Kumar ◽  
Sheela G. Bhartur ◽  
Jennifer Navarre ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Basolateral Membrane ◽  
Dominant Negative ◽  
Epithelial Cell Line ◽  
Madin Darby Canine Kidney ◽  
Recycling Endosome ◽  
Recycling System ◽  
Endosomal Compartment ◽  
Darby Canine Kidney ◽  
Canine Kidney

Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin–Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.

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 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 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.

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 A basolateral lactate/H+ co-transporter in Madin-Darby Canine Kidney (MDCK) cells

1993 ◽  
Vol 289 (1) ◽  
pp. 263-268 ◽  
Author(s):  
S O Rosenberg ◽  
T Fadil ◽  
V L Schuster
Keyword(s):  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Ionic Diffusion ◽  
Madin Darby Canine Kidney ◽  
Apical Compartment ◽  
Disulphonic Acid ◽  
Component Carrier ◽  
Darby Canine Kidney ◽  
Canine Kidney

Monolayers of Madin-Darby Canine Kidney (MDCK) cells grown on permeable filters generated lactate aerobically and accumulated it preferentially in the basolateral compartment, suggesting the presence of a lactate carrier. The mechanism of lactate transport across apical and basolateral membranes was examined by determining intracellular pH (pHi) microspectrofluorimetrically after addition of lactate to the extracellular solutions and by measuring uptake of [14C]lactate. Addition of 20 mM lactate to the apical compartment produced no change in pHi, whereas lactate added to the basolateral compartment rapidly and reversibly lowered pHi. Pyruvate produced similar results. Inhibitors of lactate/H+ co-transporters, alpha-cyano-4-hydroxycinnamate (CnCN) and quercetin, partially inhibited the fall in pHi produced by basolateral lactate. In contrast, the disulphonic stilbene. DIDS (4,4′-di-isothiocyanostilbene-2,2′-disulphonic acid) produced no inhibition at 0.5 mM. Kinetic analysis was performed by applying basolateral lactate at various concentrations and measuring the rate of entry (delta pHi/min) in the presence and absence of CnCN. Lactate flux was shown to occur by both non-ionic diffusion and a alpha-cyano-4-hydroxycinnamate-sensitive component (carrier). The latter has a Km of approximately 7 mM for the lactate anion. Propionate, but not formate, lowered pHi to the same degree as did equimolar lactate, but the propionate effect was not inhibited by CnCN. Influx of [14C]lactate was substantially greater across the basolateral membrane than across the apical membrane and occurred in the absence of Na+. We conclude that MDCK cells grown on permeable filters generate lactate aerobically and transport it across the basolateral membrane by way of a lactate/H+ cotransporter.

scholarly journals Polarized endocytosis by Madin-Darby canine kidney cells transfected with functional chicken liver glycoprotein receptor.

1989 ◽  
Vol 109 (6) ◽  
pp. 2809-2816 ◽  
Author(s):  
L Graeve ◽  
K Drickamer ◽  
E Rodriguez-Boulan
Keyword(s):  
Mdck Cells ◽  
Basolateral Membrane ◽  
Cell Surface Receptor ◽  
Madin Darby Canine Kidney ◽  
Surface Receptors ◽  
Surface Receptor ◽  
The Stability ◽  
Darby Canine Kidney ◽  
Canine Kidney

We have studied the expression of the chicken hepatic glycoprotein receptor (chicken hepatic lectin [CHL]) in Madin-Darby canine kidney (MDCK) cells, by transfection of its cDNA under the control of a retroviral promotor. Transfected cell lines stably express 87,000 surface receptors/cell with a kd = 13 nM. In confluent monolayers, approximately 40% of CHL is localized at the plasma membrane. 98% of the surface CHL is expressed at the basolateral surface where it performs polarized endocytosis and degradation of glycoproteins carrying terminal N-acetylglucosamine at a rate of 50,000 ligand molecules/h. Studies of the half-life of metabolically labeled receptor and of the stability of biotinylated cell surface receptor after internalization indicate that transfected CHL performs several rounds of uptake and recycling before it gets degraded. The successful expression of a functional basolateral receptor in MDCK cells opens the way for the characterization of the mechanisms that control targeting and recycling of proteins to the basolateral membrane of epithelial cells.

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