scholarly journals Selective Alterations in Biosynthetic and Endocytic Protein Traffic in Madin-Darby Canine Kidney Epithelial Cells Expressing Mutants of the Small GTPase Rac1

2000 ◽  
Vol 11 (1) ◽  
pp. 287-304 ◽  
Author(s):  
Tzuu-Shuh Jou ◽  
Som-Ming Leung ◽  
Linette M. Fung ◽  
Wily G. Ruiz ◽  
W. James Nelson ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Membrane Traffic ◽  
Small Gtpase ◽  
Apical Plasma Membrane ◽  
Membrane Domains ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

Madin-Darby canine kidney (MDCK) cells expressing constitutively active Rac1 (Rac1V12) accumulate a large central aggregate of membranes beneath the apical membrane that contains filamentous actin, Rac1V12, rab11, and the resident apical membrane protein GP-135. To examine the roles of Rac1 in membrane traffic and the formation of this aggregate, we analyzed endocytic and biosynthetic trafficking pathways in MDCK cells expressing Rac1V12 and dominant inactive Rac1 (Rac1N17). Rac1V12 expression decreased the rates of apical and basolateral endocytosis, whereas Rac1N17 expression increased those rates from both membrane domains. Basolateral-to-apical transcytosis of immunoglobulin A (IgA) (a ligand for the polymeric immunoglobulin receptor [pIgR]), apical recycling of pIgR-IgA, and accumulation of newly synthesized GP-135 at the apical plasma membrane were all decreased in cells expressing Rac1V12. These effects of Rac1V12 on trafficking pathways to the apical membrane were the result of the delivery and trapping of these proteins in the central aggregate. In contrast to abnormalities in apical trafficking events, basolateral recycling of transferrin, degradation of EGF internalized from the basolateral membrane, and delivery of newly synthesized pIgR from the Golgi to the basolateral membrane were all relatively unaffected by Rac1V12 expression. Rac1N17 expression had little or no effect on these postendocytic or biosynthetic trafficking pathways. These results show that in polarized MDCK cells activated Rac1 may regulate the rate of endocytosis from both membrane domains and that expression of dominant active Rac1V12 specifically alters postendocytic and biosynthetic membrane traffic directed to the apical, but not the basolateral, membrane.

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 Modulation of Endocytic Traffic in Polarized Madin-Darby Canine Kidney Cells by the Small GTPase RhoA

1999 ◽  
Vol 10 (12) ◽  
pp. 4369-4384 ◽  
Author(s):  
Som-Ming Leung ◽  
Raul Rojas ◽  
Christopher Maples ◽  
Christopher Flynn ◽  
Wily G. Ruiz ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Immunoglobulin A ◽  
Small Gtpase ◽  
Membrane Domains ◽  
Madin Darby Canine Kidney ◽  
Immunoglobulin Receptor ◽  
Early Endosomes ◽  
Biochemical Analyses ◽  
Darby Canine Kidney ◽  
Canine Kidney

Efficient postendocytic membrane traffic in polarized epithelial cells is thought to be regulated in part by the actin cytoskeleton. RhoA modulates assemblies of actin in the cell, and it has been shown to regulate pinocytosis and phagocytosis; however, its effects on postendocytic traffic are largely unexplored. To this end, we expressed wild-type RhoA (RhoAWT), dominant active RhoA (RhoAV14), and dominant inactive RhoA (RhoAN19) in Madin-Darby canine kidney (MDCK) cells expressing the polymeric immunoglobulin receptor. RhoAV14 expression stimulated the rate of apical and basolateral endocytosis, whereas RhoAN19 expression decreased the rate from both membrane domains. Polarized basolateral recycling of transferrin was disrupted in RhoAV14-expressing cells as a result of increased ligand release at the apical pole of the cell. Degradation of basolaterally internalized epidermal growth factor was slowed in RhoAV14-expressing cells. Although apical recycling of immunoglobulin A (IgA) was largely unaffected in cells expressing RhoAV14, transcytosis of basolaterally internalized IgA was severely impaired. Morphological and biochemical analyses demonstrated that a large proportion of IgA internalized from the basolateral pole of RhoAV14-expressing cells remained within basolateral early endosomes and was slow to exit these compartments. RhoAN19 and RhoAWT expression had little effect on these postendocytic pathways. These results indicate that in polarized MDCK cells activated RhoA may modulate endocytosis from both membrane domains and postendocytic traffic at the basolateral pole of the cell.

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 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.

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 FAPP2, cilium formation, and compartmentalization of the apical membrane in polarized Madin-Darby canine kidney (MDCK) cells

2006 ◽  
Vol 103 (49) ◽  
pp. 18556-18561 ◽  
Author(s):  
O. V. Vieira ◽  
K. Gaus ◽  
P. Verkade ◽  
J. Fullekrug ◽  
W. L. C. Vaz ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Mdck Cells ◽  
Madin Darby Canine Kidney ◽  
Cilium Formation ◽  
Darby Canine Kidney ◽  
Canine Kidney

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 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.

scholarly journals Selective Perturbation of Apical Membrane Traffic by Expression of Influenza M2, an Acid-activated Ion Channel, in Polarized Madin–Darby Canine Kidney Cells

1998 ◽  
Vol 9 (9) ◽  
pp. 2477-2490 ◽  
Author(s):  
Jennifer R. Henkel ◽  
Gerard Apodaca ◽  
Yoram Altschuler ◽  
Stephen Hardy ◽  
Ora A. Weisz
Keyword(s):  
Ion Channel ◽  
Mdck Cells ◽  
Endocytic Pathway ◽  
Apical Plasma Membrane ◽  
Madin Darby Canine Kidney ◽  
Recycling Endosome ◽  
Trans Golgi Network ◽  
Golgi Network ◽  
Darby Canine Kidney ◽  
Canine Kidney

The function of acidification along the endocytic pathway is not well understood, in part because the perturbants used to modify compartmental pH have global effects and in some cases alter cytoplasmic pH. We have used a new approach to study the effect of pH perturbation on postendocytic traffic in polarized Madin–Darby canine kidney (MDCK) cells. Influenza M2 is a small membrane protein that functions as an acid-activated ion channel and can elevate the pH of the trans-Golgi network and endosomes. We used recombinant adenoviruses to express the M2 protein of influenza virus in polarized MDCK cells stably transfected with the polymeric immunoglobulin (Ig) receptor. Using indirect immunofluorescence and immunoelectron microscopy, M2 was found to be concentrated at the apical plasma membrane and in subapical vesicles; intracellular M2 colocalized partly with internalized IgA in apical recycling endosomes as well as with the trans-Golgi network marker TGN-38. Expression of M2 slowed the rate of IgA transcytosis across polarized MDCK monolayers. The delay in transport occurred after IgA reached the apical recycling endosome, consistent with the localization of intracellular M2. Apical recycling of IgA was also slowed in the presence of M2, whereas basolateral recycling of transferrin and degradation of IgA were unaffected. By contrast, ammonium chloride affected both apical IgA and basolateral transferrin release. Together, our data suggest that M2 expression selectively perturbs acidification in compartments involved in apical delivery without disrupting other postendocytic transport steps.

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