scholarly journals Actin Is Required for Endocytosis at the Apical Surface of Madin-Darby Canine Kidney Cells where ARF6 and Clathrin Regulate the Actin Cytoskeleton

2006 ◽  
Vol 17 (1) ◽  
pp. 427-437 ◽  
Author(s):  
Tehila Hyman ◽  
Miri Shmuel ◽  
Yoram Altschuler
Keyword(s):  
Bound State ◽  
Cell Cortex ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Epithelial Surface ◽  
Biochemical Assays ◽  
Epithelial Cell Lines ◽  
Darby Canine Kidney ◽  
Canine Kidney

In epithelial cell lines, apical but not basolateral clathrin-mediated endocytosis has been shown to be affected by actin-disrupting drugs. Using electron and fluorescence microscopy, as well as biochemical assays, we show that the amount of actin dedicated to endocytosis is limiting at the apical surface of epithelia. In part, this contributes to the low basal rate of clathrin-dependent endocytosis observed at this epithelial surface. ARF6 in its GTP-bound state triggers the recruitment of actin from the cell cortex to the clathrin-coated pit to enable dynamin-dependent endocytosis. In addition, we show that perturbation of the apical endocytic system by expression of a clathrin heavy-chain mutant results in the collapse of microvilli. This phenotype was completely reversed by the expression of an ARF6-GTP-locked mutant. These observations indicate that concomitant to actin recruitment, the apical clathrin endocytic system is deeply involved in the morphology of the apical plasma membrane.

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 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.

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 Depletion of E-Cadherin Disrupts Establishment but Not Maintenance of Cell Junctions in Madin-Darby Canine Kidney Epithelial Cells

2007 ◽  
Vol 18 (1) ◽  
pp. 189-200 ◽  
Author(s):  
Christopher T. Capaldo ◽  
Ian G. Macara
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Morphological Changes ◽  
Cell Junctions ◽  
Cell Cortex ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
E Cadherin

E-cadherin forms calcium-dependent homophilic intercellular adhesions between epithelial cells. These contacts regulate multiple aspects of cell behavior, including the organization of intercellular tight junctions (TJs). To distinguish between the roles of E-cadherin in formation versus maintenance of junctions, Madin-Darby canine kidney (MDCK) cells were depleted of E-cadherin by RNA interference. Surprisingly, reducing E-cadherin expression had little effect on the protein levels or localization of adherens junction (AJ) or TJ markers. The cells underwent morphological changes, as the normally flat apical surface swelled into a dome. However, apical–basal polarity was not compromised, transmembrane resistance was normal, and zonula occludin protein 1 dynamics at the TJs were unchanged. Additionally, an E-cadherin/Cadherin-6 double knockdown also failed to disrupt established TJs, although β-catenin was lost from the cell cortex. Nevertheless, cells depleted of E-cadherin failed to properly reestablish cell polarity after junction disassembly. Recovery of cell–cell adhesion, transepithelial resistance, and the localization of TJ and AJ markers were all delayed. In contrast, depletion of α-catenin caused long-term disruption of junctions. These results indicate that E-cadherin and Cadherin-6 function as a scaffold for the construction of polarized structures, and they become largely dispensable in mature junctions, whereas α-catenin is essential for the maintenance of functional junctions.

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 GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells

2006 ◽  
Vol 172 (7) ◽  
pp. 1023-1034 ◽  
Author(s):  
Simona Paladino ◽  
Thomas Pocard ◽  
Maria Agata Catino ◽  
Chiara Zurzolo
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Biochemical Assays ◽  
Intracellular Sorting ◽  
Apical Sorting ◽  
Golgi Network ◽  
Darby Canine Kidney

The polarity of epithelial cells is dependent on their ability to target proteins and lipids in a directional fashion. The trans-Golgi network, the endosomal compartment, and the plasma membrane act as sorting stations for proteins and lipids. The site of intracellular sorting and pathways used for the apical delivery of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are largely unclear. Using biochemical assays and confocal and video microscopy in living cells, we show that newly synthesized GPI-APs are directly delivered to the apical surface of fully polarized Madin–Darby canine kidney cells. Impairment of basolateral membrane fusion by treatment with tannic acid does not affect the direct apical delivery of GPI-APs, but it does affect the organization of tight junctions and the integrity of the monolayer. Our data clearly demonstrate that GPI-APs are directly sorted to the apical surface without passing through the basolateral membrane. They also reinforce the hypothesis that apical sorting of GPI-APs occurs intracellularly before arrival at the plasma membrane.

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 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 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.

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