Polarized secretion of tyrosine-sulphated proteins and free tyrosine O-sulphate by filter-grown Madin-Darby canine kidney (MDCK) cells.
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ABSTRACT:
SUBMITTER: Han JR
PROVIDER: S-EPMC1151578 | biostudies-other | 1991 Oct
REPOSITORIES: biostudies-other
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Project description:We have analyzed the role of the phosphatidylinositol-4-phosphate adaptor protein-2 (FAPP2), a component of the apical transport machinery, in cilium formation in polarized Madin-Darby canine kidney (MDCK) cells. We show that ciliogenesis is defective in FAPP2 knockdown cells. Furthermore, by using fluorescence recovery after photobleaching studies of domain connectivity and the generalized polarization spectra of Laurdan, we demonstrate that FAPP2 depletion impairs the formation of condensed apical membrane domains. Laurdan staining also revealed that the ciliary membrane has a highly condensed bilayer domain at its base that could function as a fence to separate the ciliary membrane from the surrounding apical membrane. These results indicate that the compartmentalization of the apical membrane in MDCK cells into the ciliary membrane and the surrounding membrane depends on the balance of raft and nonraft domains.
| S-EPMC1693701 | biostudies-literature
Project description:The expression levels of genes involved in drug and nutrient absorption were evaluated in the Madin-Darby Canine Kidney (MDCK) in vitro drug absorption model. MDCK cells were grown on plastic surfaces (for 3 days) or on Transwell® membranes (for 3, 5, 7, and 9 days). The expression profile of genes including ABC transporters, SLC transporters, and cytochrome P450 (CYP) enzymes was determined using the Affymetrix® Canine GeneChip®. Expression of genes whose probe sets passed a stringent confirmation process was examined. Expression of a few transporter (MDR1, PEPT1 and PEPT2) genes in MDCK cells was confirmed by RT-PCR. The overall gene expression profile was strongly influenced by the type of support the cells were grown on. After 3 days of growth, expression of 28% of the genes was statistically different (1.5-fold cutoff, p < 0.05) between the cells grown on plastic and Transwell® membranes. When cells were differentiated on Transwell® membranes, large changes in gene expression profile were observed during the early stages, which then stabilized after 5-7 days. Only a small number of genes encoding drug absorption related SLC, ABC, and CYP were detected in MDCK cells, and most of them exhibited low hybridization signals. Results from this study provide valuable reference information on endogenous gene expression in MDCK cells that could assist in design of drug-transporter and/or drug-enzyme interaction studies, and help interpret the contributions of various transporters and metabolic enzymes in studies with MDCK cells.
| S-EPMC3834914 | biostudies-literature
Project description:The paracellular passage of ions in epithelial systems is dictated by the repertoire of tight junction (TJ) proteins, the number and architecture of apicolateral TJ strands, and the interaction of TJ proteins with the cytoskeleton. To investigate the relative contribution of these factors and to explore their regulation, we examined a passage number dependent phenotypic change in Madin Darby Canine Kidney (MDCK) cells iteratively passaged at low density in which dilution potentials of the monolayer abruptly decreased but transepithelial resistance (TER) progressively increased. Here, we report that the phenotypic transition involved a decrease surface expression of peanut agglutinin (PNA), a decrease in steady state levels of claudin-1 and a slight decrease in E-cadherin expression in the context of an apparently claudin-2-negative system. Late passage cells grew in more densely packed monolayers and exhibited characteristics of a competent, functional cultured monolayer including inducible vectorial ion transport in response to norepinephrine (NE) and sphere formation in three dimensional laminin gel culture. Microarray analysis of gene expression levels in early and late passage cells revealed an increase in transcript abundance for SGK and GLUT-3, and a reduction Id-3. The steady state protein expression levels of GLUT-3, however, were found to be equivalent. These findings suggest that the expression of claudin-2 in MDCK cells could be contingent on signaling via lateral cell contacts and that claudin-1, in the context of sustained claudin-8 expression, could function as a negative regulator of overall TER.
2010-02-13 | GSE14837 | GEO
Project description:Mammalian epithelial cell plasma membrane domains are separated by junctional complexes supported by actin. The extent to which actin acts elsewhere to maintain cell polarity remains poorly understood. Using latrunculin B (Lat B) to depolymerize actin filaments, several basolateral plasma membrane proteins were found to lose their polarized distribution. This loss of polarity did not reflect lateral diffusion through junctional complexes because a low-density lipoprotein receptor mutant lacking a functional endocytosis signal remained basolateral after Lat B treatment. Furthermore, Lat B treatment did not facilitate membrane diffusion across the tight junction as observed with ethylenediaminetetraacetic acid or dimethyl sulfoxide treatment. Detailed analysis of transferrin recycling confirmed Lat B depolarized recycling of transferrin from endosomes to the basolateral surface. Kinetic analysis suggested sorting was compromised at both basolateral early endosomes and perinuclear recycling endosomes. Despite loss of function, these two endosome populations remained distinct from each other and from early endosomes labeled by apically internalized ligand. Furthermore, apical and basolateral early endosomes were functionally distinct populations that directed traffic to a single common recycling endosomal compartment even after Lat B treatment. Thus, filamentous actin may help to guide receptor traffic from endosomes to the basolateral plasma membrane.
| S-EPMC65087 | biostudies-literature
Project description:Madin-Darby canine kidney (MDCK) cells were cultured on polycarbonate filters to study the synthesis and sorting of proteoglycans in polarized epithelial cells. Two strains of MDCK cells were used. MDCK I cells resemble distal tubule epithelial cells, and MDCK II cells share some characteristics with proximal tubule cells. Both strains were grown to confluency and labelled with [35S]sulphate for 24 h. The apical and basolateral media and the cell fractions were harvested and analysed by DEAE ion-exchange chromatography. A large portion of the [35S]sulphate-labelled macromolecules bound strongly to the ion-exchange columns, and could be eluted in three distinct peaks. The latest eluting peak was demonstrated to contain almost exclusively chondroitin sulphate, whereas peak 2 contained mostly heparan sulphate, demonstrated by using chondroitinase ABC and nitrous acid (pH 1.5) respectively to depolymerize the [35S]glycosaminoglycan chains. Peak 1 contained negligible amounts of proteoglycans. Large differences could be observed in proteoglycan sorting in MDCK I and II cells. Strain I secreted approx. 67% of the proteoglycans to the apical side and 17% to the basolateral side. The cell fraction contained 17% of the proteoglycans after 24 h of labelling. In contrast, 19% of the proteoglycans were sorted to the apical side of MDCK II cells and 61% to the basolateral side, whereas the cell fraction contained 20%. Furthermore, the level of [35S]proteoglycan biosynthesis (apical and basolateral media and cell fraction total) was higher in MDCK I cells than in strain II. Based on the amount of material degraded by chondroitinase ABC and nitrous acid respectively, and the total amounts of [35S]proteoglycans recovered from the cells, it was calculated that the MDCK I strain synthesized approx. 56% chondroitin sulphate and 44% heparan sulphate. In contrast, the MDCK II strain synthesized 69% heparan sulphate and 31% chondroitin sulphate. To further identify the [35S]proteoglycans synthesized by MDCK I and II cells, antibodies against perlecan, versican and syndecan were used. The antibody against mouse syndecan did not cross-react with any of the proteoglycans produced in MDCK I or II cells. Both MDCK I and II cells expressed perlecan; 57-61% could be recovered from the basolateral fractions and 18-34% from the apical medium. Versican was also found in both MDCK I and II cells. Compared with perlecan, a larger percentage of versican (43-53%) was found in the cell fractions.
| S-EPMC1136083 | biostudies-other
Project description:Rab10, a protein originally isolated from Madin-Darby Canine Kidney (MDCK) epithelial cells, belongs to a family of Rab proteins that includes Rab8 and Rab13. Although both Rab8 and Rab13 have been found to mediate polarized membrane transport, the function of Rab10 in mammalian cells has not yet been established. We have used quantitative confocal microscopy of polarized MDCK cells expressing GFP chimeras of wild-type and mutant forms of Rab10 to analyze the function of Rab10 in polarized cells. These studies demonstrate that Rab10 is specifically associated with the common endosomes of MDCK cells, accessible to endocytic probes internalized from either the apical or basolateral plasma membrane domains. Expression of mutant Rab10 defective for either GTP hydrolysis or GTP binding increased recycling from early compartments on the basolateral endocytic pathway without affecting recycling from later compartments or the apical recycling pathway. These results suggest that Rab10 mediates transport from basolateral sorting endosomes to common endosomes.
| S-EPMC1483048 | biostudies-literature
Project description:The paracellular passage of ions in epithelial systems is dictated by the repertoire of tight junction (TJ) proteins, the number and architecture of apicolateral TJ strands, and the interaction of TJ proteins with the cytoskeleton. To investigate the relative contribution of these factors and to explore their regulation, we examined a passage number dependent phenotypic change in Madin Darby Canine Kidney (MDCK) cells iteratively passaged at low density in which dilution potentials of the monolayer abruptly decreased but transepithelial resistance (TER) progressively increased. Here, we report that the phenotypic transition involved a decrease surface expression of peanut agglutinin (PNA), a decrease in steady state levels of claudin-1 and a slight decrease in E-cadherin expression in the context of an apparently claudin-2-negative system. Late passage cells grew in more densely packed monolayers and exhibited characteristics of a competent, functional cultured monolayer including inducible vectorial ion transport in response to norepinephrine (NE) and sphere formation in three dimensional laminin gel culture. Microarray analysis of gene expression levels in early and late passage cells revealed an increase in transcript abundance for SGK and GLUT-3, and a reduction Id-3. The steady state protein expression levels of GLUT-3, however, were found to be equivalent. These findings suggest that the expression of claudin-2 in MDCK cells could be contingent on signaling via lateral cell contacts and that claudin-1, in the context of sustained claudin-8 expression, could function as a negative regulator of overall TER. 3 biological replicates of early and late passage were run on Affymetrix chips. the replicates were averaged then compared.
2010-04-09 | E-GEOD-14837 | biostudies-arrayexpress
Project description:By employing an affinity-gel fractionation technique coupled to Western-blot analysis, we have identified a 175 kDa tyrosine-O-sulphate (TyrS)-binding protein present in Madin-Darby canine kidney (MDCK) cells. The binding of this TyrS-binding protein to TyrS covalently bonded to Sepharose gel was found to be pH-dependent, being strong from pH 8.0 down to pH 6.5 and increasingly weak at pH 6.0 and below. Results obtained from Triton X-114 temperature-induced phase separation and sodium carbonate buffer (pH 11) extraction experiments indicated that the TyrS-binding protein is an integral membrane protein. This 175 kDa TyrS-binding protein was found to be present in association with a major tyrosine-sulphated protein, the apically secreted 80 kDa glycoprotein (gp 80), in cell lysate prepared from MDCK cells maintained under normal growth conditions. When the cell lysate used was prepared from MDCK cells pretreated with 20 mM sodium chlorate, a metabolic sulphation inhibitor, the complex formed between the two proteins could no longer be detected, indicating that the binding of the TyrS-binding protein is through the TyrS residue(s) of gp 80. Both cell-surface biotinylation and cell-surface trypsinization studies demonstrated the predominantly, if not exclusively, intracellular location of the TyrS-binding protein. Furthermore, radioactive pulse-chase experiments revealed that the newly synthesized radiolabelled fibronectin and gp 80 were present in complexes with the TyrS-binding protein in MDCK cells pulse-labelled with [35S]methionine or [35S]sulphate. Exogenous [35S]methionine-labelled gp 80 added to the medium, on the other hand, was not found to be present in association with the TyrS-binding protein in MDCK cells over a 2-h time course. These results strongly suggested the identity of the 175 kDa TyrS-binding protein as a putative 'TyrS receptor', possibly functioning in the biosynthetic transport of tyrosine-sulphated proteins in MDCK cells.
| S-EPMC1134469 | biostudies-other
Project description:BackgroundMadin-Darby canine kidney (MDCK) cells are a cellular matrix in the production of influenza vaccines. The proliferation rate of MDCK cells is one of the critical factors that determine the vaccine production cycle. It is yet to be determined if there is a correlation between cell proliferation and alterations in metabolic levels. This study aimed to explore the metabolic differences between MDCK cells with varying proliferative capabilities through the use of both untargeted and targeted metabolomics.MethodsTo investigate the metabolic discrepancies between adherent cell groups (MDCK-M60 and MDCK-CL23) and suspension cell groups (MDCK-XF04 and MDCK-XF06), untargeted and targeted metabolomics were used. Utilizing RT-qPCR analysis, the mRNA expressions of key metabolites enzymes were identified.ResultsAn untargeted metabolomics study demonstrated the presence of 81 metabolites between MDCK-M60 and MDCK-CL23 cells, which were mainly affected by six pathways. An analysis of MDCK-XF04 and MDCK-XF06 cells revealed a total of 113 potential metabolites, the majority of which were impacted by ten pathways. Targeted metabolomics revealed a decrease in the levels of choline, tryptophan, and tyrosine in MDCK-CL23 cells, which was in accordance with the results of untargeted metabolomics. Additionally, MDCK-XF06 cells experienced a decrease in 5'-methylthioadenosine and tryptophan, while S-adenosylhomocysteine, kynurenine, 11Z-eicosenoic acid, 3-phosphoglycerate, glucose 6-phosphate, and phosphoenolpyruvic acid concentrations were increased. The mRNA levels of MAT1A, MAT2B, IDO1, and IDO2 in the two cell groups were all increased, suggesting that S-adenosylmethionine and tryptophan may have a significant role in cell metabolism.ConclusionsThis research examines the effect of metabolite fluctuations on cell proliferation, thus offering a potential way to improve the rate of MDCK cell growth.
| S-EPMC10517658 | biostudies-literature
Project description:Epithelial-mesenchymal transition (EMT) is a highly conserved morphogenic process defined by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype. EMT is associated with increased aggressiveness, invasiveness, and metastatic potential in carcinoma cells. To assess the contribution of extracellular vesicles following EMT, we conducted a proteomic analysis of exosomes released from Madin-Darby canine kidney (MDCK) cells, and MDCK cells transformed with oncogenic H-Ras (21D1 cells). Exosomes are 40-100 nm membranous vesicles originating from the inward budding of late endosomes and multivesicular bodies and are released from cells on fusion of multivesicular bodies with the plasma membrane. Exosomes from MDCK cells (MDCK-Exos) and 21D1 cells (21D1-Exos) were purified from cell culture media using density gradient centrifugation (OptiPrep™), and protein content identified by GeLC-MS/MS proteomic profiling. Both MDCK- and 21D1-Exos populations were morphologically similar by cryo-electron microscopy and contained stereotypical exosome marker proteins such as TSG101, Alix, and CD63. In this study we show that the expression levels of typical EMT hallmark proteins seen in whole cells correlate with those observed in MDCK- and 21D1-Exos, i.e. reduction of characteristic inhibitor of angiogenesis, thrombospondin-1, and epithelial markers E-cadherin, and EpCAM, with a concomitant up-regulation of mesenchymal makers such as vimentin. Further, we reveal that 21D1-Exos are enriched with several proteases (e.g. MMP-1, -14, -19, ADAM-10, and ADAMTS1), and integrins (e.g. ITGB1, ITGA3, and ITGA6) that have been recently implicated in regulating the tumor microenvironment to promote metastatic progression. A salient finding of this study was the unique presence of key transcriptional regulators (e.g. the master transcriptional regulator YBX1) and core splicing complex components (e.g. SF3B1, SF3B3, and SFRS1) in mesenchymal 21D1-Exos. Taken together, our findings reveal that exosomes from Ras-transformed MDCK cells are reprogrammed with factors which may be capable of inducing EMT in recipient cells.
| S-EPMC3734576 | biostudies-literature