Two strains of the Madin-Darby canine kidney (MDCK) cell line have distinct glycosphingolipid compositions.

Hansson, Gunnar C.; Simons, Kai; Meer, Gerrit van

doi:10.1002/j.1460-2075.1986.tb04237.x

Cited by 116 publications

(126 citation statements)

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“…In contrast, all three cell lines label intensely when fluorescent CT-B is applied after fixation and permeabilization with saponin; indicating that the overall amount of ganglioside GM1 per cell is similar (Figure 6a). Madin Darby canine kidney cells, which do not contain ganglioside GM1 (Hansson et al, 1986), do not label with fluorescent CT-B (Figure 6a). These results were quantified using flow cytometry.…”

Section: Hpv-16 E5 Upregulates Plasma Membrane Raft Componentsmentioning

confidence: 99%

HPV-16 E5 oncoprotein upregulates lipid raft components caveolin-1 and ganglioside GM1 at the plasma membrane of cervical cells

et al. 2007

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High-risk human papillomaviruses (HPVs), especially HPV-16, play a primary role in the pathogenesis of cervical cancer. HPV-16 encodes the E5, E6 and E7 oncoproteins. Although the biological functions of E5 are poorly understood, recent studies indicate that its expression correlates with papillomavirus oncogenicity. In this study we demonstrate that the HPV-16 E5 oncoprotein increases plasma membrane expression of caveolin-1, which is a constituent of lipid rafts and regulator of cell signaling, and that this phenotype is mediated by the C-terminal 10 amino acids of E5. Moreover, E5 (but not mutant E5) induces a 23-to 40-fold increase in the lipid raft component, ganglioside GM1, on the cell surface and mediates a dramatic increase in caveolin-1/GM1 association. Since gangliosides strongly inhibit cytotoxic T lymphocytes, block immune synapse formation and are expressed at high levels on the surface of many tumor cells, our results suggest a potential mechanism for immune evasion by the papillomaviruses. Additionally, surface gangliosides are known to enhance proliferative signaling by the epidermal growth factor (EGF) receptor, providing a possible mechanistic basis for observations that EGF signaling is enhanced in E5-expressing cells. Finally, the upregulation of caveolin-1 and ganglioside GM1 at the plasma membrane of E5-expressing cervical cells provides potential new therapeutic targets and diagnostic markers for high-risk HPV infections.

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Section: Hpv-16 E5 Upregulates Plasma Membrane Raft Componentsmentioning

confidence: 99%

HPV-16 E5 oncoprotein upregulates lipid raft components caveolin-1 and ganglioside GM1 at the plasma membrane of cervical cells

et al. 2007

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“…MS analysis of the extracts showed a 10-fold gain in intensity of GSP peaks in comparison with the Folch extract. We identified the specific complex GSP classes of epithelial MDCK cells (19): the ganglioside GM3, galactosylceramide-sulfate (Sulf), and the globoside derivative Forssman glycolipid (For).…”

Section: Resultsmentioning

confidence: 99%

“…These lipid classes, however, are both biologically important and abundant (19,26,27). To amend this deficiency, we first optimized the two-stage lipid extraction.…”

Section: Discussionmentioning

confidence: 99%

Membrane lipidome of an epithelial cell line

Sampaio

Gerl

Klose

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

452

406

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Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology to an epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin to glycosphingolipid, together with an increase in plasmalogen, phosphatidylethanolamine, and cholesterol content, whereas the opposite changes took place during an epithelial-to-mesenchymal transition. Moreover, during polarization, the sphingolipids became longer, more saturated, and more hydroxylated as required to generate an apical membrane domain that serves as a protective barrier for the epithelial sheet.epithelial polarity | shotgun lipidomics | high resolution mass spectrometry | Forssman glycosphingolipid | epithelial-mesenchymal transition G ain and loss of epithelial polarity are fundamental processes in tissue differentiation (1, 2). These processes have been extensively studied not only in the context of embryogenesis but in tumor progression and metastasis (1). Although cell polarization necessarily implies major changes in the structure and properties of the cell membranes, the exact changes in the molecular composition of their membrane lipidome remain unknown.Madin-Darby canine kidney (MDCK) cells are a cell culture model for epithelial polarization (3,4). When seeded at low density, MDCK cells undergo a morphological change from a fibroblast-like morphology to a well-polarized epithelium (5) and have recently also been adapted to undergo an epithelial-mesenchymal transition (EMT) (6). Epithelial cells have evolved characteristic apical and basolateral membrane domains, separated by tight junctions, with specific protein and lipid composition (7,8). The unusual robustness of the apical membrane is largely attributable to its special lipid composition (9-11).In this work, we applied shotgun MS for comprehensive and quantitative characterization of mammalian cellular membranes (12). By combining high mass resolution MS, an improved extraction procedure that increases the recovery of polar lipids (13), and the use of internal standard mixtures for absolute quantification, we expanded the lipid repertoire covered by shotgun lipidomics to glycosphingolipids (GSPs). Importantly, the analysis of the GSPs did not require the hydrolysis of glycerophospholipids (GPs) (14), and this simplification allowed us to determine the membrane lipidome with unprecedented comprehensiveness (more than 300 lipid species from 14 different lipid classes) from a single sample of 10 5 cells.We further elucidated how the lipidome is remodeled during the course of MDCK cell epithelial polarization. We found that when these c...

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“…SNAREs, annexins, Rab GTPases, etc.) and glycosphingolipids (55). Because MDCK type I cells exhibit electrophysiological and morphological properties similar to cells in the collecting duct (56,57), whereas MDCK type II cells partially resemble cells in proximal tubule (56) and thick ascending limb (58), it is conceivable that cAMP relocates CFTR to the apical membrane in cells derived from proximal tubule and/or thick ascending limb but not collecting duct.…”

Section: Table II Comparison Of I Sc In Parental and Gfp-cftr Expressmentioning

confidence: 99%

Membrane Trafficking of the Cystic Fibrosis Gene Product, Cystic Fibrosis Transmembrane Conductance Regulator, Tagged with Green Fluorescent Protein in Madin-Darby Canine Kidney Cells

Moyer¹,

Loffing²,

Schwiebert³

et al. 1998

Journal of Biological Chemistry

152

170

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The mechanism by which cAMP stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride (Cl ؊ ) secretion is cell type-specific. By using Madin-Darby canine kidney (MDCK) type I epithelial cells as a model, we tested the hypothesis that cAMP stimulates Cl ؊ secretion by stimulating CFTR Cl ؊ channel trafficking from an intracellular pool to the apical plasma membrane. To this end, we generated a green fluorescent protein (GFP)-CFTR expression vector in which GFP was linked to the N terminus of CFTR. GFP did not alter CFTR function in whole cell patchclamp or planar lipid bilayer experiments. In stably transfected MDCK type I cells, GFP-CFTR localization was substratum-dependent. In cells grown on glass coverslips, GFP-CFTR was polarized to the basolateral membrane, whereas in cells grown on permeable supports, GFP-CFTR was polarized to the apical membrane. Quantitative confocal fluorescence microscopy and surface biotinylation experiments demonstrated that cAMP did not stimulate detectable GFP-CFTR translocation from an intracellular pool to the apical membrane or regulate GFP-CFTR endocytosis. Disruption of the microtubular cytoskeleton with colchicine did not affect cAMP-stimulated Cl ؊ secretion or GFP-CFTR expression in the apical membrane. We conclude that cAMP stimulates CFTR-mediated Cl ؊ secretion in MDCK type I cells by activating channels resident in the apical plasma membrane.

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Two strains of the Madin-Darby canine kidney (MDCK) cell line have distinct glycosphingolipid compositions.

Cited by 116 publications

References 57 publications

HPV-16 E5 oncoprotein upregulates lipid raft components caveolin-1 and ganglioside GM1 at the plasma membrane of cervical cells

HPV-16 E5 oncoprotein upregulates lipid raft components caveolin-1 and ganglioside GM1 at the plasma membrane of cervical cells

Membrane lipidome of an epithelial cell line

Membrane Trafficking of the Cystic Fibrosis Gene Product, Cystic Fibrosis Transmembrane Conductance Regulator, Tagged with Green Fluorescent Protein in Madin-Darby Canine Kidney Cells

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