Membrane lipidome of an epithelial cell line

Abstract

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...

Phosphatidylethanolamine Is a Key Regulator of Membrane Fluidity in Eukaryotic Cells

“…In contrast, we found that SF9 similarly to HEK 293T cells contain all major PL species, notably PG, PS, and SM, which was described for Drosoph- (13). Nevertheless, the saturation state of acyl chains differed between both studies.…”

Sphingolipid Homeostasis in the Endoplasmic Reticulum and Beyond

“…Mass spectrometry-based methods for sphingolipid analysis have emerged as powerful tools for quantitative measurement of sphingolipid levels (Ejsing et al 2009;Shevchenko and Simons 2010;Wenk 2010) and for identification of proteinsphingolipid interactions Gallego et al 2010;Li et al 2010). In particular, lipidomic analyses can provide a global view of how sphingolipid metabolism adapts to different perturbations or external cues (Guan et al 2009;Breslow et al 2010;Sampaio et al 2011;Klose et al 2012).…”

Comprehensive Lipidome Analysis by Shotgun Lipidomics on a Hybrid Quadrupole-Orbitrap-Linear Ion Trap Mass Spectrometer

“…These analytical hallmarks support absolute quantification of lipid species annotated by sum composition without recourse to tandem mass analysis as required on triple quadrupole machines [6,16,30]. However, FTMS spectral data quality can be compromised by deviating isotopic fidelity (i.e., accuracy of relative isotopic abundances), shifting of accurate masses, and peak coalescence due to space charging effects [35,36], which can bias lipid identification and quantification.…”

“…Lipid species were annotated as previously described [3,6,30]. For example, glycerophospholipid and glycerolipid species annotated by "sum composition" were denoted as: Glipid class9Gtotal number of C in fatty acid moieties9:Gtotal number of double bonds in fatty acid moieties9(e.g., PI 34:1).…”