Lipids that determine detergent resistance of MDCK cell membrane fractions

doi:10.1016/j.chemphyslip.2015.08.011

Chemistry and Physics of Lipids

Volume 191, October 2015, Pages 68-74

https://doi.org/10.1016/j.chemphyslip.2015.08.011 Get rights and content

Highlights

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A lipidomic study of mammalian detergent-resistant membranes (DRM) from MDCK cells has been performed.
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DRM are enriched in sphingomyelins, ceramides and diacylglycerols with respect to the total cell lipids.
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DRM are enriched in saturated over unsaturated acyl chains, and in sn-1 acyl chains containing 16 carbon atoms.
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Phosphatidylethanolamines and phosphatidylinositols, that were mainly unsaturated, did not show a preference for DRM.

Abstract

A comparative lipidomic study has been performed of whole Madin-Darby canine kidney epithelial cells and of the detergent-resistant membrane fraction (DRM) obtained after treating the cells with the non-ionic detergent Triton X-100. The DRM were isolated following a standard procedure that is extensively used in cell biology studies. Significant differences were found in the lipid composition of the whole cells and of DRM. The latter were enriched in all the analyzed sphingolipid classes: sphingomyelins, ceramides and hexosylceramides. Diacylglycerols were also preferentially found in DRM. The detergent-resistant fraction was also enriched in saturated over unsaturated fatty acyl chains, and in sn-1 acyl chains containing 16 carbon atoms, over the longer and shorter ones. The glycerophospholipid species phosphatidylethanolamines and phosphatidylinositols, that were mainly unsaturated, did not show a preference for DRM. Phosphatidylcholines were an intermediate case: the saturated, but not the unsaturated species were found preferentially in DRM. The question remains on whether these DRM, recovered from detergent-membrane mixtures by floatation over a sucrose gradient, really correspond to membrane domains existing in the cell membrane prior to detergent treatment.

Introduction

The biological membrane matrix consists of many different lipids organized in a bilayer form, whose properties depend on the specific composition (Goñi, 2014). The most frequently found lipids in mammalian membranes are glycerophospholipids, but sphingolipids and cholesterol are also abundant. The specific combination of these lipids can change depending on the cell type, organelles, and physiological state of the cell, so the physical properties of each membrane can vary as well. Detergents are essential tools in membrane research, and their activity is intimately linked to the membrane lipid structure (Helenius and Simons, 1975, Heerklotz, 2008, Lichtenberg et al., 2013a, Lichtenberg et al., 2013b). An early observation of cell treatments with detergents was that a non-solubilized membrane fraction was usually obtained under most conditions (Yu et al., 1973, Gurtubay et al., 1980).

Following a different line of thought, Simons proposed the existence of membrane microdomains called “lipid rafts”, characterized by a higher degree of order than the surrounding membrane, enriched in sphingolipids and cholesterol, and with important cellular functions (Simons and Ikonen, 1997, Wymann and Simons, 2013). Due to an unfortunate chain of flawed reasonings, lipid rafts were identified with non-solubilized membrane fractions (“detergent-resistant membranes”, DRM), and often remain so in spite of clear physical evidence against (Sot et al., 2002, Sot et al., 2014, Lichtenberg et al., 2005).

Leaving aside the controversy on the relationship between DRM and lipid rafts, DRM remain an interesting object of study by themselves, that can help to unravel the still mysterious mechanism of membrane solubilization by detergents. There are many indications that solubilization is highly influenced by membrane lipid composition (Urbaneja et al., 1987, Ahyayauch et al., 2006), but no detailed studies are available of lipid distribution in the solubilized and non-solubilized fractions. The novel lipidomic techniques (Merrill et al., 2013, Ivanova et al., 2007, Frisz et al., 2013, Baker et al., 2014, van Meer et al., 2007) allow such an investigation. In the present contribution we report on the comparative lipidomic study of the total and DRM fractions of Madin-Darby canine kidney epithelial cells (MDCK). Both fractions have been prepared according to a standard procedure (Lingwood and Simons, 2007), extensively used in cell biology studies. A preliminary investigation of this system was published by Schuck et al. (2003) when lipidomic techniques were less developed than in our day.

Section snippets

Materials

Triton X-100 (regular, batch 48H0208) was from Sigma (St. Louis, MO). Dulbecco’s Modified Eagle’s medium (DMEM) was obtained from Biowest SAS (Nuaillé, France). Fetal bovine serum (FBS) was from LONZA Biologics (Slough, UK); Glutamine/streptomycin/penicillin was from GIBCO (Carlsbad, CA, USA).

Cell culture and DRM isolation

Madin-Darby canine kidney epithelial cells (MDCK) were grown at 37 °C in DMEM supplemented with 10% FBS and penicillin–streptomycin–glutamine. Detergent-resistant membranes (DRM) were obtained as described (

Results

Detergent-resistant membranes (DRM) were isolated from MDCK cells following an established procedure (Lingwood and Simons, 2007). Protein in the DRM was 7.4% of total cell protein. The lipidomic method does not allow an absolute quantification of the various lipid species, instead a semi-quantitative comparison of the lipid concentrations in DRM and in whole cells was achieved. When required, the sum of the normalized areas under the chromatographic peaks of each metabolite provided data

Discussion

The amazing amount of data provided by lipidomics forces us to present here but a short summary of our results. MDCK cell membrane fractions resistant to solubilization are clearly enriched in sphingolipids, SM, Cer, and CMH, as well as in DAG (Fig. 1). Other studies that compared the lipidome of MDCK apical membrane, whole-cell membranes and the influenza virus envelope also described an increase in sphingolipids (Cer and SM) and a decrease in glycerophospholipids (PI, PC and PE) in apical

Acknowledgements

This work was supported in part by BFU 2012-36241, from the Spanish Ministry of Economy, and by grant IT 849-13 from the Basque Government. M.M. was a predoctoral student supported by the Basque Government.

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Lipids that determine detergent resistance of MDCK cell membrane fractions

Highlights

Abstract

Introduction

Section snippets

Materials

Cell culture and DRM isolation

Results

Discussion

Acknowledgements

Biochim. Biophys. Acta

FEBS Lett.

J. Lipid Res.

J. Biol. Chem.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Methods Enzymol.

Trends Biochem. Sci.

Trends Biochem. Sci.

Biophys. J.

Neurosci. Lett.

Adv. Nutr.

Biochemistry

J. Liposome Res.

Chem. Phys. Lipids

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

Biochim. Biophys. Acta

FEBS J.