Ultrastructure and lipid composition of detergent

Authors: Renske A van Gestel Jos F Brouwers Anton Ultee J Bernd Helms Bart M Gadella

Publish Date: 2015/09/16

Volume: 363, Issue: 1, Pages: 129-145

PDF Link

Abstract

Lipid rafts are microdomains of ordered lipids Lo phase in biological membranes The Lo phase of cellular membranes can be isolated from disordered lipids Ld phase after treatment with 1  Triton  X100 at 4 °C in which the Lo phase forms the detergentresistant membrane DRM fraction The lipid composition of DRM derived from MadinDarby canine kidney MDCK cells McArdle cells and porcine sperm is compared with that of the whole cell Remarkably the unsaturation and chain length degree of aliphatic chains attached to phospholipids is virtually the same between DRM and whole cells Cholesterol and sphingomyelin were enriched in DRMs but to a cellspecific molar ratio Sulfatides sphingolipids from MDCK cells were enriched in the DRM while a seminolipid an alkylacylglycerolipid from sperm was depleted from the DRM Treatment with 5 mM methylßcyclodextrin MBCD caused cholesterol removal from the DRM without affecting the composition and amount of the phospholipid while higher levels disrupted the DRM The substantial amount of polyunsaturated phospholipids in DRMs as well as a low stoichiometric amount of cholesterol suggest that lipid rafts in biological membranes are more fluid and dynamic than previously anticipated Using negative staining ultrastructural features of DRM were monitored and in all three cell types the DRMs appeared as multilamellar vesicular structures with a similar morphology The detergent resistance is a result of protein–cholesterol and sphingolipid interactions allowing a relatively passive attraction of phospholipids to maintain the Lo phase For this special issue the relevance of our findings is discussed in a sperm physiological contextLipid rafts are commonly defined as lipidordered Lo phase microdomains in the membrane that concentrate specific eg signaling molecules while excluding others Helms and Zurzolo 2004 Brown 2006 Fan et al 2010 Lingwood and Simons 2010 Nyholm 2015 They thereby favor specific protein–protein interactions enhancing the activation or inactivation of signaling cascades Simons and Toomre 2000 Brown and London 2000 Kusumi et al 2004 Surma et al 2012 Lipid rafts have been shown to accumulate lipidmodified proteins like glycosylphosphatidylinositol GPIanchored proteins in the outer leaflet and doubly acylated tyrosine kinases of the Src family in the inner leaflet Simons and Toomre 2000 Lipid rafts have been demonstrated in other subcellular organelles than plasma membranes including endosomes phagosomes mitochondria and Golgi membranes Dermine et al 2001 Gkantiragas et al 2001 Gruenberg 2001 Surma et al 2012 Garofalo et al 2015 Lipid rafts are enriched in cholesterol and sphingolipids and glycolipids such as gangliosides Chen et al 2008 and the forces driving the formation of lipid rafts may be the intrinsic ability of these lipids to cluster into domains with a close lipid packing as has been shown in biophysical studies Simons and Ikonen 1997 Rietveld and Simons 1998 Brown and London 2000 The association of cholesterol with the sphingolipids is most likely strengthened by hydrogen bonding between the 3OH group of the sterol and the amide function of the sphingolipid ceramide backbone Simons and Ikonen 2000 Phospholipids containing highly unsaturated fatty acids are not expected to be in lipid rafts since they would loosen lipid packing and thus inhibit lipid domain formation In contrast phospholipids containing saturated fatty acids have the ability of more condensed packing and of promoting domain formation Therefore phospholipids with saturated fatty acids are expected to be highly enriched in lipid rafts Simons and Vaz 2004The assumption that rafts from biological membranes are enriched in phospholipids with saturated fatty acids is based on extrapolation from model membranes Ahmed et al 1997 Anderson and Jacobson 2002 de Almeida et al 2003 Scherfeld et al 2003 Crane and Tamm 2004 but remains to be experimentally validated However these model membranes may show an oversimplified picture since mostly binary and ternary lipid mixtures in most cases cholesterol sphingomyelin SM and dipalmitoyl phosphatidyl choline were used in these model systems and the composition of these model membranes do not reflect the large complexity of biological membranes Moreover biological membranes contain diverse membrane proteins that may play a role in membrane microdomain propertiesIn this study we isolated the DRM fraction from different cell types with a cold Triton X100 method in which cells are treated with 1  Triton  X100 at 4 °C and the resulting suspension is placed into a discontinuous sucrose gradient for separating the Triton X100 soluble and insoluble fractions The latter floats to a low sucrose density and is defined as the detergentresistant membrane DRM supposed to be enriched with the Lo phase of the original lipid rafts The DRM that appears as an opaque white band in the interphase of 30 and 5  sucrose in the gradient was processed for negative staining and ultrastructural properties were determined using transmission electron microscopy Although the question remains whether the DRM fraction resembles the in vivo situation Simons and Ikonen 1997 Hattersley et al 2013 we chose to use the Triton X100 method to make a comparison with most other studies possible In this respect an extensive study of Schuck and colleagues on the resistance of cell membranes to different detergents showed that Triton X100 used in the present study and CHAPS are the most reliable detergents for analyzing raft association Schuck et al 2003 They concluded that despite its disadvantages “detergent isolation remains the starting point for defining membrane subdomains”In the current study the lipid content and composition of extracted lipids from the DRM versus the whole cell were compared We examined whether the polyunsaturated phospholipid species are indeed excluded from DRMs derived from biological membranes and we determined which specific phospholipid species are enriched in DRMs We qualitatively and quantitatively analyzed the lipids that are present in the DRM fraction of biological membranes derived from three cell types with considerable differences in total lipid composition Evans et al 1980 Lynch et al 1986 DeLong et al 1999 MadinDarby canine kidney MDCK cells McArdle cells and porcine sperm MDCK cells have a relative saturated lipid composition while McArdle cells are somewhat more unsaturated The rationale for comparing these cells with sperm is that sperm almost exclusively contains polyunsaturated phospholipids but the presence of lipid rafts in sperm cells has been reported in the literature Travis et al 2001 Selvaraj et al 2009 We chose MDCK cells as a reference as this cell type is established as a model cell line for studying lipid rafts Verkade et al 2000 Gallegos et al 2006 McArdle cells were chosen as an additional comparative cell model since Pike et al 2002 demonstrated in HeLa cells cervixderived epithelial cell line that the DRM fraction contained substantial amounts of polyunsaturated fatty acid containing phospholipids Beyond the study of cholesterol and phospholipids the partitioning of glycolipids from sperm and MDCK cells into the Triton soluble and insoluble membrane fraction representing the Ld and Lo phase was also compared Both cell types are enriched in glycolipids Gadella et al 1993 Pescio et al 2012 In MDCK the glycolipid fraction exclusively contains ceramides glycosylated predominantly with galactosyl3sulfate sulfatides while sperm exclusively contains alkylacylglycerol which is only glycosylated with galactosyl3sulfate seminolipid for review see Vos et al 1994 McArdle cells were not used for this purpose as they only contain trace amounts of glycolipidsImplications of the observed differences of DRM versus whole cell lipid composition with respect to what this may imply for lipid raft composition in terms of membrane fluidity and membrane heterogeneity dynamics are discussed including the role of cholesterol cholesterol interacting proteins and glycolipids with special reference to sperm physiologySemen was obtained from the Cooperative Centre for Artificial Insemination in Pigs “Utrecht en de Hollanden” Bunnik the Netherlands Semen was filtered through gauze to remove gelatinous material Sperm cells were washed on a discontinuous Percoll Amersham Biosciences Uppsala Sweden gradient as described Flesch et al 1998 All solutions used were isoosmotic 285–315 mOsm/kg and at room temperature unless stated otherwiseMadinDarby canine kidney MDCK strain II cells were maintained at 37 °C in 5  CO2 in MEM Gibco Paisley UK supplemented with 10  foetal bovine serum FBS Gibco and MEM nonessential amino acids Gibco Experiments were performed on confluent or subconfluent cells cultured in 175 cm2 culture flasks McArdle cells McARH7777 ATCC no CRL1601 were cultured in Dulbecco’s modified Eagle’s medium Gibco supplemented with 10  FBS and 10  horse serum HS Gibco and maintained in 175 cm2 culture flasks at 37 °C 5  CO2 under humidified atmosphere

Keywords: