Ultrastructure and lipid composition of detergent-resistant membranes derived from mammalian sperm and two types of epithelial cells.

Renske A Van Gestel,Jos F Brouwers,J Bernd Helms,Bart M Gadella,Anton Ultee

doi:10.1007/s00441-015-2272-y

Renske A Van Gestel, Jos F Brouwers + Show 3 more

Open Access

https://doi.org/10.1007/s00441-015-2272-y

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Abstract

Lipid rafts are micro-domains 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 X-100 at 4 °C in which the Lo phase forms the detergent-resistant membrane (DRM) fraction. The lipid composition of DRM derived from Madin-Darby 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 cell-specific 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 methyl-ß-cyclodextrin (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 (poly)unsaturated 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 multi-lamellar 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 context.

Highlights

Lipid rafts are commonly defined as lipid-ordered (Lo phase) micro-domains in the membrane that concentrate specific molecules while excluding others (Helms and Zurzolo 2004; Brown 2006; Fan et al 2010; Lingwood and Simons 2010; Nyholm 2015)
Depletion of cholesterol left the phospholipid content in detergent-resistant membrane (DRM) largely unaffected, severe cholesterol depletion led to phospholipid solubilization
Our data indicate that the now-existing models for lipid rafts have to be refined, since many models assume that lipid–lipid interactions between saturated phospholipids and, for instance, cholesterol are the driving force for the constitution of DRMs (Simons and Vaz 2004)

Summary

Introduction

Lipid rafts are commonly defined as lipid-ordered (Lo phase) micro-domains in the membrane that concentrate specific (e.g., 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). Phospholipids with saturated fatty acids are expected to be highly enriched in lipid rafts (Simons and Vaz 2004)

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