Polyunsaturated fatty acid-cholesterol interactions: Domain formation in membranes

Polyunsaturated fatty acids (PUFA) constitute an influential group of molecules that promote health by an as yet unknown mechanism. They are structurally distinguished from less unsaturated fatty acids by the presence of a repeating CH-CH₂-CH unit that produces an extremely flexible chain rapidly reorienting through conformational states. The most highly unsaturated case in point is docosahexaenoic acid (DHA) with 6 double bonds. This review will summarize how the high disorder of DHA affects the properties of the membrane phospholipids into which the PUFA incorporates, focusing upon the profound impact on the interaction with cholesterol. Results obtained with model membranes using an array of biophysical techniques will be presented. They demonstrate DHA and the sterol possesses a mutual aversion that drives the lateral segregation of DHA-containing phospholipids into highly disordered domains away from cholesterol. These domains are compositionally and organizationally the opposite of lipid rafts, the ordered domain enriched in predominantly saturated sphingolipids “glued” together by cholesterol that is believed to serve as the platform for signaling proteins. We hypothesize that DHA-rich domains also form in the plasma membrane and are responsible, in part, for the diverse range of health benefits associated with DHA.     

Use of Bodipy-labeled sphingolipid and cholesterol analogs to examine membrane microdomains in cells

Much evidence has accumulated to show that cellular membranes such as the plasma membrane, contain multiple "microdomains" of differing lipid and protein composition and function. These domains are sometimes enriched in cholesterol and sphingolipids and are believed to be important structures for the regulation of many biological and pathological processes. This review focuses on the use of fluorescent (Bodipy) labeled analogs of sphingolipids and cholesterol to study such domains. We discuss the similarities between the behavior of Bodipy-cholesterol and natural cholesterol in artificial bilayers and in cultured cells, and the use of Bodipy-sphingolipid analogs to visualize membrane domains in living cells based on the concentration-dependent monomer-excimer fluorescence properties of the Bodipy-fluorophore. The use of Bodipy-D-erythro-lactosylceramide is highlighted for detection of domains on the plasma membrane and endosome membranes, and the importance of the sphingolipid stereochemistry in modulating domain formation is discussed. Finally, we suggest that Bodipy-sphingolipids may be useful in future studies to examine the relationship between membrane domains at the cell surface and domains enriched in other lipids and proteins on the inner leaflet of the plasma membrane.     

The temperature-pressure phase diagram of a DPPC-ergosterol fungal model membrane -- a SAXS and FT-IR spectroscopy study

What distinguishes polyunsaturated fatty acids (PUFAs) from less unsaturated fatty acids is the presence of a repeating CH–CH₂–CH unit that produces an extremely flexible structure rapidly isomerizing through conformational states. Docosahexaenoic acid (DHA) with 6 double bonds is the most extreme example. The focus of this review is the profound impact that the high disorder of DHA has on its interaction with cholesterol when the PUFA is incorporated into membrane phospholipids. Results from a battery of biophysical techniques are described. They demonstrate an aversion of DHA for the sterol that drives the lateral segregation of DHA-containing phospholipids into liquid disordered (l_d) domains that are depleted in cholesterol. These domains are compositionally and organizationally the antithesis of lipid rafts, the much-studied liquid ordered (l_o) domain that is enriched in predominantly saturated sphingolipids and cholesterol. We hypothesize that the introduction of DHA-rich domains into the plasma membrane where they coexist with lipid rafts is the origin, in part, of the astonishing diversity of health benefits that accrue from dietary consumption of DHA. According to our model, changes in the conformation of signaling proteins when they move between these disparate domains have the potential to modulate cell function.     

Lipid rafts-protein association and the regulation of protein activity

Lipid rafts are membrane microdomains enriched in saturated phospholipids, sphingolipids, and cholesterol. They have a varied but distinct protein composition and have been implicated in diverse cellular processes including polarized traffic, signal transduction, endo- and exo-cytoses, entrance of obligate intracellular pathogens, and generation of pathological forms of proteins associated with Alzheimer's and prion diseases. Raft proteins can be permanently or temporarily associated to lipid rafts. Here, we review recent advances on the biochemical and cell biological characterization of rafts, and on the emerging concept of the temporary residency of proteins in rafts as a regulatory mechanism of their biological activity.     

Lipid rafts in plants

About two decades ago a provocative hypothesis evolved suggesting that the plasma membrane (PM) of mammalian and probably other eukaryotic cells constitutes a mosaic of patches comprising particular molecular compositions. These scattered lipid bilayer microdomains are supposedly enriched in sterols as well as sphingolipids and depleted in unsaturated phospholipids. In addition, the PM microdomains are proposed to host glycosyl-phosphatidylinositol-anchored polypeptides and a subset of integral and peripheral cell surface proteins while excluding others. Though the actual in vivo existence of such “lipid rafts” remains controversial, a range of fundamental biological functions has been put forward for these PM microenvironments. A variety of recent studies provide preliminary evidence that lipid rafts may also occur in plant cells.     

Lipid membrane domains in cell surface and vacuolar systems

Detergent insoluble sphingolipid-cholesterol enriched 'raft'-like membrane microdomains have been implicated in a variety of biological processes including sorting, trafficking, and signaling. Mutant cells and knockout animals of sphingolipid biosynthesis are clearly useful to understand the biological roles of lipid components in raft-like domains. It is suggested that raft-like domains distribute in internal vacuolar membranes as well as plasma membranes. In addition to sphingolipid-cholesterol-rich membrane domains, recent studies suggest the existence of another lipid-membrane domain in the endocytic pathway. This domain is enriched with a unique phospholipid, lysobisphosphatidic acid (LBPA) and localized in the internal membrane of multivesicular endosome. LBPA-rich membrane domains are involved in lipid and protein sorting within the endosomal system. Possible interaction between sphingolipids and LBPA in sphingolipid-storage disease is discussed.     

Lipid rafts are enriched in arachidonic acid and plasmenylethanolamine and their composition is independent of caveolin-1 expression: a quantitative electrospray ionization/mass spectrometric analysis

Lipid rafts are specialized cholesterol-enriched membrane domains that participate in cellular signaling processes. Caveolae are related domains that become invaginated due to the presence of the structural protein, caveolin-1. In this paper, we use electrospray ionization mass spectrometry (ESI/MS) to quantitatively compare the phospholipids present in plasma membranes and nondetergent lipid rafts from caveolin-1-expressing and nonexpressing cells. Lipid rafts are enriched in cholesterol and sphingomyelin as compared to the plasma membrane fraction. Expression of caveolin-1 increases the amount of cholesterol recovered in the lipid raft fraction but does not affect the relative proportions of the various phospholipid classes. Surprisingly, ESI/MS demonstrated that lipid rafts are enriched in plasmenylethanolamines, particularly those containing arachidonic acid. While the total content of anionic phospholipids was similar in plasma membranes and nondetergent lipid rafts, the latter were highly enriched in phosphatidylserine but relatively depleted in phosphatidylinositol. Detergent-resistant membranes made from the same cells showed a higher cholesterol content than nondetergent lipid rafts but were depleted in anionic phospholipids. In addition, these detergent-resistant membranes were not enriched in arachidonic acid-containing ethanolamine plasmalogens. These data provide insight into the structure of lipid rafts and identify potential new roles for these domains in signal transduction.     

Sphingolipid Domains in the Plasma Membranes of Fibroblasts Are Not Enriched with Cholesterol

The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. Thus, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize the cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton.     

Lipid membrane domains in cell surface and vacuolar systems

Detergent insoluble sphingolipid-cholesterol enriched raft-like membrane microdomains have been implicated in a variety of biological processes including sorting, trafficking, and signaling. Mutant cells and knockout animals of sphingolipid biosynthesis are clearly useful to understand the biological roles of lipid components in raft-like domains. It is suggested that raft-like domains distribute in internal vacuolar membranes as well as plasma membranes. In addition to sphingolipid-cholesterol-rich membrane domains, recent studies suggest the existence of another lipid-membrane domain in the endocytic pathway. This domain is enriched with a unique phospholipid, lysobisphosphatidic acid (LBPA) and localized in the internal membrane of multivesicular endosome. LBPA-rich membrane domains are involved in lipid and protein sorting within the endosomal system. Possible interaction between sphingolipids and LBPA in sphingolipid-storage disease is discussed.     

Reactivity of Phospholipid Hydroperoxide Glutathione Peroxidase with Membrane and Lipoprotein Lipid Hydroperoxides

A comparative study has been carried out on the general reactivity of lipid hydroperoxides in liposornes, biological membranes and lipoproteins with two Se-dependent peroxidases: Glutathione Peroxidase (GPX) and Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPX). While PHGPX reduces all hydroperoxides derived from phospholipids, cholesterol and cholesterol esters, GPX reduces only fatty acid hydroperoxides released after treatment of phospholipid hydroperoxides with phospholipase A,. These findings highlight the role of PHGPX in protecting biomembranes from peroxidative damage and add new insight into how cholesterol hydroperoxides are detossified in cells.     

Role of cholesterol in lipid raft formation: lessons from lipid model systems

Biochemical and cell-biological experiments have identified cholesterol as an important component of lipid ‘rafts’ and related structures (e.g., caveolae) in mammalian cell membranes, and membrane cholesterol levels as a key factor in determining raft stability and organization. Studies using cholesterol-containing bilayers as model systems have provided important insights into the roles that cholesterol plays in determining lipid raft behavior. This review will discuss recent progress in understanding two aspects of lipid-cholesterol interactions that are particularly relevant to understanding the formation and properties of lipid rafts. First, we will consider evidence that cholesterol interacts differentially with different membrane lipids, associating particularly strongly with saturated, high-melting phospho- and sphingolipids and particularly weakly with highly unsaturated lipid species. Second, we will review recent progress in reconstituting and directly observing segregated raft-like (liquid-ordered) domains in model membranes that mimic the lipid compositions of natural membranes incorporating raft domains.     

Cholesterol is not crucial for the existence of microdomains in kidney brush-border membrane models.

The external membrane leaflet plays a key role in the organization of the cell plasma membrane as a mosaic of ordered microdomains enriched in sphingolipids and cholesterol and of fluid domains. In this study, the thermotropic behavior and the topology of bilayers made of a phosphatidylcholine/sphingomyelin mixture, which mimicks the lipid composition of the external leaflet of renal brush-border membranes, were examined by differential scanning calorimetry and atomic force microscopy. In the absence of cholesterol, a broad phase separation process occurred where ordered gel phase domains of size varying from the mesoscopic to the microscopic scale, enriched in sphingomyelin, occupied half of the bilayer surface at room temperature. Increasing amounts of cholesterol progressively decreased the enthalpy of the transition and modified the topology of membranes domains up to a concentration of 33 mol % for which no membrane domains were detected. These results strongly suggest that, in membranes highly enriched in sphingolipids like renal and intestinal brush borders, there is a threshold close to the physiological concentration above which cholesterol acts as a suppressor rather than as a promoter of membrane domains. They also suggest that cholesterol depletion does not abolish the lateral heterogenity in brush-border membranes.     

Reactivity of Phospholipid Hydroperoxide Glutathione Peroxidase with Membrane and Lipoprotein Lipid Hydroperoxides

A comparative study has been carried out on the general reactivity of lipid hydroperoxides in liposornes, biological membranes and lipoproteins with two Se-dependent peroxidases: Glutathione Peroxidase (GPX) and Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPX). While PHGPX reduces all hydroperoxides derived from phospholipids, cholesterol and cholesterol esters, GPX reduces only fatty acid hydroperoxides released after treatment of phospholipid hydroperoxides with phospholipase A,. These findings highlight the role of PHGPX in protecting biomembranes from peroxidative damage and add new insight into how cholesterol hydroperoxides are detossified in cells.     

Stable Cell Surface Expression of GPI‐Anchored Proteins,but not Intracellular Transport,Depends on their Fatty Acid Structure

Glycosylphosphatidylinositol‐anchored proteins (GPI‐APs) are a class of lipid anchored proteins expressed on the cell surface of eukaryotes. The potential interaction of GPI‐APs with ordered lipid domains enriched in cholesterol and sphingolipids has been proposed to function in the intracellular transport of these lipid anchored proteins. Here, we examined the biological importance of two saturated fatty acids present in the phosphatidylinositol moiety of GPI‐APs. These fatty acids are introduced by the action of lipid remodeling enzymes and required for the GPI‐AP association within ordered lipid domains. We found that the fatty acid remodeling is not required for either efficient Golgi‐to‐plasma membrane transport or selective endocytosis via GPI‐enriched early endosomal compartment (GEEC)/ clathrin‐independent carrier (CLIC) pathway, whereas cholesterol depletion significantly affects both pathways independent of their fatty acid structure. Therefore, the mechanism of cholesterol dependence does not appear to be related to the interaction with ordered lipid domains mediated by two saturated fatty acids. Furthermore, cholesterol extraction drastically releases the unremodeled GPI‐APs carrying an unsaturated fatty acid from the cell surface, but not remodeled GPI‐APs carrying two saturated fatty acids. This underscores the essential role of lipid remodeling to ensure a stable membrane association of GPI‐APs particularly under potential membrane lipid perturbation.      

Enantiomers of phospholipids and cholesterol: A key to decipher lipid-lipid interplay in membrane

Most phospholipids constituting biological membranes are chiral molecules with a hydrophilic head group and hydrophobic alkyl chains, rendering biphasic property characteristic of membrane lipids. Some lipids assemble into small domains via chirality-dependent homophilic and heterophilic interactions, the latter of which sometimes include cholesterol to form lipid rafts and other microdomains. On the other hand, lipid mediators and hormones derived from chiral lipids are recognized by specific membrane or nuclear receptors to induce downstream signaling. It is crucial to clarify the physicochemical properties of the lipid self-assembly for the study of the functions and behavior of biological membranes, which often become elusive due to effects of membrane proteins and other biological events. Three major lipids with different skeletal structures were discussed: sphingolipids including ceramides, phosphoglycerolipids, and cholesterol. The physicochemical properties of membranes and physiological functions of lipid enantiomers and diastereomers were described in comparison to natural lipids. When each enantiomer formed a self-assembly or interacted with achiral lipids, both lipid enantiomers exhibited identical membrane physicochemical properties, while when the enantiomer interacted with chiral lipids or with the opposite enantiomer, mixed membranes exhibited different properties. For example, racemic membranes comprising native sphingomyelin and its antipode exhibited phase segregation due to their strong homophilic interactions. Therefore, lipid enantiomers and diastereomers can be good probes to investigate stereospecific lipid-lipid and lipid-protein interactions occurring in biological membranes.     

Cholesterol modulation of membrane resistance to Triton X-100 explored by atomic force microscopy

Biomembranes are not homogeneous, they present a lateral segregation of lipids and proteins which leads to the formation of detergent-resistant domains, also called “rafts”. These rafts are particularly enriched in sphingolipids and cholesterol. Despite the huge body of literature on raft insolubility in non-ionic detergents, the mechanisms governing their resistance at the nanometer scale still remain poorly documented. Herein, we report a real-time atomic force microscopy (AFM) study of model lipid bilayers exposed to Triton X-100 (TX-100) at different concentrations. Different kinds of supported bilayers were prepared with dioleoylphosphatidylcholine (DOPC), sphingomyelin (SM) and cholesterol (Chol). The DOPC/SM 1:1 (mol/mol) membrane served as the non-resistant control, and DOPC/SM/Chol 2:1:1 (mol/mol/mol) corresponded to the raft-mimicking composition. For all the lipid compositions tested, AFM imaging revealed that TX-100 immediately solubilized the DOPC fluid phase leaving resistant patches of membrane. For the DOPC/SM bilayers, the remaining SM-enriched patches were slowly perforated leaving crumbled features reminiscent of the initial domains. For the raft model mixture, no holes appeared in the remaining SM/Chol patches and some erosion occurred. This work provides new, nanoscale information on the biomembranes' resistance to the TX-100-mediated solubilization, and especially about the influence of Chol.     

Detection of lipid domains in model and cell membranes by fluorescence lifetime imaging microscopy

The discovery that the lipids constituting the plasma membrane are not randomly distributed, but instead are able to form laterally segregated lipid domains with different properties has given hints how the formation of such lipid domains influences and regulates many processes occurring at the plasma membrane. While in model systems these lipid domains can be easily accessed and their properties studied, it is still challenging to determine the properties of cholesterol rich lipid domains, the so called “Rafts”, in the plasma membrane of living cells due to their small size and transient nature. One promising technique to address such issues is fluorescence lifetime imaging (FLIM) microscopy, as spatially resolved images make the visualization of the lateral lipid distribution possible, while at the same time the fluorescence lifetime of a membrane probe yields information about the bilayer structure and organization of the lipids in lipid domains and various properties like preferential protein-protein interactions or the enrichment of membrane probes. This review aims to give an overview of the techniques underlying FLIM probes which can be applied to investigate the formation of lipid domains and their respective properties in model membrane and biological systems. Also a short technical introduction into the techniques of a FLIM microscope is given.     

Hemagglutinin Clusters in the Plasma Membrane Are Not Enriched with Cholesterol and Sphingolipids

The clusters of the influenza envelope protein, hemagglutinin, within the plasma membrane are hypothesized to be enriched with cholesterol and sphingolipids. Here, we directly tested this hypothesis by using high-resolution secondary ion mass spectrometry to image the distributions of antibody-labeled hemagglutinin and isotope-labeled cholesterol and sphingolipids in the plasma membranes of fibroblast cells that stably express hemagglutinin. We found that the hemagglutinin clusters were neither enriched with cholesterol nor colocalized with sphingolipid domains. Thus, hemagglutinin clustering and localization in the plasma membrane is not controlled by cohesive interactions between hemagglutinin and liquid-ordered domains enriched with cholesterol and sphingolipids, or from specific binding interactions between hemagglutinin, cholesterol, and/or the majority of sphingolipid species in the plasma membrane.     

Physical arrangement of membrane lipids susceptible to being used in the process of cell sorting of proteins]

Detection of immiscible lipid domains in biological membranes offers an alternative support to protein sorting. Liquid ordered domains ("rafts") comprising cholesterol and saturated sphingolipids incorporate saturated glycosyl-phosphatidylinositol (GPI)-anchored or acylated (palmitoyl- and myristoyl-) proteins or particular transmembrane protein sequences. These lipid domains can be isolated in the form of Detergent resistant membranes (DRM) from biological plasma membrane preparations. Caveolae appear to be a differentiated fraction of plasma membranes comprising such numerous cross-linked microdomains associated with caveolin in different cell types. While the biological relevance of such membrane domains is evidenced in vivo by co-patching of proteins sharing the identical affinity for sphingolipids and by the disruption of co-patching following cell cholesterol depletion, only a few physical studies confort the principle of membrane heterogeneity. Results are now presented where cholesterol addition in a tertiary lipid mixture forces outphase-separation, as a realistic model where the lipid segregation can promote protein sorting to the segregated Lo phase. A lipid mixture comprising phosphatidylserine, phosphatidylethanolamine and sphingomyelin of natural origin in the ratio (1/4/3: mole/mole) has been rendered neatly heterogeneous after the addition of cholesterol (27 mole%). Xray diffraction (Small angle Xray scattering) showed the splitting of two neatly resolved lamellar diffractions in the presence of cholesterol. Above 37 degrees C the heterogeneity was traceable by a broadened diffraction spot up to the complete get-to-liquid transition of sphingomyelin at temperatures > 40 degrees C where the spot became again symmetrical and narrow. The large temperature range where the immiscible lamellar phases are detected, the specific requirement for cholesterol association with sphingomyelin, the positive influence of calcium and the reversibility of domain formation support the occurrence for such domains at the inner side of the plasma membrane whereon lipids-bound proteins concentrate.     

A simplified method for the preparation of detergent-free lipid rafts

Lipid rafts are small plasma membrane domains that contain high levels of cholesterol and sphingolipids. Traditional methods for the biochemical isolation of lipid rafts involve the extraction of cells with nonionic detergents followed by the separation of a low-density, detergent-resistant membrane fraction on density gradients. Because of concerns regarding the possible introduction of artifacts through the use of detergents, it is important to develop procedures for the isolation of lipid rafts that do not involve detergent extraction. We report here a simplified method for the purification of detergent-free lipid rafts that requires only one short density gradient centrifugation, but yields a membrane fraction that is highly enriched in cholesterol and protein markers of lipid rafts, with no contamination from nonraft plasma membrane or intracellular membranes.