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.     

Lipid rafts: signaling and sorting platforms of cells and their roles in cancer

Lipid rafts are defined as microdomains within the lipid bilayer of cellular membranes that assemble subsets of transmembrane or glycosylphosphatidylinisotol-anchored proteins and lipids (cholesterol and sphingolipids) and experimentally resist extraction in cold detergent (detergent-resistant membrane). These highly dynamic raft domains are essential in signaling processes and also form sorting platforms for targeted protein traffic. Lipid rafts are involved in protein endocytosis that occurs via caveolae or flotillin-dependent pathways. Non-constitutive protein components of rafts fluctuate dramatically in cancer with impacts on cell proliferation, signaling, protein trafficking, adhesion and apoptosis. This article focuses on the identification of candidate cancer-associated biomarkers in carcinoma cells using state-of-the-art proteomics.     

Cellular lipid dynamics

During the past years, the notion of microdomains at the surface of cellular membranes has been developed. These are constituted by lipid rafts which involve sphingoglycolipids and cholesterol. To these rafts are associated proteins which have a lipid anchor or are transmembrane proteins. These lipid rafts target specific proteins at the plasma membrane surface and can remain associated with them. They are present in surface receptors and endocytosis occurs upon binding of the specific ligands. Thus these rafts participate to major aspects of cellular dynamics. These rafts are complex structures, insoluble in non-ionic detergents. According to the detergent used, many types of rafts can be isolated. Any alteration of cholesterol, sphingoglycolipids, or abnormalities of the proteins themselves, can lead to abnormal targeting at the membrane surface. It is possible that specific sphingoglycolipids are necessary to target specific proteins at the membrane surface. This may explain the complexity of the sphingoglycolipid molecules, both in relation to their oligosaccharide and to their ceramide structures. There is both a cellular and a tissue specificity of these constituents. Complex sphingoglycolipids are involved in cellular differentiation, cellular polarization, and modified in relation to cancer. Virus and bacteria can be linked to the sphingoglycolipids of these microdomains and alter cellular signaling and function. Sphingoglycolipids are involved in autoimmune diseases as antibody targets and in neurolipidoses which are genetic diseases involving their catabolism. The dynamics of the lipid rafts, in relation to cholesterol, can be altered in Niemann-Pick's disease type C and in Alzheimer's disease. Thus these microdomains are involved in many aspects related to normal and pathological cellular dynamics.     

Plasma Membranes Are Subcompartmentalized into a Plethora of Coexisting and Diverse Microdomains in Arabidopsis and Nicotiana benthamiana

Eukaryotic plasma membranes are highly compartmentalized structures. So far, only a few individual proteins that function in a wide range of cellular processes have been shown to segregate into microdomains. However, the biological roles of most microdomain-associated proteins are unknown. Here, we investigated the heterogeneity of distinct microdomains and the complexity of their coexistence. This diversity was determined in living cells of intact multicellular tissues using 20 different marker proteins from Arabidopsis thaliana, mostly belonging to the Remorin protein family. These proteins associate with microdomains at the cytosolic leaflet of the plasma membrane. We characterized these membrane domains and determined their lateral dynamics by extensive quantitative image analysis. Systematic colocalization experiments with an extended subset of marker proteins tested in 45 different combinations revealed the coexistence of highly distinct membrane domains on individual cell surfaces. These data provide valuable tools to study the lateral segregation of membrane proteins and their biological functions in living plant cells. They also demonstrate that widely used biochemical approaches such as detergent-resistant membranes cannot resolve this biological complexity of membrane compartmentalization in vivo.     

Interactions of Ras proteins with the plasma membrane and their roles in signaling

The complex dynamic structure of the plasma membrane plays critical roles in cellular signaling; interactions with the membrane lipid milieu, spatial segregation within and between cellular membranes and/or targeting to specific membrane-associated scaffolds are intimately involved in many signal transduction pathways. In this review, we focus on the membrane interactions of Ras proteins. These small GTPases play central roles in the regulation of cell growth and proliferation, and their excessive activation is commonly encountered in human tumors. Ras proteins associate with the membrane continuously via C-terminal lipidation and additional interactions in both their inactive and active forms; this association, as well as the targeting of specific Ras isoforms to plasma membrane microdomains and to intracellular organelles, have recently been implicated in Ras signaling and oncogenic potential. We discuss biochemical and biophysical evidence for the roles of specific domains of Ras proteins in mediating their association with the plasma membrane, and consider the potential effects of lateral segregation and interactions with membrane-associated protein assemblies on the signaling outcomes.     

Sphingolipid trafficking--sorted out?

Studies of intracellular membrane traffic have traditionally focused on the protein components of membranes, but what about lipids? Recent findings have drawn attention to the transport of one type of lipid, the sphingolipids. Their unique physical properties may allow them to aggregate into microdomains in membranes that concentrate sphingolipids into specific transport pathways. Gerrit van Meer and Koert Burger consider here the routes of sphingolipid biosynthesis and transport, and the role of proteins in their targeting. The following article by Deborah Brown turns the tables to review the evidence suggesting that sphingolipid domains are important in specific targeting of GPI-anchored proteins to the plasma membrane.     

Sphingolipid topology and the dynamic organization and function of membrane proteins

When acquiring internal membranes and vesicular transport, eukaryotic cells started to synthesize sphingolipids and sterols. The physical differences between these and the glycerophospholipids must have enabled the cells to segregate lipids in the membrane plane. Localizing this event to the Golgi then allowed them to create membranes of different lipid composition, notably a thin, flexible ER membrane, consisting of glycerolipids, and a sturdy plasma membrane containing at least 50% sphingolipids and sterols. Besides sorting membrane proteins, in the course of evolution the simple sphingolipids obtained key positions in cellular physiology by developing specific interactions with (membrane) proteins involved in the execution and control of signaling. The few signaling sphingolipids in mammals must provide basic transmission principles that evolution has built upon for organizing the specific regulatory pathways tuned to the needs of the different cell types in the body.     

Association of sterol- and glycosylphosphatidylinositol-linked proteins with Drosophila raft lipid microdomains

In vertebrates, the formation of raft lipid microdomains plays an important part in both polarized protein sorting and signal transduction. To establish a system in which raft-dependent processes could be studied genetically, we have analyzed the protein and lipid composition of these microdomains in Drosophila melanogaster. Using mass spectrometry, we identified the phospholipids, sphingolipids, and sterols present in Drosophila membranes. Despite chemical differences between Drosophila and mammalian lipids, their structure suggests that the biophysical properties that allow raft formation have been preserved. Consistent with this, we have identified a detergent-insoluble fraction of Drosophila membranes that, like mammalian rafts, is rich in sterol, sphingolipids, and glycosylphosphatidylinositol-linked proteins. We show that the sterol-linked Hedgehog N-terminal fragment associates specifically with this detergent-insoluble membrane fraction. Our findings demonstrate that raft formation is preserved across widely separated phyla in organisms with different lipid structures. They further suggest sterol modification as a novel mechanism for targeting proteins to raft membranes and raise the possibility that signaling and polarized intracellular transport of Hedgehog are based on raft association.     

Transport of lipids by ABC proteins: Interactions and implications for cellular toxicity, viability and function

Members of the ATP-binding cassette (ABC) family of membrane-bound transporters are involved in multiple aspects of transport and redistribution of various lipids and their conjugates. Most ABC transporters localize to the plasma membrane; some are associated with liquid-ordered cholesterol-/sphingolipid-rich microdomains, and to a lesser extent the membranes of the Golgi and endoplasmic reticulum. Hence, ABC transporters are well placed to regulate plasma membrane lipid composition and the efflux and redistribution of structural phospholipids and sphingolipids during periods of cellular stress and recovery. ABC transporters can also modulate cellular sensitivity to extrinsic pro-apoptotic signals through regulation of sphingomyelin-ceramide biosynthesis and metabolism. The functionality of ABC transporters is, in turn, modulated by the lipid content of the microdomains in which they reside. Cholesterol, a major membrane microdomain component, is not only a substrate of several ABC transporters, but also regulates ABC activity through its effects on microdomain structure. Several important bioactive lipid mediators and toxic lipid metabolites are also effluxed by ABC transporters. In this review, the complex interactions between ABC transporters and their lipid/sterol substrates will be discussed and analyzed in the context of their relevance to cellular function, toxicity and apoptosis.     

The SPFH domain-containing proteins: more than lipid raft markers

Membrane microdomains with distinct lipid compositions, called lipid rafts, represent a potential mechanism for compartmentalizing cellular functions within the plane of biological membranes. SPFH domain-containing proteins are found in lipid raft microdomains in diverse cellular membranes. The functions of these proteins are just beginning to be elucidated. Recent advances in the understanding of structural features and their roles within lipid rafts include a potential function for SPFH proteins in the formation of membrane microdomains and lipid raft-associated processes, such as endocytosis and mechanosensation.     

G(s) signaling is intact after disruption of lipid rafts.

Membrane microdomains enriched in cholesterol and sphingolipids modulate a number of signal transduction pathways and provide a residence for heterotrimeric G proteins, their receptors, and their effectors. We investigated whether signaling through G(s) was dependent on these membrane domains, characterized by their resistance to detergents, by depleting cells of cholesterol and sphingolipids. For cholesterol depletion, rat salivary epithelial A5 cells were cultured under low-cholesterol conditions, and then treated with the cholesterol chelator methyl-beta-cyclodextrin. For sphingolipid depletion, LY-B cells, a mutant CHO cell line that is unable to synthesize sphingolipids, were incubated under low-sphingolipid conditions. Depletion of cholesterol or sphingolipid led to a loss or decrease, respectively, in the amount of Galpha(s) from the detergent-resistant membranes without any change in the cellular or membrane-bound amounts of Galpha(s). The cAMP accumulation in response to a receptor agonist was intact and the level slightly increased in cells depleted of cholesterol or sphingolipids compared to that in control cells. These results indicate that localization of Galpha(s) to detergent-resistant membranes was not required for G(s) signaling. Analysis of the role of lipid rafts on the kinetics of protein associations in the membrane suggests that compartmentalization in lipid rafts may be more effective in inhibiting protein interactions and, depending on the pathway, ultimately inhibit or promote signaling.     

Sphingolipids and gangliosides of the nervous system in membrane function and dysfunction

Simple sphingolipids such as ceramide and sphingomyelin (SM) as well as more complex glycosphingolipids play very important roles in cell function under physiological conditions and during disease development and progression. Sphingolipids are particularly abundant in the nervous system. Due to their amphiphilic nature they localize to cellular membranes and many of their roles in health and disease result from membrane reorganization and from lipid interaction with proteins within cellular membranes. In this review we discuss some of the functions of sphingolipids in processes that entail cellular membranes and their role in neurodegenerative diseases, with an emphasis on SM, ceramide and gangliosides.     

Lipids as modulators of membrane fusion mediated by viral fusion proteins

Enveloped viruses infect host cells by fusion of viral and target membranes. This fusion event is triggered by specific glycoproteins in the viral envelope. Fusion glycoproteins belong to either class I, class II or the newly described third class, depending upon their arrangement at the surface of the virion, their tri-dimensional structure and the location within the protein of a short stretch of hydrophobic amino acids called the fusion peptide, which is able to induce the initial lipid destabilization at the onset of fusion. Viral fusion occurs either with the plasma membrane for pH-independent viruses, or with the endosomal membranes for pH-dependent viruses. Although, viral fusion proteins are parted in three classes and the subcellular localization of fusion might vary, these proteins have to act, in common, on lipid assemblies. Lipids contribute to fusion through their physical, mechanical and/or chemical properties. Lipids can thus play a role as chemically defined entities, or through their preferential partitioning into membrane microdomains called "rafts", or by modulating the curvature of the membranes involved in the fusion process. The purpose of this review is to make a state of the art on recent findings on the contribution of cholesterol, sphingolipids and glycolipids in cell entry and membrane fusion of a number of viral families, whose members bear either class I or class II fusion proteins, or fusion proteins of the recently discovered third class.     

Membrane microdomains in hepatocytes: potential target areas for proteins involved in canalicular bile secretion

The formation of hepatic bile requires that water be transported across liver epithelia. Rat hepatocytes express three aquaporins (AQPs): AQP8, AQP9, and AQP0. Recognizing that cholesterol and sphingolipids are thought to promote the assembly of proteins into specialized membrane microdomains, we hypothesized that canalicular bile secretion involves the trafficking of vesicles to and from localized lipid-enriched microdomains in the canalicular plasma membrane. Hepatocyte plasma membranes were sonicated in Triton and centrifuged overnight on a sucrose gradient to yield a Triton-soluble pellet and a Triton-insoluble, sphingolipid-enriched microdomain fraction at the 5%/30% sucrose interface. The detergent-insoluble portion of the hepatocyte plasma membrane was enriched in alkaline phosphatase (a microdomain-positive marker) and devoid of amino-peptidase N (a microdomain-negative marker), enriched in caveolin, both AQP8 and AQP9, but negative for clathrin. The microdomain fractions contained chloride-bicarbonate anion exchanger isoform 2 and multidrug resistance-associated protein 2. Exposure of isolated hepatocytes to glucagon increased the expression of AQP8 but not AQP9 in the microdomain fractions. Sphingolipid analysis of the insoluble fraction showed the predominant species to be sphingomyelin. These data support the presence of sphingolipid-enriched microdomains of the hepatocyte membrane that represent potential localized target areas for the clustering of AQPs and functionally related proteins involved in canalicular bile secretion.     

Lipid remodeling of GPI-anchored proteins and its function

Many proteins are attached to the cell surface via a conserved post-translational modification, the glycosylphosphatidylinositol (GPI) anchor. GPI-anchored proteins are functionally diverse, but one of their most striking features is their association with lipid microdomains, which consist mainly of sphingolipids and sterols. GPI-anchored proteins modulate various biological functions when they are incorporated into these specialized domains. The biosynthesis of GPI and its attachment to proteins occurs in the endoplasmic reticulum. The lipid moieties of GPI-anchored proteins are further modified during their transport to the cell surface, and these remodeling processes are essential for the association of proteins with lipid microdomains. Recently, several genes required for GPI lipid remodeling have been identified in yeast and mammalian cells. In this review, we describe the pathways for lipid remodeling of GPI-anchored proteins in yeast and mammalian cells, and discuss how lipid remodeling affects the association of GPI-anchored proteins with microdomains in cellular events.     

The γ-aminobutyric acid receptor B, but not the metabotropic glutamate receptor type-1, associates with lipid rafts in the rat cerebellum

Recent evidence suggests that specialized microdomains, called lipid rafts, exist within plasma membranes. These domains are enriched in cholesterol and sphingolipids and are resistant to non-ionic detergent-extraction at 4 degrees C. They contain specific populations of membrane proteins, and can change their size and composition in response to cellular signals, resulting in activation of signalling cascades. Here, we demonstrate that both the metabotropic gamma-aminobutyric acid receptor B (GABA(B) receptor) and the metabotropic glutamate receptor-1 from rat cerebellum are insoluble in the non-ionic detergent Triton X-100. However, only the GABA(B) receptor associates with raft fractions isolated from rat brain by sucrose gradient centrifugation. Moreover, increasing the stringency of isolation by decreasing the protein : detergent ratio caused an enrichment of the GABA(B) receptor in raft fractions. In contrast, depletion of cholesterol from cerebellar membranes by either saponin or methyl-beta-cyclodextrin treatment, which solubilize known raft markers, also increased the solubility of the GABA(B) receptor. These properties are all consistent with an association of the GABA(B) receptor with lipid raft microdomains.     

Organelle biogenesis and intracellular lipid transport in eukaryotes.

The inter- and intramembrane transport of phospholipids, sphingolipids, and sterols involves the most fundamental processes of membrane biogenesis. Identification of the mechanisms involved in these lipid transport reactions has lagged significantly behind that for intermembrane protein traffic until recently. Application of methods that include fluorescently labeled and spin-labeled lipid analogs, new cellular fractionation techniques, topographically specific chemical modification techniques, the identification of organelle-specific metabolism, permeabilized cell methodology, and yeast molecular genetics has contributed to revealing a diverse biochemical array of transport processes for lipids. Compelling evidence now exists for ATP-dependent, ATP-independent, vesicle-dependent, and vesicle-independent transport processes that are lipid and membrane specific. ATP-dependent transport processes include the transbilayer movement of phosphatidylserine and phosphatidylethanolamine at the plasma membrane and the transport of phosphatidylserine from its site of synthesis to the mitochondria. ATP-independent processes include the transbilayer movement of virtually all lipids at the endoplasmic reticulum, the movement of phosphatidylserine between the inner and outer mitochondrial membranes, and the transfer of nascent phosphatidylcholine and phosphatidylethanolamine to the plasma membrane. The ATP-independent movement of lipids between organelles is believed to be due to the action of lipid transfer proteins, but this still remains to be proved. Vesicle-based transport mechanisms (which are also inherently ATP dependent) include the transport of nascent cholesterol, sphingomyelin, and glycosphingolipids from the Golgi apparatus to the plasma membrane and the recycling of sphingolipids and selected pools of phosphatidylcholine from the plasma membrane to the cell interior. The vesicles involved in cholesterol transport to the plasma membrane are different from those involved in bulk protein transport to the cell surface. The vesicles involved in recycling sphingomyelin to and from the cell surface are different from those involved in the assembly of newly synthesized sphingolipids into the plasma membrane. The preliminary characterization of these lipid translocation processes suggests divergent rather than unifying mechanisms for lipid transport in organelle assembly.     

Imaging lipid rafts

Lipid rafts are plasma membrane microdomains enriched in sphingolipids and cholesterol. These domains have been suggested to serve as platforms for various cellular events, such as signaling and membrane trafficking. However, little is known about the distribution and dynamics of lipids in these microdomains. Here we report investigations carried out using recently developed probes for the lipid components of lipid rafts: lysenin, a sphingomyelin-binding protein obtained from the coelomic fluid of the earthworm Eisenia foetida; and the fluorescein ester of poly(ethyleneglycol) cholesteryl ether (fPEG-Chol), which partitions into cholesterol-rich membranes. Lysenin reveals that the organization of sphingomyelin differs between different cell types and even between different membrane domains within the same cell. When added to live cells, fPEG-Chol is distributed exclusively on the outer leaflet of the plasma membrane and is clustered dynamically upon activation of receptor signaling. The surface-bound fPEG-Chol is slowly internalized via a clathrin-independent pathway into endosomes with lipid raft markers.     

The modulation of gap-junctional intercellular communication by lipid rafts

Lipid rafts are specific microdomains of plasma membrane which are enriched in cholesterol and sphingolipids. These domains seem to favour the interactions of particular proteins and the regulation of signalling pathways in the cells. Recent data have shown that among the proteins, which are preferentially localized in lipid rafts, are connexins that are the structural proteins of gap junctions. Since gap junctional intercellular communication is involved in various cellular processes and pathologies such as cancer, we were interested to review the various observations concerning this specific localization of connexins in lipid rafts and its consequences on gap junctional intercellular communication capacity. In particular, we will focus our discussion on the role of the lipid raft-connexin connection in cancer progression. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.