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Upon fusion of multivesicular bodies (MVBs) with the plasma membrane, intraluminal vesicles (ILVs) are released into the extracellular space as exosomes. Since the lipid composition of the exosomal membrane resembles that of raft microdomains, the inward budding process involves the raft-like region of the MVB limiting membrane. Although published research suggests that cellular RNAs may be selectively sorted into exosomes, the molecular mechanisms remain elusive. In this review, we suggest that there is a continuous interaction of cellular RNAs with the outer (cytoplasmic) surface of MVBs and that the selection for incorporation of these RNAs into ILVs is based on their affinity to the raft-like region in the outer layer of the MVB membrane.

In this review we describe the history of the development of the raft concept for membrane sub-compartmentalization. From its early beginnings as a mechanism for apical sorting in epithelial cells the concept has evolved to a general principle for membrane organisation. After a shaky start with crude methodology based on detergent extraction the field has become increasingly sophisticated, employing a host of different methods that support the existence of dynamic raft domains in membranes. These are composed of fluctuating nanoscale assemblies of sphingolipid, cholesterol and proteins that can be stabilized to coalesce, forming platforms that function in membrane signalling and trafficking.

According to the raft hypothesis, sphingolipid-cholesterol (CHOL) microdomains are involved in numerous cellular functions. Here, we have prepared liposomes to simulate the lipid composition of rafts/caveolae using phosphatidylchone, sphingomyelin (SPM)-CHOL in vitro. Experiments of both 1,6-diphenyl-1,3,5-hexatriene and merocyanine-540 fluorescence showed that a phase transition from l(d) to l(o) can be observed clearly. In particular, we investigated the behavior of a membrane protein, plasma membrane Ca(2+)-ATPase (PMCA), in lipid rafts (l(o) phase). Three complementary approaches to characterize the physical appearance of PMCA were employed in the present study. Tryptophan intrinsic fluorescence increase, fluorescence quenching by both acrylamid and hypocrellin B decrease, and MIANS fluorescence decrease, indicate that the conformation of PMCA embedded in lipid l(o) phase is more compact than in lipid l(d) phase. Also, our results showed that PMCA activity decreased with the increase of SPM-CHOL content, in other words, with the increase of l(o) phase. This suggests that the specific domains containing high SPM-CHOL concentration are not a favorable place for PMCA activity. Finally, a possible explanation about PMCA molecules concentrated in caveolae/rafts was discussed.

We have investigated whether raft lipids of Madin–Darby canine kidney (MDCK) cells play any role in microvilli maintenance using a combination of atomic force microscopy (AFM) and laser scanning confocal microscopy. MDCK cells were treated to reduce the amount of sphingolipids, cholesterol, or both and subsequently imaged, in buffer solution, using AFM. It was observed that inhibition of either sphingolipid or cholesterol biosynthesis led to a reduction in the number of microvilli on the surface of MDCK cells. However, this effect was not uniform across the monolayer, with some cells resembling those in untreated controls. The subsequent extraction of cholesterol from cells grown in the presence of inhibitors led to a further reduction in microvilli on the surface of the cells and, in some cases, resulted in monolayers devoid of full length microvilli. Significantly, smaller spikes were observed on the surface of the smoother cells.

The relative insolubility of lipid rafts in cold non-ionic detergents is the most widely used method to purify these fascinating membrane domains from intact cells or membranes. Most of what we know about lipid raft function has been derived from experiments utilising detergent insolubility as the basis for raft purification. Recently, a wider range of detergents have been used to purify ‘rafts’, and rafts have been subclassified based on their differential solubility in different detergents. This minireview critically examines the use of detergents as tools for raft isolation and for the subclassification of rafts.

Niemann–Pick type C disease (NPC) is a neurovisceral (or, extremely rarely, only visceral) lipidosis caused by mutations in the NPC1 gene or, in a few patients, the HE1 gene, which encode sterol regulating proteins. NPC is characterised by a complex lipid anomaly including a disturbed cellular trafficking of cholesterol but also multi-lipid storage in visceral organs and brain. Lipids were studied using conventional methods in enlarged spleens that had been removed from five patients for different therapeutic and diagnostic reasons and found to have microscopic signs of lysosomal storage disease not suspected clinically. The spleen lipid findings with a concurrent accumulation of cholesterol, sphingomyelin and glucosylceramide (Acc-CSG) allowed us to suggest NPC diagnoses for these patients, who were free of neurologic symptoms. From two patients no material for confirmatory studies was available, but in two other patients NPC diagnoses could be confirmed with the filipin cytochemical cholesterol assay and NPC1 gene analysis, respectively. However, these tests and also HE1 gene analysis were negative in a third patient. Since the Acc-CSG lipid pattern seems to indicate a multi-lipid trafficking defect rather than being highly specific for NPC, this patient, if not affected with very atypical NPC, may be a candidate for a different lipid trafficking disorder. The Acc-CSG pattern was considered to be similar to the lipid pattern known for the lipid rafts, these functional cell structures being probably disorganised and accumulated in late endosomes and lysosomes of NPC cells.

Doppel is a paralog of the normal prion protein, PrP(C). It has been suggested that Doppel can compensate for the absence of PrP(C) in PrP(0/0) mice. In this work, we tested whether Doppel and PrP(C) share the same cell location, thereby sharing the same neighboring cell components, probably required to share the same cell function. Our results show that, at detergent conditions in which membrane rafts were intact, neither PrP(C) and Doppel co-immunoprecipitate with the appropriate antibodies, nor was Doppel retained by a Cu(2+)IMAC resin, as PrP(C) does. This indicates that, although Doppel is a raft-associated protein as is PrP(C), both proteins are not present in the same membrane microenvironment, and they probably do not perform the same function.

The aim of this study was to gain insight into the mechanism through which transmembrane proteins are targeted to liquid ordered (Lo) phase domains or rafts. This was investigated by analyzing the Triton X-100 resistance of designed transmembrane peptides in model membranes of 1,2-dioleoyl-sn-glycero-3-phosphocholine, sphingomyelin and cholesterol (1/1/1, molar ratio), which contain both Lo phase domains and fluid bilayers. By using peptides with one or two palmitate chains covalently linked to their N-terminus or with variable hydrophobic lengths, the roles of protein palmitoylation and of mismatch between the transmembrane segment of the protein and the bilayer thickness, respectively, were investigated. The results show that neither hydrophobic matching nor palmitoylation is sufficient for partitioning of peptides into Lo phase domains. It is concluded that the Lo phase itself, due to the tight packing of the lipids, constitutes an unfavorable environment for accommodation of protein transmembrane segments.

Membrane microdomains denoted commonly as lipid rafts (or membrane rafts) have been implicated in T-cell receptor (TCR), and more generally immunoreceptor, signaling for over 25years. However, this area of research has been complicated by doubts about the real nature (and even existence) of these membrane entities, especially because of methodological problems connected with possible detergent artefacts. Recent progress in biophysical approaches and functional studies of raft resident proteins apparently clarified many controversial aspects in this area. At present, the prevailing view is that these membrane microdomains are indeed involved in many aspects of cell biology, including immunoreceptor signaling. Moreover, several other types of raft-like microdomains (perhaps better termed nanodomains) have been described, which apparently also play important biological roles.

Inclusion of proteins into membrane-rafts favours interactions required for virus assembly but has also been proposed to facilitate vesicular transport of proteins. The hemagglutinin (HA) of influenza virus contains a raft-targeting sequence in the outer leaflet of its transmembrane region. We report that its mutation enhances co-localization of HA with a cis-Golgi marker and retards Golgi-localized processing, such as acquisition of Endo-H resistant carbohydrates and proteolytic cleavage. In contrast, trimerization of the molecule in the ER and transport to the apical membrane were not affected. The second signal for raft-targeting, S-acylation at cytoplasmic cysteines, did not retard HA transport.

Compositional lipid microdomains (“lipid rafts”) in plasma membranes are believed to be important components of many cellular processes. The biophysical mechanisms by which cells regulate the size, lifetime, and spatial localization of these domains are rather poorly understood at the moment. Over the years, experimental studies of raft formation have inspired several phenomenological theories and speculations incorporating a wide variety of thermodynamic assumptions regarding lipid–lipid and lipid–protein interactions, and the potential role of active cellular processes on membrane structure. Here we critically review and discuss these theories, models, and speculations, and present our view on future directions.

International audience; Contactin and TAG-1 are glycan phosphatidyl inositol (GPI)-anchored cell adhesion molecules that play a crucial role in the organization of axonal subdomains at the node of Ranvier of myelinating fibers. Contactin and TAG-1 mediate axo-glial selective interactions in association with Caspr-family molecules at paranodes and juxtaparanodes, respectively. How membrane proteins can be confined in these neighbouring domains along the axon has been the subject of intense investigations. This review will specifically examine the properties conferred by the lipid microenvironment to regulate trafficking and selective association of these axo-glial complexes. Increasing evidences from genetic and neuropathological models point to a role of lipid rafts in the formation or stabilization of the paranodal junctions.

MRP2(/ABCC2) excretes amphiphilic organic anions into bile, and associates with detergent-resistant bile canalicular membrane domains (DRM). Here, we have evaluated sensitivities of MRP2 transport function and DRM association by titrating the cellular cholesterol content. We demonstrate that the role of cholesterol in the partitioning of MRP2 to DRM can be separated from the role of cholesterol in the function of MRP2, such that (i) cholesterol is not necessary for the polarized distribution of MRP2 at the canalicular membrane, (ii) partitioning into DRM is not required for MRP2 function, yet (iii) the presence of cholesterol is necessary for transport activity. (c) 2008 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Membrane rafts are regions of increased lipid acyl chain order that differ in their lipid and protein composition from the surrounding membrane. By providing an additional level of compartmentalization they have been proposed to serve many functions in cellular signal transduction and trafficking. We will review their potential involvement in different forms of membrane traffic, explicitly excluding signalling, and discuss select aspects of the raft hypothesis in its current form.

Exocytosis of mast cell granules requires a vesicular- and plasma membrane-associated fusion machinery. We examined the distribution of SNARE membrane fusion and Munc18 accessory proteins in lipid rafts of RBL mast cells. SNAREs were found either excluded (syntaxin2), equally distributed between raft and non-raft fractions (syntaxin4, VAMP-8, VAMP-2), or selectively enriched in rafts (syntaxin3, SNAP-23). Syntaxin4-binding Munc18-3 was absent, whereas small amounts of the syntaxin3-interacting partner Munc18-2 consistently distributed into rafts. Cognate SNARE complexes of syntaxin3 with SNAP-23 and VAMP-8 were enriched in rafts, whereas Munc18-2/syntaxin3 complexes were excluded. This demonstrates a spatial separation between these two types of complexes and suggests that Munc18-2 acts in a step different from SNARE complex formation and fusion.

Recent biophysical data suggest that the properties of ceramide observed in model membranes may apply to biological systems. In particular, the ability of ceramide to form microdomains, which coalesce into larger platforms or macrodomains, appears to be important for some cellular signaling processes. Several laboratories have now demonstrated similar reorganization of plasma membrane sphingolipid rafts, via ceramide generation, into macrodomains. This event appeared necessary for signaling upon activation of a specific set of cell surface receptors. In this article, we review the properties and functions of rafts, and the role of sphingomyelinase and ceramide in the biogenesis and re-modeling of these rafts. As clustering of some cell surface receptors in these domains may be critical for signal transduction, we propose a new model for transmembrane signal transmission.

The role of specific lipid structures in biological membranes has been elusive. There are hundreds of them in nature. Why has nature made them? How do they aid in the functioning of membrane proteins? Genetics with its ‘knock out’ organisms declares that functions persist in the absence of any particular lipid. Nonetheless some lipids, such as cardiolipin (CL), are associated with particular functions in the cell. It may merely expand the variety of culture conditions (pH, temperature, etc.) under which the wild-type organism survives. This article explores a unique role of CL as a proton trap within membranes that conduct oxidative phosphorylation and therefore the synthesis of ATP. CL’s pK2 (above 8.0) provides a role for it as a headgroup proton trap for oxidative phosphorylation. It suggests why CL is found in membranes that pump protons. The high pK2 also indicates that the headgroup has but one negative charge in the neutral pH range. Data on the binding of CL to all of the oxidative phosphorylation proteins suggest that the CL may aggregate the oxidative phosphorylation proteins into a patch while it restricts pumped protons within its headgroup domain – supplying protons to the ATP synthase with minimal changes in the bulk phase pH.

Phosphoprotein associated with GEMs (PAG), also known as Csk-binding protein (Cbp), is a broadly expressed palmitoylated transmembrane adapter protein found in membrane rafts, also called GEMs (glycosphingolipid-enriched membrane microdomains). PAG is known to bind and activate the essential regulator of Src-family kinases, cytoplasmic protein tyrosine kinase Csk. In the present study we used the yeast 2-hybrid system to search for additional proteins which might bind to PAG. We have identified the abundant cytoplasmic adapter protein EBP50 (ezrin/radixin/moesin (ERM)-binding phosphoprotein of 50 kDa), also known as NHERF (Na+/H+ exchanger regulatory factor), as a specific PAG-binding partner. The interaction involves the C-terminal sequence (TRL) of PAG and N-terminal PDZ domain(s) of EBP50. As EBP50 is known to interact via its C-terminal domain with the ERM-family proteins, which in turn bind to actin cytoskeleton, the PAG–EBP50 interaction may be important for connecting membrane rafts to the actin cytoskeleton.

Evidence is accumulating that in cell membranes microdomains exist, also referred to as rafts or detergent resistant membranes. In this study, atomic force microscopy is used to study supported lipid bilayers, consisting of a fluid phosphatidylcholine, sphingomyelin and cholesterol. Domains were visualized of which the morphology and size depended on the cholesterol concentration. The presence of cholesterol was found to induce bilayer coupling. At 30 mol% cholesterol, a change in percolation phase was observed, and at 50 mol%, when both fluid lipids and solid lipids are saturated with cholesterol, phase separation was still observed. In addition, we were able to directly visualize the resistance of domains against non-ionic detergent.

Type II diabetes mellitus (T2DM) is a disease characterized by progressive deposition of amyloid in the extracellular matrix of β-cells. We investigated the interaction of the islet amyloid polypeptide (IAPP) with lipid model raft mixtures and INS-1E cells using fluorescence microscopy techniques. Following preferential partitioning of IAPP into the fluid lipid phase, the membrane suffers irreversible damage and predominantly circularly-shaped lipid-containing IAPP amyloid is formed. Interaction studies with the pancreatic β-cell line INS-1E revealed that growing IAPP fibrils also incorporate substantial amounts of cellular membranes in vivo. Additionally, the inhibitory effect of the red wine compound resveratrol on IAPP fibril formation has been studied, alluding to its potential use in developing therapeutic strategies against T2DM.

Caveolins have been identified as key components of caveolae, specialized cholesterol-enriched raft domains visible as small flask-shaped invaginations of the plasma membrane. In polarized MDCK cells caveolin-1 and -2 are found together on basolateral caveolae whereas the apical membrane, where only caveolin-1 is present, lacks caveolae. Expression of a caveolin mutant prevented the formation of the large caveolin-1/-2 hetero-oligomeric complexes, and led to intracellular retention of caveolin-2 and disappearance of caveolae from the basolateral membrane. Correspondingly, in MDCK cells over-expressing caveolin-2 the basolateral membrane exhibited an increased number of caveolae. These results indicate the involvement of caveolin-2 in caveolar biogenesis.

Many mono or multicellular organisms secrete soluble proteins, referred to as protein toxins, which alter the behavior of foreign, or target cells, possibly leading to their death. These toxins affect either the cell membrane by forming pores or modifying lipids, or some intracellular target. To reach this target, they must cross one of the cellular membranes, generally that of an intracellular organelle. As described in this minireview, lipids play crucial roles in the intoxication process of most if not all toxins, by allowing/promoting binding, endocytosis, trafficking and/or translocation into the cytoplasm.

Protein kinase (PK) Ctheta and Akt/PKBalpha cooperate in T cell receptor/CD28-induced T cell signaling. We here demonstrate the recruitment of endogenous Akt1 and PKCtheta to lipid rafts in CD3-stimulated T cells. Further we show that Myr-PKCtheta mediates translocation of endogenous Akt1 to the plasma membrane as well as to lipid rafts, most likely explained by the observed complex formation of both protein kinases. In addition, in peripheral mouse T cells, the PKC inhibitor Gö6850 could partially block Akt1 activation in CD3-induced signaling, placing PKC isotype(s) upstream of Akt1. However, T cells derived from PKCtheta knockout mice were not impaired in CD3- or phorbol ester-induced Akt1 activity. Taken together, the results of this study give new insights into the functional link of Akt1 and PKCtheta in T cell signaling, demonstrating the co-recruitment of the two kinases and showing a novel pathway leading to Akt1 transactivation where PKC isotype(s) are involved but PKCtheta is not essential.

Previously, it was shown that the recruitment of RET into lipid rafts by glial cell line-derived neurotrophic factor (GDNF)/GFRalpha1 is crucial for efficient signal transduction. Here, we show that the mouse GFRalpha4 is a functional, N-glycosylated, glycosylphosphatidylinositol (GPI)-anchored protein, which mediates persephin (PSPN)-induced phosphorylation of RET, but has an almost undetectable capacity to recruit RET into the 0.1% Triton X-100 insoluble membrane fraction. In spite of this, PSPN/mGFRalpha4 promotes neurite outgrowth in PC6-3 cells and survival of cerebellar granule neurons. As we show that also human PSPN/GFRalpha4 is unable to recruit RET into lipid rafts, we propose that the mammalian GFRalpha4 in this respect differs from GFRalpha1.

Large unilamellar vesicles consisting of phospholipids with or without cholesterol have been prepared containing GPI and/or gangliosides asymmetrically located in the outer leaflet of the bilayer. Such asymmetric distribution of GPI and gangliosides is found in 'rafts' and caveolae. Using these vesicles, GPI can be readily hydrolysed by phospholipases. Both cholesterol and ganglioside are seen to inhibit, in an additive way, the hydrolytic activity of GPI-specific phospholipase D. Copyright (C) 1999 Federation of European Biochemical Societies., This work was supported in part by funds from CICYT (Grant PB96/0171), the Basque Government (Grants EX98/28 and PI98/32), and the University of the Basque Country (G03/98). A.-V.V. is the recipient of a scholarship from the University of the Basque Country.

Efficient post-Golgi trafficking depends on microtubules, but actin filaments and actin-associated proteins are also postulated. Here we examined, by inverse fluorescence recovery after photobleaching, the role of actin dynamics in the exit from the TGN of fluorescent-tagged apical or basolateral and raft or non-raft-associated cargoes. Either the actin-stabilizing jasplakinolide or the actin-depolymerising latrunculin B variably but significantly inhibited post-Golgi traffic of non-raft associated apical p75NTR and basolateral VSV-G cargoes. The TGN-exit of the apical-destined VSV-G mutant was impaired only by latrunculin B. Strikingly, the raft-associated GPI-anchor protein was not affected by either actin toxin. Results indicate that actin dynamics participates in the TGN egress of both apical- and basolateral-targeted proteins but is not needed for apical raft-associated cargo.