Phosphatidylinositol-Dependent Membrane Fusion Induced by a Putative Fusogenic Sequence of Ebola Virus

The membrane-interacting abilities of three sequences representing the putative fusogenic subdomain of the Ebola virus transmembrane protein have been investigated. In the presence of calcium, the sequence EBO_GE (GAAIGLAWIPYFGPAAE) efficiently fused unilamellar vesicles composed of phosphatidylcholine, phosphatidylethanolamine, cholesterol, and phosphatidylinositol (molar ratio, 2:1:1:0.5), a mixture that roughly resembles the lipid composition of the hepatocyte plasma membrane. Analysis of the lipid dependence of the process demonstrated that the fusion activity of EBO_GE was promoted by phosphatidylinositol but not by other acidic phospholipids. In comparison, EBO_EA (EGAAIGLAWIPYFGPAA) and EBO_EE (EGAAIGLAWIPYFGPAAE) sequences, which are similar to EBO_GE except that they bear the negatively charged glutamate residue at the N terminus and at both the N and C termini, respectively, induced fusion to a lesser extent. As revealed by binding experiments, the glutamate residue at the N terminus severely impaired peptide-vesicle interaction. In addition, the fusion-competent EBO_GE sequence did not associate significantly with vesicles lacking phosphatidylinositol. Tryptophan fluorescence quenching by vesicles containing brominated phospholipids indicated that the EBO_GE peptide penetrated to the acyl chain level only when the membranes contained phosphatidylinositol. We conclude that binding and further penetration of the Ebola virus putative fusion peptide into membranes might be governed by the nature of the N-terminal residue and by the presence of phosphatidylinositol in the target membrane. Moreover, since insertion of such a peptide leads to membrane destabilization and fusion, the present data would be compatible with the involvement of this sequence in Ebola virus fusion.Ebola virus belongs to the Filoviridae family (23). This human pathogen occasionally causes epidemics of African hemorrhagic fever with a high rate of mortality (8, 23, 37). Little is known about the viral infectivity mechanism, and there is no specific treatment for Ebola virus hemorrhagic fever as yet. The most prominent pathology of Ebola virus infection includes necrosis of liver parenchyma as a direct consequence of virus replication (23). Ebola virus virions are composed of a helical nucleocapsid containing one linear, negative-sense, single-stranded RNA and surrounded by a lipidic envelope derived from the host cell plasma membrane (8, 23). The envelope contains solely one type of highly glycosylated protein (Ebola GP) arranged into oligomers, most probably trimers, which constitute the spikes that protrude from the virion surface (8, 30, 38, 39).The mode of entry of Ebola virus into target cells remains unknown. However it seems likely that the single surface protein Ebola GP is responsible for both receptor binding and membrane fusion during entry into the host cells. Homology analysis of its coding gene-derived sequence has identified several structural features that Ebola GP shares with other envelope fusion proteins derived from oncogenic retroviruses (12, 39). Just recently a detailed analysis has detected a high degree of structural homology between Ebola GP and the Rous sarcoma virus transmembrane protein (12). Several structural elements that might be involved in the ectodomain fusogenic function are shared by these viruses. In particular, there exists in both viruses an amino acid region bounded by cysteines that has at its center a sequence of approximately 16 uncharged and hydrophobic residues. Its location with respect to the viral membrane, the presence of a canonical fusion tripeptide (YFG in Ebola virus), and the fact that this sequence exhibits a high degree of identity among the Filoviridae members suggest that this region might constitute in Ebola virus the fusion peptide that is critical for virion-membrane fusion in the Retroviridae and other families (11, 40, 41).According to the most widely accepted mechanistic model proposed for the initial phase of the viral fusion process, activation of the viral spikes induces the exposure of previously buried hydrophobic fusion peptides in the vicinity of the target cell (5, 43). Further interaction of the viral fusion peptides with the cell membrane would depend mainly on the capacity for binding of these peptides to the membrane lipid components and could eventually trigger the process that brings about the actual merging of the viral and cell membranes via a currently unknown mechanism (41). This fact has justified the development of in vitro studies on the membrane-destabilizing effects of fusion peptides by using representative synthetic peptides of different viruses and model membranes (7, 15, 19, 29).The membrane environment into which the fusion peptide should partition obviously plays an important role in the process. Previous work from this laboratory has focused on the effect of the target membrane composition on viral fusion. Reports from this and other laboratories indicate the existence of conformational changes induced by lipidic components in the membrane-bound human immunodeficiency virus type 1 (HIV-1) fusion peptide (25, 28, 29), and we have identified a fusogenic conformation of the peptide represented by an extended β-type structure (25, 26, 28). The fusogenic interaction of the HIV-1 fusion peptide is, moreover, sensitive to factors that affect gp41 activity in vivo (27). Modulation of viral fusion by lipids has also been observed for complete virions and reconstituted systems fusing with model membranes (6, 24, 42). These observations indicate that enveloped viruses may optimize host interactions during the entry process, not only at the level of the selective binding to cell receptors but also at the level of the envelope fusion and subsequent capsid penetration.Our primary objective in this study was to confirm that the proposed fusogenic sequence for Ebola virus might interact with membranes, destabilize them, and eventually induce fusion. Because Ebola virus infects and replicates very efficiently in the liver, we initially employed as target membranes large unilamellar vesicles (LUV) made of a lipidic mixture that represents the hepatocyte plasma membrane composition (18). Our results demonstrate that this Ebola virus peptide interacts with phosphatidylinositol (PI)-containing membranes and induces vesicle fusion. Moreover, we show that the sequence lacking the negatively charged Glu residue at the N terminus interacts more efficiently with membranes. These data suggest that, similarly to the HIV-1 fusion peptide (26–28), the Ebola virus peptide segment under study may be important in viral fusion in vivo.     

Sphingolipid and cholesterol dependence of alphavirus membrane fusion. Lack of correlation with lipid raft formation in target liposomes

Semliki Forest virus (SFV) and Sindbis virus (SIN) are enveloped viruses that infect their host cells by receptor-mediated endocytosis and subsequent fusion from within acidic endosomes. Fusion of the viral envelope requires the presence of both cholesterol and sphingolipids in the target membrane. This is suggestive of a possible involvement of sphingolipid-cholesterol microdomains, or "lipid rafts," in the membrane fusion and cell entry process of the virus. In this study, large unilamellar vesicles (LUVs) were prepared from synthetic sphingolipids and sterols that vary with respect to their capacity to promote microdomain formation, as assessed by gradient flotation analysis in the presence of Triton X-100. SFV and SIN fused with LUVs irrespective of the presence or absence of Triton X-100-insoluble microdomains. These results suggest that SFV and SIN do not require the presence of lipid rafts for fusion with target membranes. Furthermore, it is not necessary for sphingolipids to reside in a detergent-insoluble complex with cholesterol to promote SFV or SIN fusion.     

Murine coronavirus requires lipid rafts for virus entry and cell-cell fusion but not for virus release

Thorp and Gallagher first reported that depletion of cholesterol inhibited virus entry and cell-cell fusion of mouse hepatitis virus (MHV), suggesting the importance of lipid rafts in MHV replication (E. B. Thorp and T. M. Gallagher, J. Virol. 78:2682-2692, 2004). However, the MHV receptor is not present in lipid rafts, and anchoring of the MHV receptor to lipid rafts did not enhance MHV infection; thus, the mechanism of lipid rafts involvement is not clear. In this study, we defined the mechanism and extent of lipid raft involvement in MHV replication. We showed that cholesterol depletion by methyl beta-cyclodextrin or filipin did not affect virus binding but reduced virus entry. Furthermore, MHV spike protein bound to nonraftraft membrane at 4 degrees C but shifted to lipid rafts at 37 degrees C, indicating a redistribution of membrane following virus binding. Thus, the lipid raft involvement in MHV entry occurs at a step following virus binding. We also found that the viral spike protein in the plasma membrane of the infected cells was associated with lipid rafts, whereas that in the Golgi membrane, where MHV matures, was not. Moreover, the buoyant density of the virion was not changed when MHV was produced from the cholesterol-depleted cells, suggesting that MHV does not incorporate lipid rafts into the virion. These results indicate that MHV release does not involve lipid rafts. However, MHV spike protein has an inherent ability to associate with lipid rafts. Correspondingly, cell-cell fusion induced by MHV was retarded by cholesterol depletion, consistent with the association of the spike protein with lipid rafts in the plasma membrane. These findings suggest that MHV entry requires specific interactions between the spike protein and lipid rafts, probably during the virus internalization step.     

Structure of the Ebola fusion peptide in a membrane-mimetic environment and the interaction with lipid rafts

The fusion peptide EBO(16) (GAAIGLAWIPYFGPAA) comprises the fusion domain of an internal sequence located in the envelope fusion glycoprotein (GP2) of the Ebola virus. This region interacts with the cellular membrane of the host and leads to membrane fusion. To gain insight into the mechanism of the peptide-membrane interaction and fusion, insertion of the peptide was modeled by experiments in which the tryptophan fluorescence and (1)H NMR were monitored in the presence of sodium dodecyl sulfate micelles or in the presence of detergent-resistant membrane fractions. In the presence of SDS micelles, EBO(16) undergoes a random coil-helix transition, showing a tendency to self-associate. The three-dimensional structure displays a 3(10)-helix in the central part of molecule, similar to the fusion peptides of many known membrane fusion proteins. Our results also reveal that EBO(16) can interact with detergent-resistant membrane fractions and strongly suggest that Trp-8 and Phe-12 are important for structure maintenance within the membrane bilayer. Replacement of tryptophan 8 with alanine (W8A) resulted in dramatic loss of helical structure, proving the importance of the aromatic ring in stabilizing the helix. Molecular dynamics studies of the interaction between the peptide and the target membrane also corroborated the crucial participation of these aromatic residues. The aromatic-aromatic interaction may provide a mechanism for the free energy coupling between random coil-helical transition and membrane anchoring. Our data shed light on the structural "domains" of fusion peptides and provide a clue for the development of a drug that might block the early steps of viral infection.     

The vesicle- and target-SNARE proteins that mediate Glut4 vesicle fusion are localized in detergent-insoluble lipid rafts present on distinct intracellular membranes

Insulin stimulates the fusion of intracellular vesicles containing the glucose transporter Glut4 with the plasma membrane in adipocytes and muscle cells. Glut4 vesicle fusion is thought to be catalyzed by the interaction of the vesicle soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptor VAMP2 with the target soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors SNAP-23 and syntaxin 4. Here, we use combined membrane fractionation, detergent solubility, and sucrose gradient flotation to demonstrate that the large majority (>70%) of SNAP-23 and a significant proportion of syntaxin 4 ( approximately 35%) are associated with plasma membrane lipid rafts in 3T3-L1 adipocytes. Furthermore, VAMP2 is shown to be concentrated in lipid rafts isolated from intracellular membranes. Insulin stimulation had no effect on the plasma membrane raft association of SNAP-23 or syntaxin 4 but promoted VAMP2 insertion into plasma membrane rafts. Immunofluorescence analysis revealed that SNAP-23 was clustered at the plasma membrane and almost completely segregated from the transferrin receptor. SNAP-23 distribution seemed to be distinct from caveolin-1, and clusters of SNAP-23 were dispersed after cholesterol extraction with methyl-beta-cyclodextrin, suggesting that the majority of SNAP-23 is associated with non-caveolar, cholesterol-rich lipid rafts. The results described implicate lipid rafts as important platforms for Glut4 vesicle fusion and suggest the hypothesis that such rafts may represent a spatial integration point of insulin signaling and membrane traffic.     

Resistance of Human Erythrocyte Membranes to Triton X-100 and C<Subscript>12</Subscript>E<Subscript>8</Subscript>

Lipid rafts are microdomains enriched in cholesterol and sphingolipids that contain specific membrane proteins. The resistance of domains to extraction by nonionic detergents at 4°C is the commonly used method to characterize these structures that are operationally defined as detergent-resistant membranes (DRMs). Because the selectivity of different detergents in defining membrane rafts has been questioned, we have compared DRMs from human erythrocytes prepared with two detergents: Triton X-100 and C₁₂E₈. The DRMs obtained presented a cholesterol/protein mass ratio three times higher than in the whole membrane. Flotillin-2 was revealed in trace amounts in DRMs obtained with C₁₂E₈, but it was almost completely confined within the DRM fraction with Triton X-100. Differently, stomatin was found distributed in DRM and non-DRM fractions for both detergents. We have also measured the order parameter (S) of nitroxide spin labels inserted into DRMs by means of electron paramagnetic resonance. The 5- and 16-stearic acid spin label revealed significantly higher S values for DRMs obtained with either Triton X-100 or C₁₂E₈ in comparison to intact cells, while the difference in the S values between Triton X-100 and C₁₂E₈ DRMs was not statistically significant. Our results suggest that although the acyl chain packing is similar in DRMs prepared with either Triton X-100 or C₁₂E₈ detergent, protein content is dissimilar, with flotillin-2 being selectively enriched in Triton X-100 DRMs.     

Specific association of glycoprotein B with lipid rafts during herpes simplex virus entry

Herpes simplex virus (HSV) entry requires the interaction of glycoprotein D (gD) with a cellular receptor such as herpesvirus entry mediator (HVEM or HveA) or nectin-1 (HveC). However, the fusion mechanism is still not understood. Since cholesterol-enriched cell membrane lipid rafts are involved in the entry of other enveloped viruses such as human immunodeficiency virus and Ebola virus, we tested whether HSV entry proceeds similarly. Vero cells and cells expressing either HVEM or nectin-1 were treated with cholesterol-sequestering drugs such as methyl-beta-cyclodextrin or nystatin and then exposed to virus. In all cases, virus entry was inhibited in a dose-dependent manner, and the inhibitory effect was fully reversible by replenishment of cholesterol. To examine the association of HVEM and nectin-1 with lipid rafts, we analyzed whether they partitioned into nonionic detergent-insoluble glycolipid-enriched membranes (DIG). There was no constitutive association of either receptor with DIG. Binding of soluble gD or virus to cells did not result in association of nectin-1 with the raft-containing fractions. However, during infection, a fraction of gB but not gC, gD, or gH associated with DIG. Similarly, when cells were incubated with truncated soluble glycoproteins, soluble gB but not gC was found associated with DIG. Together, these data favor a model in which HSV uses gB to rapidly mobilize lipid rafts that may serve as a platform for entry and cell signaling. It also suggests that gB may interact with a cellular molecule associated with lipid rafts.     

Membrane partitioning of various delta-opioid receptor forms before and after agonist activations: the effect of cholesterol

Lipid rafts depicted as densely packed and thicker membrane microdomains, based on the dynamic clustering of cholesterol and sphingolipids, may help as platforms involved in a wide variety of cellular processes. The reasons why proteins segregate into rafts are yet to be clarified. The human delta opioid receptor (hDOR) reconstituted in a model system has been characterised after ligand binding by an elongation of its transmembrane part, inducing rearrangement of its lipid microenvironment [Alves, Salamon, Hruby, and Tollin (2005) Biochemistry 44, 9168-9178]. We used hDOR to understand better the correlation between its function and its membrane microdomain localisation. A fusion protein of hDOR with the Green Fluorescent Protein (DOR*) allows precise receptor membrane quantification. Here we report that (i) a fraction of the total receptor pool requires cholesterol for binding activity, (ii) G-proteins stabilize a high affinity state conformation which does not seem modulated by cholesterol. In relation to its distribution, and (iii) a fraction of DOR* is constitutively associated with detergent-resistant membranes (DRM) characterised by an enrichment in lipids and proteins raft markers. (iv) An increase in the quantity of DOR* was observed upon agonist addition. (v) This DRM relocation is prevented by uncoupling the receptor-G-protein interaction.     

Light- and guanosine 5'-3-O-(thio)triphosphate-sensitive localization of a G protein and its effector on detergent-resistant membrane rafts in rod photoreceptor outer segments

Detergent-resistant membrane microdomains in the plasma membrane, known as lipid rafts, have been implicated in various cellular processes. We report here that a low-density Triton X-100-insoluble membrane (detergent-resistant membrane; DRM) fraction is present in bovine rod photoreceptor outer segments (ROS). In dark-adapted ROS, transducin and most of cGMP-phosphodiesterase (PDE) were detergent-soluble. When ROS membranes were exposed to light, however, a large portion of transducin localized in the DRM fraction. Furthermore, on addition of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) to light-bleached ROS, transducin became detergent-soluble again. PDE was not recruited to the DRM fraction after light stimulus alone, but simultaneous stimulation by light and GTPgammaS induced a massive translocation of all PDE subunits to the DRM. A cholesterol-removing reagent, methyl-beta-cyclodextrin, selectively but partially solubilized PDE from the DRM, suggesting that cholesterol contributes, at least in part, to the association of PDE with the DRM. By contrast, transducin was not extracted by the depletion of cholesterol. These data suggest that transducin and PDE are likely to perform their functions in phototransduction by changing their localization between two distinct lipid phases, rafts and surrounding fluid membrane, on disc membranes in an activation-dependent manner.     

Structure and cholesterol dynamics of caveolae/raft and nonraft plasma membrane domains

Despite recognition that the plasma membrane (PM) is comprised of lipid raft domains that are key organizing sites of multiple signaling pathways and other cell functions, limited information is available regarding the structure and function in sterol dynamics of these microdomains. To begin to resolve these issues, MDCK membranes were subfractionated by three different techniques to produce (i) detergent-resistant membranes (DRM) and detergent-soluble membranes (DSM), (ii) nondetergent caveolae/rafts (NDCR), and (iii) nondetergent, affinity-purified caveolae/rafts (ACR) and noncaveolae/nonrafts (NR). ACR exhibited the least cross contamination with other PM domains or intracellular membranes, in marked contrast to DRM that contained the highest level of cross contaminants. Spectral properties of dehydroergosterol (DHE), a naturally occurring fluorescent sterol, showed that ACR, NDCR, and NR did not contain crystalline sterol, consistent with the lack of crystalline sterol in PM of intact cells. In contrast, DRM contained significant levels of crystalline sterol. Fluorescence polarization of membrane probes showed that ACR were the least fluid and had the highest transbilayer fluidity gradient, the most liquid ordered phase, and the sterol dynamics most responsive to sterol carrier protein-2 (SCP-2). In contrast, DRM had structural properties similar to those of NR, anomalous (very fast) spontaneous sterol dynamics, and sterol dynamics that were unresponsive to SCP-2. Differences between the structural and functional properties of DRM and those of the nondetergent preparations (ACR and NDCR) were not due to the presence of detergent. A nondetergent, affinity-purified (ACR) lipid domain fraction isolated from MDCK cells for the first time revealed unique structural (noncrystalline sterol, liquid-ordered, high transbilayer fluidity gradient) and functional (cholesterol dynamics) properties of lipid rafts as compared to nonrafts (NR). In summary, this study showed membrane microdomains (rafts/caveolae) isolated by three different methodologies have unique structural, functional, and organizational characteristics.     

Asymmetric requirement for cholesterol in receptor-bearing but not envelope-bearing membranes for fusion mediated by ecotropic murine leukemia virus

We show that fusion mediated by ecotropic murine leukemia virus envelope is dependent on cholesterol in receptor-bearing membranes. The effect is >10 times larger in insect cells than mammalian cells, probably because the former can be more extensively depleted of cholesterol. The fact that cholesterol is apparently not needed in envelope-bearing membranes suggests that it plays a role in an asymmetric step in membrane fusion and argues against a class of models in which cholesterol is important in symmetric fusion intermediates. The insect cell system has promise for clarifying the role of membrane rafts in other aspects of cell physiology.     

Membrane partitioning of various δ-opioid receptor forms before and after agonist activations: The effect of cholesterol

Lipid rafts depicted as densely packed and thicker membrane microdomains, based on the dynamic clustering of cholesterol and sphingolipids, may help as platforms involved in a wide variety of cellular processes. The reasons why proteins segregate into rafts are yet to be clarified. The human delta opioid receptor (hDOR) reconstituted in a model system has been characterised after ligand binding by an elongation of its transmembrane part, inducing rearrangement of its lipid microenvironment [Alves, Salamon, Hruby, and Tollin (2005) Biochemistry 44, 9168-9178]. We used hDOR to understand better the correlation between its function and its membrane microdomain localisation. A fusion protein of hDOR with the Green Fluorescent Protein (DOR?) allows precise receptor membrane quantification. Here we report that (i) a fraction of the total receptor pool requires cholesterol for binding activity, (ii) G-proteins stabilize a high affinity state conformation which does not seem modulated by cholesterol. In relation to its distribution, and (iii) a fraction of DOR? is constitutively associated with detergent-resistant membranes (DRM) characterised by an enrichment in lipids and proteins raft markers. (iv) An increase in the quantity of DOR? was observed upon agonist addition. (v) This DRM relocation is prevented by uncoupling the receptor-G-protein interaction.     

The lipid raft proteome of Borrelia burgdorferi

Eukaryotic lipid rafts are membrane microdomains that have significant amounts of cholesterol and a selective set of proteins that have been associated with multiple biological functions. The Lyme disease agent, Borrelia burgdorferi, is one of an increasing number of bacterial pathogens that incorporates cholesterol onto its membrane, and form cholesterol glycolipid domains that possess all the hallmarks of eukaryotic lipid rafts. In this study, we isolated lipid rafts from cultured B. burgdorferi as a detergent resistant membrane (DRM) fraction on density gradients, and characterized those molecules that partitioned exclusively or are highly enriched in these domains. Cholesterol glycolipids, the previously known raft‐associated lipoproteins OspA and OpsB, and cholera toxin partitioned into the lipid rafts fraction indicating compatibility with components of the DRM. The proteome of lipid rafts was analyzed by a combination of LC‐MS/MS or MudPIT. Identified proteins were analyzed in silico for parameters that included localization, isoelectric point, molecular mass and biological function. The proteome provided a consistent pattern of lipoproteins, proteases and their substrates, sensing molecules and prokaryotic homologs of eukaryotic lipid rafts. This study provides the first analysis of a prokaryotic lipid raft and has relevance for the biology of Borrelia, other pathogenic bacteria, as well as for the evolution of these structures. All MS data have been deposited in the ProteomeXchange with identifier PXD002365 ( http://proteomecentral.proteomexchange.org/dataset/PXD002365 ).     

Effect of Cholesterol Depletion and Temperature on the Isolation of Detergent-Resistant Membranes from Human Erythrocytes

Transient lateral microdomains or lipid rafts play important roles in many physiological membrane-mediated cell processes. Detergent-resistant membranes (DRMs) are good models for the study of lipid rafts. Here we report that DRMs can be obtained by treating human erythrocytes with the nonionic detergents Triton X-100 or octaethylene glycol monododecyl ether (C₁₂E₈) at 37°C, and by treatment at 4°C of cholesterol-depleted erythrocytes. Electron paramagnetic resonance with spin labels inserted at different membrane depths (5- and 16-doxyl stearic acids, 5-SASL and 16-SASL) were used to measure the order parameter (S) of the cell membranes and DRMs. We previously reported significantly higher S values in DRMs with respect to intact erythrocyte membranes. Here we show that higher S values were still measurable in DRMs prepared from intact erythrocytes at 37°C, or from cholesterol-depleted cells at 4°C, for both detergents. For 5-SASL only, increased S values were measured in 4°C DRMs obtained from cholesterol-depleted versus intact erythrocytes. Flotillin-2, a protein marker of lipid rafts, was found in DRMs from intact cells in trace amounts but it was sensitively increased in C₁₂E₈ DRMs prepared at 4°C from cholesterol-depleted erythrocytes, while the membrane-skeletal proteins spectrin and actin were excluded from both Triton X-100 and C₁₂E₈ DRMs. However, contrary to the 4°C treatment results, flotillin-2 and stomatin were not resistant to Triton X-100 and C₁₂E₈ treatment at physiological temperature. The role of cholesterol in DRMs formation is discussed and the results presented provide further support for the use of C₁₂E₈ to the study of DRMs.     

Nuclear magnetic resonance evidence for retention of a lamellar membrane phase with curvature in the presence of large quantities of the HIV fusion peptide

The HIV fusion peptide (HFP) is a biologically relevant model system to understand virus/host cell fusion. ²H and ³¹P NMR spectroscopies were applied to probe the structure and motion of membranes with bound HFP and with a lipid headgroup and cholesterol composition comparable to that of membranes of host cells of HIV. The lamellar phase was retained for a variety of highly fusogenic HFP constructs as well as a non-fusogenic HFP construct and for the influenza virus fusion peptide. The lamellar phase is therefore a reasonable structure for modeling the location of HFP in lipid/cholesterol dispersions. Relative to no HFP, membrane dispersions with HFP had faster ³¹P transverse relaxation and faster transverse relaxation of acyl chain ²H nuclei closest to the lipid headgroups. Relative to no HFP, mechanically aligned membrane samples with HFP had broader ³¹P signals with a larger fraction of unoriented membrane. The relaxation and aligned sample data are consistent with bilayer curvature induced by the HFP which may be related to its fusion catalytic function. In some contrast to the subtle effects of HFP on a host-cell-like membrane composition, an isotropic phase was observed in dispersions rich in phosphatidylethanolamine lipids and with bound HFP.     

Aspirin inhibits formation of cholesterol rafts in fluid lipid membranes

Aspirin and other non-steroidal anti-inflammatory drugs have a high affinity for phospholipid membranes, altering their structure and biophysical properties. Aspirin has been shown to partition into the lipid head groups, thereby increasing membrane fluidity. Cholesterol is another well known mediator of membrane fluidity, in turn increasing membrane stiffness. As well, cholesterol is believed to distribute unevenly within lipid membranes leading to the formation of lipid rafts or plaques. In many studies, aspirin has increased positive outcomes for patients with high cholesterol. We are interested if these effects may be, at least partially, the result of a non-specific interaction between aspirin and cholesterol in lipid membranes.We have studied the effect of aspirin on the organization of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) membranes containing cholesterol. Through Langmuir–Blodgett experiments we show that aspirin increases the area per lipid and decreases compressibility at 32.5 mol% cholesterol, leading to a significant increase of fluidity of the membranes. Differential scanning calorimetry provides evidence for the formation of meta-stable structures in the presence of aspirin. The molecular organization of lipids, cholesterol and aspirin was studied using neutron diffraction. While the formation of rafts has been reported in binary DPPC/cholesterol membranes, aspirin was found to locally disrupt membrane organization and lead to the frustration of raft formation. Our results suggest that aspirin is able to directly oppose the formation of cholesterol structures through non-specific interactions with lipid membranes.     

Effect of cholesterol on the interaction of the HIV GP41 fusion peptide with model membranes. Importance of the membrane dipole potential

Fusion of viral and cell membranes is a key event in the process by which the human immunodeficiency virus (HIV) enters the target cell. Membrane fusion is facilitated by the interaction of the viral gp41 fusion peptide with the cell membrane. Using synthetic peptides and model membrane systems, it has been established that the sequence of events implies the binding of the peptide to the membrane, followed by a conformational change (transformation of unordered and helical structures into beta-aggregates) which precedes lipid mixing. It is known that this process can be influenced by the membrane lipid composition. In the present work we have undertaken a systematic study in order to determine the influence of cholesterol (abundant in the viral membrane) in the sequence of events leading to lipid mixing. Besides its effect on membrane fluidity, cholesterol can affect a less known physical parameter, the membrane dipole potential. Using the dipole potential fluorescent sensor di-8-ANEPPS together with other biophysical techniques, we show that cholesterol increases the affinity of the fusion peptide for the model membranes, and although it lowers the extent of lipid mixing, it increases the mixing rate. The influence of cholesterol on the peptide affinity and the lipid mixing rate are shown to be mainly due to its influence of the membrane dipole potential, whereas the lipid mixing extent and peptide conformational changes seem to be more dependent on other membrane parameters such as membrane fluidity and hydration.     

Contribution of PIP-5 kinase Iα to raft-based FcγRIIA signaling

Receptor FcγIIA (FcγRIIA) associates with plasma membrane rafts upon activation to trigger signaling cascades leading to actin polymerization. We examined whether compartmentalization of PI(4,5)P₂ and PI(4,5)P₂-synthesizing PIP5-kinase Iα to rafts contributes to FcγRIIA signaling. A fraction of PIP5-kinase Iα was detected in raft-originating detergent-resistant membranes (DRM) isolated from U937 monocytes and other cells. The DRM of U937 monocytes contained also a major fraction of PI(4,5)P₂. PIP5-kinase Iα bound PI(4,5)P₂, and depletion of the lipid displaced PIP5-kinase Iα from the DRM. Activation of FcγRIIA in BHK transfectants led to recruitment of the kinase to the plasma membrane and enrichment of DRM in PI(4,5)P₂. Immunofluorescence studies revealed that in resting cells the kinase was associated with the plasma membrane, cytoplasmic vesicles and the nucleus. After FcγRIIA activation, PIP5-kinase Iα and PI(4,5)P₂ co-localized transiently with the activated receptor at distinct cellular locations. Immunoelectron microscopy studies revealed that PIP5-kinase Iα and PI(4,5)P₂ were present at the edges of electron-dense assemblies containing activated FcγRIIA in their core. The data suggest that activation of FcγRIIA leads to membrane rafts coalescing into signaling platforms containing PIP5-kinase Iα and PI(4,5)P₂.