Microscopy Of Giant Unilamellar Vesicles Research Articles

Membrane lipid organization and nicotinic acetylcholine receptor function: A two-way physiological relationship

Nicotinic acetylcholine receptors (nAChRs) are involved in a great range of physiological and pathological conditions. Since they are transmembrane proteins, they interact strongly with the lipids surrounding them. Thus, the plasma membrane composition and heterogeneity play an essential role for the correct nAChR function, on the one hand, and the nAChR influences its immediate lipid environment, on the other hand. The aim of this work was to investigate in more detail the role of the biophysical properties of the membrane in nAChR function and vice versa, focusing on the relationship between Chol and nAChRs. To this end, we worked with different model systems which were treated either with (i) more Chol, (ii) cholesteryl hemisuccinate, or (iii) the enzyme cholesterol oxidase to generate different membrane sterol conditions and in the absence and presence of γTM4 peptide as a representative model of the nAChR.Fluorescence measurements with crystal violet and patch-clamp recordings were used to study nAChR conformation and function, respectively. Using confocal microscopy of giant unilamellar vesicles we probed the membrane phase state/order and organization (coexistence of lipid domains) and lipid-nAChR interaction. Our results show a feedback relationship between membrane organization and nAChR function, i.e. whereas the presence of a model of nAChRs conditions membrane organization, changing its lipid microenvironment, membrane organization and composition perturb nAChRs function. We postulate that nAChRs have a gain of function in disordered membrane environments but a loss of function in ordered ones, and that Chol molecules at the outer leaflet in annular sites and at the inner leaflet in non-annular sites are related to nAChR gating and desensitization, respectively. Thus, depending on the membrane composition, organization, and/or order, the nAChR adopts different conformations and locates in distinct lipid domains and this has a direct effect on its function.

Phase behaviour of C18-N-acyl sphingolipids, the prevalent species in human brain

Lipidomic analysis of the N-acyl components of sphingolipids in different mammalian tissues had revealed that brain tissue differed from all the other samples in that SM contained mainly C18:0 and C24:1N-acyl chains, and that the most abundant Cer species was C18:0. Only in the nervous system was C18:0 found in sizable proportions. The high levels of C18:0 and C16:0, respectively in brain and non-brain SM, were important because SM is by far the most abundant sphingolipid in the plasma membrane. In view of these observations, the present paper is devoted to a comparative study of the properties of C16:0 and C18:0 sphingolipids (SM and Cer) pure and in mixtures of increasing complexities, using differential scanning calorimetry, confocal microscopy of giant unilamellar vesicles, and correlative fluorescence microscopy and atomic force microscopy of supported lipid bilayers. Membrane rigidity was measured by force spectroscopy. It was found that in mixtures containing dioleoyl phosphatidylcholine, sphingomyelin and cholesterol, i.e. representing the lipids predominant in the outer monolayer of cell membranes, lateral inhomogeneities occurred, with the formation of rigid domains within a continuous fluid phase. Inclusion of saturated Cer in the system was always found to increase the rigidity of the segregated domains. C18:0-based sphingolipids exhibit hydrocarbon chain-length asymmetry, and some singularities observed with this N-acyl chain, e.g. complex calorimetric endotherms, could be attributed to this property. Moreover, C18:0-based sphingolipids, that are typical of the excitable cells, were less miscible with the fluid phase than their C16:0 counterparts. The results could be interpreted as suggesting that the predominance of C18:0 Cer in the nervous system would contribute to the tightness of its plasma membranes, thus facilitating maintenance of the ion gradients.

Contrasting roles of oxidized lipids in modulating membrane microdomains

Lipid rafts display a lateral heterogeneity forming membrane microdomains that hold a fundamental role on biological membranes and are indispensable to physiological functions of cells. Oxidative stress in cellular environments may cause lipid oxidation, changing membrane composition and organization, thus implying in effects in cell signaling and even loss of homeostasis. The individual contribution of oxidized lipid species to the formation or disruption of lipid rafts in membranes still remains unknown. Here, we investigate the role of different structures of oxidized phospholipids on rafts microdomains by carefully controlling the membrane composition. Our experimental approach based on fluorescence microscopy of giant unilamellar vesicles (GUV) enables the direct visualization of the impact of hydroperoxidized POPC lipid (referred to as POPCOOH) and shortened chain lipid PazePC (1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine) on phase separation. We found that the molecular structure of oxidized lipid is of paramount importance on lipid mixing and/or demixing. The hydrophobic mismatch promoted by POPCOOH coupled to its cylindrical molecular shape favor microdomains formation. In contrast, the conical shape of PazePC causes disarrangement of lipid 2D organized platforms. Our findings contribute to better unraveling how oxidized phospholipids can trigger formation or disruption of lipid rafts. As a consequence, phospholipid oxidation may indirectly affect association or dissociation of key biomolecules in the rafts thus altering cell signaling and homeostasis.

Synthesis, biophysical and functional studies of two BP100 analogues modified by a hydrophobic chain and a cyclic peptide

Antimicrobial peptides (AMPs) work as a primary defense against pathogenic microorganisms. BP100, (KKLFKKILKYL-NH2), a rationally designed short, highly cationic AMP, acts against many bacteria, displaying low toxicity to eukaryotic cells. Previously we found that its mechanism of action depends on membrane surface charge and on peptide-to-lipid ratio. Here we present the synthesis of two BP100 analogs: BP100‑alanyl‑hexadecyl‑1‑amine (BP100-Ala-NH-C16H33) and cyclo(1‑4)‑d‑Cys1, Ile2, Leu3, Cys4-BP100 (Cyclo(1‑4)‑cILC-BP100). We examined their binding to large unilamellar vesicles (LUV), conformational and functional properties, and compared with those of BP100. The analogs bound to membranes with higher affinity and a lesser dependence on electrostatic forces than BP100. In the presence of LUV, BP100 and BP100-Ala-NH-C16H33 acquired α-helical conformation, while Cyclo(1‑4)‑cILC-BP100) was partly α-helical and partly β-turn. Taking in conjunction: 1. particle sizes and zeta potential, 2. effects on lipid flip-flop, 3. leakage of LUVs internal contents, and 4. optical microscopy of giant unilamellar vesicles, we concluded that at high concentrations, all three peptides acted by a carpet mechanism, while at low concentrations the peptides acted by disorganizing the lipid bilayer, probably causing membrane thinning. The higher activity and lesser membrane surface charge dependence of the analogs was probably due to their greater hydrophobicity. The MIC values of both analogs towards Gram-positive and Gram-negative bacteria were similar to those of BP100 but both analogues were more hemolytic. Confocal microscopy showed Gram-positive B. subtilis killing with concomitant extensive membrane damage suggestive of lipid clustering, or peptide-lipid aggregation. These results were in agreement with those found in model membranes.

Biophysical approaches in the study of biomembrane solubilization: quantitative assessment and the role of lateral inhomogeneity.

Detergents are amphiphilic molecules widely used to solubilize biological membranes and/or extract their components. Nevertheless, because of the complex composition of biomembranes, their solubilization by detergents has not been systematically studied. In this review, we address the solubilization of erythrocytes, which provide a relatively simple, robust and easy to handle biomembrane, and of biomimetic models, to stress the role of the lipid composition on the solubilization process. First, results of a systematic study on the solubilization of human erythrocyte membranes by different series of non-ionic (Triton, CxEy, Brij, Renex, Tween), anionic (bile salts) and zwitterionic (ASB, CHAPS) detergents are shown. Such quantitative approach allowed us to propose Resat-the effective detergent/lipid molar ratio in the membrane for the onset of hemolysis as a new parameter to classify the solubilization efficiency of detergents. Second, detergent-resistant membranes (DRMs) obtained as a result of the partial solubilization of erythrocytes by TX-100, C12E8 and Brij detergents are examined. DRMs were characterized by their cholesterol, sphingolipid and specific proteins content, as well as lipid packing. Finally, lipid bilayers of tuned lipid composition forming liposomes were used to investigate the solubilization process of membranes of different compositions/phases induced by Triton X-100. Optical microscopy of giant unilamellar vesicles revealed that pure phospholipid membranes are fully solubilized, whereas the presence of cholesterol renders the mixture partially or even fully insoluble, depending on the composition. Additionally, Triton X-100 induced phase separation in raft-like mixtures, and selective solubilization of the fluid phase only.

Molecular organization, localization and orientation of antifungal antibiotic amphotericin B in a single lipid bilayer.

Amphotericin B is a popular antifungal antibiotic, a gold standard in treatment of systemic mycotic infections, due to its high effectiveness. On the other hand, applicability of the drug is limited by its considerable toxicity to patients. Biomembranes are a primary target of physiological activity of amphotericin B and both the pharmacologically desired and toxic side effects of the drug relay on its molecular organization in the lipid phase. In the present work, molecular organization, localization and orientation of amphotericin B, in a single lipid bilayer system, was analysed simultaneously, thanks to application of a confocal fluorescence lifetime imaging microscopy of giant unilamellar vesicles. The results show that the presence of sterols, in the lipid phase, promotes formation of supramolecular structures of amphotericin B and their penetration into the membrane hydrophobic core. The fact that such an effect is substantially less pronounced in the case of cholesterol than ergosterol, the sterol of fungal membranes, provides molecular insight into the selectivity of the drug.

Role of Aminophospholipids in the Formation of Lipid Rafts in Model Membranes

The phase separation of aminophospholipids in glycerophospholipid matrix and the effect of cholesterol were studied by means of fluorescence microscopy of giant unilamellar vesicles (GUV). GUVs were composed of binary mixtures, egg yolk phosphatidylcholine (eggPC)/egg yolk phosphatidylethanolamine (eggPE) and egg yolk phosphatidylcholine (eggPC)/brain phosphatidylserine (brainPS), and ternary ones with both aminophospholipids (eggPC/eggPE/brainPS). Gel/liquid-disordered phase coexistence was detected in these mixtures, where aminophospholipids segregate in gel leaf-like domains. When cholesterol (CHOL) was added, the phase separation was shifted at lower temperatures. CHOL increases miscibility of aminophospholipids in PC matrix. Addition of PE and PS to the ternary mixtures (eggPC/eggSM/CHOL) induced liquid-ordered domain formation at higher temperatures. Based on these results, one can conclude that aminophospholipids promote the formation of Lo domains.

End-Product Diacylglycerol Enhances the Activity of PI-PLC through Changes in Membrane Domain Structure

Diacylglycerol (DAG)-induced activation of phosphatidylinositol-phospholipase C (PI-PLC) was studied with vesicles containing PI, either pure or in mixtures with dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine, sphingomyelin, or galactosylceramide, used as substrates. At 22°C, DAG at 33 mol % increased PI-PLC activity in all of the mixtures, but not in pure PI bilayers. DAG also caused an overall decrease in diphenylhexatriene fluorescence polarization (decreased molecular order) in all samples, and increased overall enzyme binding. Confocal fluorescence microscopy of giant unilamellar vesicles of all of the compositions under study, with or without DAG, and quantitative evaluation of the phase behavior using Laurdan generalized polarization, and of enzyme binding to the various domains, indicated that DAG activates PI-PLC whenever it can generate fluid domains to which the enzyme can bind with high affinity. In the specific case of PI/dimyristoyl phosphatidylcholine bilayers at 22°C, DAG induced/increased enzyme binding and activation, but no microscopic domain separation was observed. The presence of DAG-generated nanodomains, or of DAG-induced lipid packing defects, is proposed instead for this system. In PI/galactosylceramide mixtures, DAG may exert its activation role through the generation of small vesicles, which PI-PLC is known to degrade at higher rates. In general, our results indicate that global measurements obtained using fluorescent probes in vesicle suspensions in a cuvette are not sufficient to elucidate DAG effects that take place at the domain level. The above data reinforce the idea that DAG functions as an important physical agent in regulating membrane and cell properties.

Membrane Resistance to Detergent-Induced Solubilization as a Matter of Physical Phase in Binary Lipid Mixtures

Molecular biology protocols routinely use detergents for extraction of membrane or intracellular components. However, detergent-induced solubilization usually results in the presence of membrane debris, with similar composition to membrane rafts and liquid-ordered (Lo) phase. In the present work we have studied the solubilization by the detergent Triton X-100 (TX-100) of membranes composed of different lipid mixtures and phases. The membrane compositions evaluated were palmitoyl-oleoyl-phosphatidylcholine (POPC) and POPC:cholesterol (7:3) (liquid disordered phase, Ld), Sphingomyelin (SM, Gel phase) and SM:cholesterol (7:3) (Lo phase). The solubilization was followed by optical microscopy of giant unilamellar vesicles (GUVs), showing that POPC and SM membranes are totally solubilized by TX-100, while POPC:cholesterol membranes are partially solubilized and SM:cholesterol membranes are completely insoluble. The incorporation of TX-100 in GUVs was measured trough area increase for vesicles in fluid phases, showing that for POPC membranes, the addition of cholesterol diminishes the incorporation of TX-100 and for vesicles of SM:cholesterol the area increase very low. Light scattering of large unilamellar vesicles (LUVs) titrated with TX-100 confirmed the results of optical microscopy. The isothermal titration calorimetry (ITC) of LUVs with detergent showed that the addition of cholesterol diminishes the partition coefficient of TX-100 in both POPC and SM membranes. In addition, enthalpic changes are observed in the same detergent-lipid molar ratios associated with the onset and completion of the solubilization process. Differential scanning calorimetry of SM LUVs with detergent show detergent-induced fluidification of the gel phase, which could account for part of the large endothermic contribution measured with ITC. Our result suggests that cholesterol increases membrane resistance to detergent-induced solubilization and confirmed that the Lo phase is virtually insoluble. Supported by: FAPESP and CNPq

Solubilization of binary lipid mixtures by the detergent Triton X-100: the role of cholesterol.

The solubilization of lipid bilayers of different composition and phase by the detergent Triton X-100 (Triton X-100) was investigated using optical and fluorescence microscopy of giant unilamellar vesicles (GUVs) and light scattering of large unilamellar vesicles (LUVs). The compositions explored were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), in the liquid-disordered (Ld) phase, sphingomyelin (SM), in the gel phase, and binary mixtures of these phospholipids with 30 mol % cholesterol (chol), resulting in bilayers in the Ld and liquid-ordered (Lo) phases, respectively. We show that the phospholipid bilayers are completely soluble in TX-100, but optical microscopy reveals that whereas fluid POPC is gradually solubilized by TX-100, gel SM is first shattered in bilayer fragments. Incorporation of TX-100 in the membrane leads to increase in GUV area, which was quantified and expressed as bound detergent-to-lipid molar ratio. The partition of TX-100 in POPC is very high, decreases in POPC/chol, and is negligible in SM/chol. Fluorescence microscopy shows that TX-100 induces Lo/Ld phase separation in previously homogeneous POPC/chol GUVs, and insoluble bilayer fragments/vesicles are detected with optical microscopy and light scattering. Vesicles of SM/chol, in the Lo phase, are virtually insoluble in TX-100. Taken together, our results show that the presence of cholesterol is the origin of membrane resistance to solubilization, which depending on the specific lipid mixture can result in either partially (POPC/chol) or completely (SM/chol) insoluble mixtures.

Ether- versus Ester-Linked Phospholipid Bilayers Containing either Linear or Branched Apolar Chains

We studied the properties of bilayers formed by ether-and ester-containing phospholipids, whose hydrocarbon chains can be either linear or branched, using sn-1,2 dipalmitoyl, dihexadecyl, diphytanoyl, and diphytanyl phosphatidylcholines (DPPC, DHPC, DPhoPC, and DPhPC, respectively) either pure or in binary mixtures. Differential scanning calorimetry and confocal fluorescence microscopy of giant unilamellar vesicles concurred in showing that equimolar mixtures of linear and branched lipids gave rise to gel/fluid phase coexistence at room temperature. Mixtures containing DHPC evolved in time (0.5 h) from initial reticulated domains to extended solid ones when an equilibrium was achieved. The nanomechanical properties of supported planar bilayers formed by each of the four lipids studied by atomic force microscopy revealed average breakdown forces Fb decreasing in the order DHPC ≥ DPPC > DPhoPC >> DPhPC. Moreover, except for DPPC, two different Fb values were found for each lipid. Atomic force microscopy imaging of DHPC was peculiar in showing two coexisting phases of different heights, probably corresponding to an interdigitated gel phase that gradually transformed, over a period of 0.5 h, into a regular tilted gel phase. Permeability to nonelectrolytes showed that linear-chain phospholipids allowed a higher rate of solute + water diffusion than branched-chain phospholipids, yet the former supported a smaller extent of swelling of the corresponding vesicles. Ether or ester bonds appeared to have only a minor effect on permeability.

Structural features of ultradeformable archaeosomes for topical delivery of ovalbumin

The ultradeformable archaeosomes (UDA, made of total polar archaeolipids (TPA) extracted from the extreme halophile archaea Halorubrum tebenquichense:soybean phosphatidylcholine (SPC):sodium cholate (NaChol), 3:3:1 w:w), are promising topical adjuvants showing high deformability, an essential property for intact skin penetration up to the viable epidermis/dermis. To gain insights on UDA structure, the interactions between TPA, SPC and the edge activator NaChol, were assessed by electrospray ionization mass spectroscopy (ESI-MS) and confocal fluorescence microscopy of giant unilamellar vesicles (GUV). The non covalent heterodimers NaChol–SPC, NaChol–phosphatidylglycerophosphate methyl ether (PGPMe), NaChol–sulfated diglycosyl diphytanyl–glycerol diether (SDGD5) and SPC–PGPMe detected in the gas phase by ESI-MS after direct infusion of UDA, together with the homogeneous partition of FASTDiO and DiIC18 in GUV suggested that in these proportions, lipids and NaChol were miscible. We propose therefore, a model where in UDA the SPC diluted sufficient enough in the rich PGPMe TPA, so as to the low lateral mobility of molecules (typical of rich in PGPMe bilayers) was no longer experienced. We also found that 50μm deep within in vitro human skin canyons, the fluorescence of Alexa fluor 647-ovalbumin in UDL was ∼1.5 folds higher than in UDA, indicating a potential steric hindrance of the voluminous structure of PGPMe UDA bilayer, to the penetration of a particulate cargo such as the 7nm diameter ovalbumin. According to these observations, a further reduction in PGPMe – a lipid playing no immune role – content could help to improve the performance of UDA as topical adjuvants.

The Interaction Between the Antimicrobial Peptide K-Hya1 and Model Membranes: Distinct Action in Neutral or Negatively Charged Bilayers

Antimicrobial peptides (AMPs) are important molecules for the innate immune defense system of plants and animals. The antimicrobial peptide Hylina1 (Hya1) was isolated from arboreal South African frog Hypsiboas albopunctatus. In this work, we investigate the interaction of an analogue peptide, K-Hya1 (KIFGAIWPLALGALKNLIK-NH2) with model membranes composed of DPPC (dipalmitoyl phosphatidylcholine), DPPG dipalmitoyl phosphatidylglycerol) and DPPC:DPPG 1:1 or of POPC (palmitoyl oleoyl phosphatidylcholine) and POPC:POPG (palmitoyl oleoyl phosphatidylglycerol) with different techniques. Differential Scanning Calorimetry (DSC) profiles show distinct environments in the bilayer with anionic lipids, suggesting a disturbed region due to peptide adsorption, and an unaltered region. On the other hand, in neutral membranes an average perturbation is observed, which suggests a superficial interaction. Steady-state fluorescence spectroscopy of the intrinsic Trp residue shows a deeper insertion of this residue into charged bilayers as compared with neutral membranes. Dye-leakage experiments show that membrane charge also modulates the kinetics of membrane permeabilization, which is much faster for charged bilayers. Optical microscopy of giant unilamellar vesicles (GUVs) in the fluid phase revealed a different mechanism of action of the peptide in the presence or absence of negatively charged lipids. K-Hya1 induces small perturbations in POPC vesicles, causing membrane permeabilization without morphological changes. On the other hand, the peptide induces permeabilization accompanied by a large increase in surface area of POPC:POPG vesicles, suggesting the opening of several pores in the bilayer. Taken together, the results clearly show a peptide-bilayer interaction modulated by the presence of negatively charged lipids. Data can be interpreted as a different orientation of the peptide in the bilayer: parallel to the surface in neutral membranes and stable and crossed in anionic membranes.

Effect of N-Propyl Gallate on Lipid Peroxidation in Heterogenous Model Membranes

ABSTRACTThe antioxidant n-propyl gallate (nPG) is widely used in the food industry, pharmaceutics and cosmetics. Therefore, we should be well acquainted with its exact mechanism of action and its effects on the human organism, especially at the molecular level. Because of its slight solubility in water, it could be expected that, in cells, nPG would enter the membrane structures, thus altering their properties. Up to date little is known about its interaction with heterogenous lipid bilayers. That is why we focused our present study on the influence of nPG on the phase behavior of phosphatidylcholine/sphingomyelin/cholesterol (PC/SM/CHOL) ternary mixtures. Fluorescence microscopy of giant unilamellar vesicles (GUVs) was used as an experimental approach. Two phosphatidylcholine species were compared: palmitoyl-oleoyl phosphatidylcholine (POPC) and palmitoyl-docosahexaenoyl (ω-3) phosphatidylcholine (PDPC) differing in the number of double bonds at the sn-2 position (1 for POPC and 6 for PDPC). Fluorescence microscopy observations showed that the presence of docosahexaenoic acid induced the formation of micron-scale liquid-ordered (Lo) domains (model of cellular “rafts”) at physiological and higher temperatures compared to the monounsaturated oleic acid. nPG decreased the liquid-ordered (Lo)/liquid-disordered (Ld) miscibility transition temperature, Tm, for both types of vesicles in a concentration- dependent manner. Its effect was more pronounced in GUVs composed of PDPC/SM/CHOL.

Fluorescence Microscopy Monitoring of Nisin Binding to Bactoprenol Bound Cell Wall Precursors

The antimicrobial peptide nisin displays its activity by a unique dual mechanism. It forms pores within cell membranes of Gram-positive bacteria by binding to the cell wall precursor lipid II and inhibits the cell wall synthesis [1]. We examined membrane-nisin interactions by confocal microscopy of giant unilamellar vesicles (GUVs) similarly as recently for TAT peptides [2].Interaction of nisin with pure phospholipid GUVs was controlled by electrostatic attraction. Presence of lipid II in the GUV membrane elicited a quite different response. When 0.2 mol% lipid II were integrated in phosphatidylcholine membranes, specific interactions with lipid II led to immediate binding of nisin and formation of large lipid II:nisin aggregates. Simultaneously, pore formation occurred. The same effects of specific binding were observed with lipid I, the precursor of lipid II in the cell wall biosynthesis cycle.In contrast, none of these effects was observed for GUV membranes containing undecaprenyl-phosphate (C55-P) and undecaprenyl-diphosphate (C55-PP), the carrier lipids for the cell wall building block. This finding was according to expectation for C55-P, but unexpected for C55-PP since its pyrophosphate unit was previously described as nisin binding site [3]. Our results show that the interaction of nisin with lipid II resulting in aggregation and pore formation requires further structural elements in addition to the bactoprenoldiphosphate unit.[1] H. E. Hasper, N. E. Kramer, J. L. Smith, J. D. Hillman, C. Zachariah, O. P. Kuipers, B. de Kruijff, E. Breukink, Science 2006, 313, 1636.[2] C. Ciobanasu, J.-P. Siebrasse, U. Kubitscheck, Biophys. J. 2010, 99, 153.[3] S.-T. D. Hsu, E. Breukink, E. Tischenko, M. A. G. Lutters, B. de Kruijff, R. Kaptein, A. M. J. J. Bovin, N. A. J. van Nuland, Nat. Struct. Mol. Biol., 2004, 11, 963.

Effects Caused by Triton X-100 on Lipid Bilayers of Different Composition

Detergents, such as Triton X-100, are widely used as solubilizing agents of biological membranes. Due to their amphiphilic character, detergents are incorporated in lipid bilayers at low concentrations, but are able to solubilize the membrane at higher concentrations. Here we study the incorporation of Triton X-100 into model membranes of different lipid composition and the subsequent membrane solubilization. The membranes were composed of pure POPC (palmitoyl oleoyl phoshatidylcholine), pure SM (sphingomyelin) and binary mixtures of these lipids with 30 mol% cholesterol. The process of incorporation/solubilization was followed at room temperature by optical microscopy of giant unilamellar vesicles (GUVs), isothermal titration calorimetry (ITC) and turbidity measurements. Optical microscopy revealed different responses of GUVs to Triton X-100, depending on membrane composition. Incorporation of Triton X-100 in the bilayer caused an increase in the surface area of GUVs composed of POPC, changes in the spontaneous curvature of GUVs of POPC/chol, and no morphological effects in GUVs made of SM and SM/chol. These observations were discussed in terms of a modulation of the flip-flop rate of bilayer-incorporated Triton X-100 by membrane phase and packing. Eventually, Triton X-100 was able to solubilize GUVs composed of POPC, POPC/chol and SM. During the solubilization process several holes opened and the bilayer gradually vanished. On the other hand, GUVs composed of SM/chol were insoluble in Triton X-100. ITC and turbidity measurements were used to determine the incorporation and solubility thresholds of Triton X-100 in the different membrane compositions. Financial support: INCT-FCx and FAPESP.

Sterol Properties Required for Microdomain Formation: From Model Systems to Living Yeast and Mammalian Cells

This work presents new insights into the evolution of sterol-enriched domains in the plasma membrane (PM) of eukaryotes. The recent finding of sphingolipid-enriched gel domains in the PM of Saccharomyces cerevisiae(1) demands novel strategies to study sterol-enriched liquid ordered domains in this organism. Therefore, we employed di-4-ANEPPS fluorescence, here shown to be sensitive to S. cerevisiae sterols and Fluorescence Lifetime Imaging Microscopy (FLIM) for the first time in S. cerevisiae PM. In vivo studies were performed with S. cerevisiae (wild type and erg6Δ), and animal (CHO) cells, modeled by liposomes containing ergosterol, zymosterol, or cholesterol, respectively. Different fluorescent probes were used, showing that these sterols had comparable effects regarding lipid bilayer order and complete solubilization of gel domains. However, confocal fluorescence microscopy of Giant Unilamellar Vesicles showed that zymosterol, a biosynthetic precursor of ergosterol and cholesterol, has no ability to form lipid rafts.In living cells, sterol-induced emission blue-shift and longer fluorescence lifetime of di-4-ANEPPS increased as: wild type<erg6Δ<CHO. Concomitantly, in model systems, cholesterol and zymosterol had much larger effects than ergosterol in di-4-ANEPPS fluorescence. However, zymosterol was unique because it yielded overlapping results for both saturated and unsaturated lipids. The two other sterols produced much stronger alterations with a saturated lipid, even though the absolute effect in di-4-ANEPPS fluorescence was often smaller than that of zymosterol. Hence, the differential regarding the interaction of sterols with saturated versus unsaturated lipids emerges as a requisite for lipid raft formation and as an important factor in sterol evolution.(1) F. Aresta-Branco, A.M. Cordeiro, H.S. Marinho, L. Cyrne, F. Antunes, R.F.M. de Almeida (2011) “Gel domains in the plasma membrane of Saccharomyces cerevisiae: highly ordered, ergosterol-free, sphingolipid-enriched lipid rafts.” J. Biol. Chem. 286:5043-5054.Acknowledgements: PTDC/QUI-BIQ/104311/2008 and Ciencia2007 (FCT, Portugal).

Analysis of HIV-1 fusion peptide inhibition by synthetic peptides from E1 protein of GB virus C

The aim of this study was to identify proteins that could inhibit the activity of the peptide sequence representing the N-terminal of the surface protein gp41 of HIV, corresponding to the fusion peptide of the virus (HIV-1 FP). To do this we synthesized and studied 58 peptides corresponding to the envelope protein E1 of the hepatitis G virus (GBV-C).Five of the E1 synthetic peptides: NCCAPEDIGFCLEGGCLV (P7), APEDIGFCLEGGCLVALG (P8), FCLEGGCLVALGCTICTD (P10), QAGLAVRPGKSAAQLVGE (P18) and AQLVGELGSLYGPLSVSA (P22) were capable of inhibiting the leakage of vesicular contents caused by HIV-1 FP. A series of experiments were carried out to determine how these E1 peptides interact with HIV-1 FP. Our studies analyzed the interactions with and without the presence of lipid membranes. Isothermal titration calorimetry revealed that the binding of P7, P18 and P22 peptides to HIV-1 FP is strongly endothermic, and that binding is entropy-driven. Gibbs energy for the process indicates a spontaneous binding between E1 peptides and HIV-1 FP. Moreover, confocal microscopy of Giant Unilamellar Vesicles revealed that the disruption of the lipid bilayer by HIV-1 FP alone was inhibited by the presence of any of the five selected peptides.Our results highlight that these E1 synthetic peptides could be involved in preventing the entry of HIV-1 by binding to the HIV-1 FP. Therefore, the continued study into the interaction between GBV-C peptides and HIV-1 FP could lead to the development of new therapeutic agents for the treatment of AIDS.

Observing the Solubilization of Lipid Bilayers by Detergents with Optical Microscopy of GUVs

The solubilization of lipid bilayers by detergents was studied with optical microscopy of giant unilamellar vesicles (GUVs) composed of palmitoyl oleoyl phosphatidylcholine (POPC). A solution of the detergents Triton X-100 (TX-100) and sodium dodecyl sulfate (SDS) was injected with a micropipette close to single GUVs. The solubilization process was observed with phase contrast and fluorescence microscopy and found to be dependent on the detergent nature. In the presence of TX-100, GUVs initially showed an increase in their surface area, due to insertion of TX-100 with rapid equilibration between the two leaflets of the bilayer. Then, above a solubility threshold, several holes opened, rendering the bilayer a lace fabric appearance, and the bilayer gradually vanished. On the other hand, injection of SDS caused initially an increase in the membrane spontaneous curvature, which is mainly associated with incorporation of SDS in the outer layer only. This created a stress in the membrane, which caused either opening of transient macropores with substantial decrease in vesicle size or complete vesicle bursting. In another experimental setup, the extent of solubilization/destruction of a collection of GUVs was measured as a function of either TX-100 or SDS concentration.

Cholesterol Displaces Palmitoylceramide from Its Tight Packing with Palmitoylsphingomyelin in the Absence of a Liquid-Disordered Phase

A set of different biophysical approaches has been used to explore the phase behavior of palmitoylsphingomyelin (pSM)/cholesterol (Chol) model membranes in the presence and absence of palmitoylceramide (pCer). Fluorescence spectroscopy of di-4-ANEPPDHQ-stained pSM/Chol vesicles and atomic force microscopy of supported planar bilayers show gel Lβ/liquid-ordered (Lo) phase coexistence within the range XChol = 0–0.25 at 22°C. At the latter compositional point and beyond, a single Lo pSM/Chol phase is detected. In ternary pSM/Chol/pCer mixtures, differential scanning calorimetry of multilamellar vesicles and confocal fluorescence microscopy of giant unilamellar vesicles concur in showing immiscibility, but no displacement, between Lo cholesterol-enriched (pSM/Chol) and gel-like ceramide-enriched (pSM/pCer) phases at high pSM/(Chol + pCer) ratios. At higher cholesterol content, pCer is unable to displace cholesterol at any extent, even at XChol < 0.25. It is interesting that an opposite strong cholesterol-mediated pCer displacement from its tight packing with pSM is clearly detected, completely abolishing the pCer ability to generate large microdomains and giving rise instead to a single ternary phase. These observations in model membranes in the absence of the lipids commonly used to form a liquid-disordered phase support the role of cholesterol as the key determinant in controlling its own displacement from Lo domains by ceramide upon sphingomyelinase activity.