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This work intends to describe the physical properties of red blood cell (RBC) membranes in obese adults. The hypothesis driving this research is that obesity, in addition to increasing the amount of body fat, will also modify the lipid composition of membranes in cells other than adipocytes. Forty-nine control volunteers (16 male, 33 female, BMI 21.8 ± 5.6 and 21.5 ± 4.2 kg/m2, respectively) and 52 obese subjects (16 male and 36 female, BMI 38.2± 11.0 and 40.7 ± 8.7 kg/m2, respectively) were examined. The two physical techniques applied were atomic force microscopy (AFM) in the force spectroscopy mode, which allows the micromechanical measurement of penetration forces, and fluorescence anisotropy of trimethylammonium diphenylhexatriene (TMA-DPH), which provides information on lipid order at the membrane polar–nonpolar interface. These techniques, in combination with lipidomic studies, revealed a decreased rigidity in the interfacial region of the RBC membranes of obese as compared to control patients, related to parallel changes in lipid composition. Lipidomic data show an increase in the cholesterol/phospholipid mole ratio and a decrease in sphingomyelin contents in obese membranes. ω-3 fatty acids (e.g., docosahexaenoic acid) appear to be less prevalent in obese patient RBCs, and this is the case for both the global fatty acid distribution and for the individual major lipids in the membrane phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS). Moreover, some ω-6 fatty acids (e.g., arachidonic acid) are increased in obese patient RBCs. The switch from ω-3 to ω-6 lipids in obese subjects could be a major factor explaining the higher interfacial fluidity in obese patient RBC membranes., This work was supported in part by the Basque Government Department of Economic Development, grant No. KK-2019/00028 (OBINTER); the Basque Government Department of Education, grants No. IT1264-19, IT1281-19, IT1270-19, and IT1625-22; the Basque Government Department of Health, grants No. 2019-222030, 2020-333023; Fundación Ramón Areces; and by Centre for the Development of Industrial Technology (CDTI) of the Spanish Ministry of Science and Innovation under the grant agreement: TECNOMIFOOD project (CER-20191010) and Basque Government: IT1625-22.

Lipid bilayers of different phospholipid compositions have been prepared, in the form of vesicles, or of supported lipid bilayers, and doped with Aurora™ at 0.1 mol%. Aurora™ consists of an Au55 gold nanoparticle (about 1.4 nm in diameter) capped with triphenylphosphine ligands and a single diglyceride (distearoyl glycerol) ligand. Gold nanoparticles have been incorporated in the past inside liposomes, or grafted onto their surfaces, with diagnostic or therapeutic aims. Including the gold nanoparticles in a stable form within the lipid bilayers has serious technical difficulties. We have tested the hypothesis that, because of the diglyceride ligand, Aurora™ would allow the easy incorporation of gold nanoclusters into cell membranes or lipid bilayers. Our results show that Aurora™ readily incorporates into lipid bilayers, particularly when they are in the fluid phase, i.e. the state in which cell membranes exist. Calorimetric, fluorescence polarization or fluorescence confocal microscopy concur in showing that bilayer-embedded Aurora™hardly changes the physical properties of the bilayers, nor does it perturb the phase equilibrium in lipid mixtures giving rise to lateral phase separation in the plane of the membrane. Atomic force microscopy shows, in fluid bilayers, well-resolved particles, 1.2–2.9 nm in height, that are interpreted as single Aurora™conjugates. Cryo-transmission electron microscopy allows the clear observation of lipid bilayers with an enhanced contrast due to the Aurora™ gold nanoparticles; the single particles can be resolved at high magnification. Our studies support the applicability of Aurora™ as a membrane-friendly form of nano-gold particles for biological research or clinical applications.

The push-pull solvatochromic pyrene derivatives PA and PK have been applied to the study of model membrane vesicles, cells and purified human serum lipoproteins, using both confocal fluorescence microscopy and fluorescence spectroscopy. These polarity-sensitive probes provide information similar to that obtained by Laurdan or Prodan, i.e. mainly lipid order in biomembranes, but they have the essential advantage of being excitable by a standard 405 nm laser light, bypassing the use of multiphoton excitation. In addition, they are brighter and much more photostable than those dimethylamino naphthalene derivatives. Our results with model membrane spectroscopy (multilamellar vesicles) and with microscopy (giant unilamellar vesicles) showed the capacity of PA and PK to report differently on liquid-disordered, liquid-ordered and gel phase bilayers. Moreover, a ratiometric parameter, the Red/Blue Intensity Ratio (RBIR) could be used for inter-domain, inter-vesicle and even inter-technique comparison, and the appropriate microscopy-spectroscopy conversion coefficients could be estimated. In studies at the cellular level, PA probe stained almost exclusively the plasma membrane of red blood cells, revealing its high degree of lipid order. Using Chinese Hamster Ovary cells PA was shown to be an excellent probe for the detection of cytoplasmic lipid droplets, superior to Nile Red in that PA provides simultaneously a detailed information of membrane order in the whole cell, in which the lipid droplets appear with a very good contrast. Moreover, spectrofluorometric data of PA-stained serum lipoproteins indicated an essentially identical value of RBIR for lipid droplets and for high-density lipoproteins., This work was supported in part by grants from the Spanish Ministry of Science (FEDER MINECO PGC2018-099857-B-I00) and the Basque Government (grants No. IT1264-19 and IT1270-19), by the Fundación Biofísica Bizkaia and by the Basque Excellence Research Centre (BERC) program of the Basque Government.

BACKGROUND Arachidyl amido cholanoic acid (Aramchol) is a potent downregulator of hepatic stearoyl-CoA desaturase 1 (SCD1) protein expression that reduces liver triglycerides and fibrosis in animal models of steatohepatitis. In a phase IIb clinical trial in patients with nonalcoholic steatohepatitis (NASH), 52 wk of treatment with Aramchol reduced blood levels of glycated hemoglobin A1c, an indicator of glycemic control. AIM To assess lipid and glucose metabolism in mouse hepatocytes and in a NASH mouse model [induced with a 0.1% methionine and choline deficient diet (0.1MCD)] after treatment with Aramchol. METHODS Isolated primary mouse hepatocytes were incubated with 20 mu mol/L Aramchol or vehicle for 48 h. Subsequently, analyses were performed including Western blot, proteomics by mass spectrometry, and fluxomic analysis with(13)C-uniformly labeled glucose. For thein vivopart of the study, male C57BL/6J mice were randomly fed a control or 0.1MCD for 4 wk and received 1 or 5 mg/kg/d Aramchol or vehicle by intragastric gavage for the last 2 wk. Liver metabolomics were assessed using ultra-high-performance liquid chromatography-time of flight-MS for the determination of glucose metabolism-related metabolites. RESULTS Combination of proteomics and Western blot analyses showed increased AMPK activity while the activity of nutrient sensor mTORC1 was decreased by Aramchol in hepatocytes. This translated into changes in the content of their downstream targets including proteins involved in fatty acid (FA) synthesis and oxidation [P-ACC alpha/beta(S79), SCD1, CPT1A/B, HADHA, and HADHB], oxidative phosphorylation (NDUFA9, NDUFB11, NDUFS1, NDUFV1, ETFDH, and UQCRC2), tricarboxylic acid (TCA) cycle (MDH2, SUCLA2, and SUCLG2), and ribosome (P-p70S6K[T389] and P-S6[S235/S236]). Flux experiments with(13)C-uniformely labeled glucose showed that TCA cycle cataplerosis was reduced by Aramchol in hepatocytes, as indicated by the increase in the number of rounds that malate remained in the TCA cycle. Finally, liver metabolomic analysis showed that glucose homeostasis was improved by Aramchol in 0.1MCD fed mice in a dose-dependent manner, showing normalization of glucose, G6P, F6P, UDP-glucose, and Rbl5P/Xyl5P. CONCLUSION Aramchol exerts its effect on glucose and lipid metabolism in NASH through activation of AMPK and inhibition of mTORC1, which in turn activate FA beta-oxidation and oxidative phosphorylation. Supported by the National Institutes of Health Grant, No. R01CA172086; Plan Nacional of I+D, No. SAF2017-88041-R; Ministerio de Economia y Competitividad de Espana, No. SAF2017-87301-R; Asociacion Espanola contra el Cancer, No. AECC17/302; Ayudas Fundacion BBVA a equipos de Investigacion Cientifica 2018; Fondo Europeo de Desarrollo Regional, Ministerio de Economia y Competitividad de Espana, No. PGC2018-099857-BI00; Basque Government Grants, No. IT1264-19; Ministerio de Economia y Competitividad de Espana for the Severo Ochoa Excellence Accreditation, No. SEV2016-0644. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The biophysical properties of sphingolipids containing lignoceric (C24:0) or nervonic (C24:1) fatty acyl residues have been studied in multicomponent lipid bilayers containing cholesterol (Chol), by means of confocal microscopy, differential scanning calorimetry and atomic force microscopy. Lipid membranes composed of dioleoyl phosphatidylcholine and cholesterol were prepared, with the addition of different combinations of ceramides (C24:0 and/or C24:1) and sphingomyelins (C24:0 and/or C24:1). Results point to C24:0 sphingolipids, namely lignoceroyl sphingomyelin (lSM) and lignoceroyl ceramide (lCer), having higher membrane rigidifying properties than their C24:1 homologues (nervonoyl SM, nSM, or nervonoyl Cer, nCer), although with a similar strong capacity to induce segregated gel phases. In the case of the lSM-lCer multicomponent system, the segregated phases have a peculiar fibrillar or fern-like morphology. Moreover, the combination of C24:0 and C24:1 sphingolipids generates interesting events, such as a generalized bilayer dynamism/instability of supported planar bilayers. In some cases, these sphingolipids give rise to exothermic curves in thermograms. These peculiar features were not present in previous studies of C24:1 combined with C16:0 sphingolipids. Conclusions of our study point to nSM as a key factor governing the relative distribution of ceramides when both lCer and nCer are present. The data indicate that lCer could be easier to accommodate in multicomponent bilayers than its C16:0 counterpart. These results are relevant for events of membrane platform formation, in the context of sphingolipid-based signaling cascades. EGR and AGA were postdoctoral scientists supported by the University of the Basque Country (UPV/EHU). This work was supported in part by the Spanish Ministerio de Ciencia e Innovacion (MCI), Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) (Grant No. PGC2018-099857-B-I00), by the Basque Government (Grants Nos. IT1264-19, IT1270-19), by the Fundacion Biofisica Bizkaia and by the Basque Excellence Research Centre (BERC) program of the Basque Government. Dedicated to Professor J. C. Gomez-Fernandez, on occasion of his 70th birthday.

In this work, based on several studies, we develop an artificial lipid membrane to mimic the HeLa cell membrane using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- l -serine (POPS) and cholesterol (CHOL). This is then a means to further study the fusion process of specific engineered liposomes. To characterize the mimicked HeLa cell membrane, we determined a series of surface pressure–area (π-A) isotherms and the isothermal compression modulus was calculated together with the dipole moment normal to the plane of the monolayer. The existence of laterally segregated domains was assessed using a fluorescence technique (Laurdan) and two microscopy techniques: Brewster angle microscopy (BAM) and atomic force microscopy (AFM) of Langmuir-Blodgett films (LBs) extracted at 30 mN m−1. To examine the nature and composition of the observed domains, force spectroscopy (FS) based on AFM was applied to the LBs. Finally, two engineered liposome formulations were tested in a fusion assay against mimicked HeLa cell membrane LBs, showing good results and thereby opening the door to further assays and uses.

Programmed cell death or apoptosis is a central biological process that is dysregulated in many diseases, including inflammatory conditions and cancer. The detection and quantification of apoptotic cells in vivo is hampered by the need for fixatives or washing steps for non-fluorogenic reagents, and by the low levels of free calcium in diseased tissues that restrict the use of annexins. In this manuscript, we report the rational design of a highly stable fluorogenic peptide (termed Apo-15) that selectively stains apoptotic cells in vitro and in vivo in a calcium-independent manner and under wash-free conditions. Furthermore, using a combination of chemical and biophysical methods, we identify phosphatidylserine as a molecular target of Apo-15. We demonstrate that Apo-15 can be used for the quantification and imaging of drug-induced apoptosis in preclinical mouse models, thus creating opportunities for assessing the in vivo efficacy of anti-inflammatory and anti-cancer therapeutics., Programmed cell death or apoptosis is an essential biological process that is impaired in some diseases and can be used to assess the effectiveness of drugs. Here the authors design Apo-15 as a fluorogenic peptide for the detection and real-time imaging of apoptotic cells.

13 pags., 8 figs., α-Synuclein amyloid self-assembly is the hallmark of a number of neurodegenerative disorders, including Parkinson's disease, although there is still very limited understanding about the factors and mechanisms that trigger this process. Primary nucleation has been observed to be initiated in vitro at hydrophobic/hydrophilic interfaces by heterogeneous nucleation generating parallel β-sheet aggregates, although no such interfaces have yet been identified in vivo. In this work, we have discovered that α-synuclein can self-assemble into amyloid aggregates by homogeneous nucleation, without the need of an active surface, and with a preference for an antiparallel β-sheet arrangement. This particular structure has been previously proposed to be distinctive of stable toxic oligomers and we here demonstrate that it indeed represents the most stable structure of the preferred amyloid pathway triggered by homogeneous nucleation under limited hydration conditions, including those encountered inside α-synuclein droplets generated by liquid-liquid phase separation. In addition, our results highlight the key role that water plays not only in modulating the transition free energy of amyloid nucleation, and thus governing the initiation of the process, but also in dictating the type of preferred primary nucleation and the type of amyloid polymorph generated depending on the extent of protein hydration. These findings are particularly relevant in the context of in vivo α-synuclein aggregation where the protein can encounter a variety of hydration conditions in different cellular microenvironments, including the vicinity of lipid membranes or the interior of membraneless compartments, which could lead to the formation of remarkably different amyloid polymorphs by either heterogeneous or homogeneous nucleation., This work was supported by MINECO (RYC-2012-12068, NC), MINECO/FEDER, EU (BFU2015-64119-P, NC), MICIU/FEDER, EU (PGC2018-096335-B-100, NC), MICIU/FEDER, EU (RTI2018- 099019-A-I00, VS), MCI/AEI/FEDER, UE (PGC2018-099857-BI00, FMG) and Basque Government (IT1264-19, FMG and IT1196-19, JLRA), and Fundacion Biofısica Bizkaia and the Basque Excellence Research Centre (BERC) program of the Basque Government (FMG, JLRA). We thank the Proteomics Unit of Servicios Cientifico Teecnicos del CIBA (IACS-Universidad de Zaragoza)

Sphingosine [(2S,3R,4E)-2-amino-4-octadecene-1,3-diol] is the most common sphingoid base in mammals. Ceramides are N-acyl sphingosines. Numerous small variations on this canonical structure are known, including the 1-deoxy, the 4,5-dihydro, and many others. However, whenever there is a Δ4 double bond, it adopts the trans (or E) configuration. We synthesized a ceramide containing 4Z-sphingosine and palmitic acid (cis-pCer) and studied its behavior in the form of monolayers extended on an air-water interface. cis-pCer acted very differently from the trans isomer in that, upon lateral compression of the monolayer, a solid-solid transition was clearly observed at a mean molecular area ≤44 Å2·molecule-1, whose characteristics depended on the rate of compression. The solid-solid transition, as well as states of domain coexistence, could be imaged by atomic force microscopy and by Brewster-angle microscopy. Atomistic molecular dynamics simulations provided results compatible with the experimentally observed differences between the cis and trans isomers. The data can help in the exploration of other solid-solid transitions in lipids, both in vitro and in vivo, that have gone up to now undetected because of their less obvious change in surface properties along the transition, as compared to cis-pCer. Fil: Fanani, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Busto, Jon V.. Universidad del País Vasco; España. Consejo Superior de Investigaciones Científicas; España Fil: Sot, Jesús. Consejo Superior de Investigaciones Científicas; España. Universidad del País Vasco; España Fil: Abad, José L.. Consejo Superior de Investigaciones Científicas. Instituto de Química Avanzada de Catalunya; España Fil: Fabrías, Gemma. Consejo Superior de Investigaciones Científicas. Instituto de Química Avanzada de Catalunya; España Fil: Saiz, Leonor. University of California; Estados Unidos. Massachusetts Institute of Technology; Estados Unidos Fil: Vilar, Jose M. G.. Universidad del País Vasco; España. Consejo Superior de Investigaciones Científicas; España Fil: Goñi, Félix M.. Consejo Superior de Investigaciones Científicas; España. Universidad del País Vasco; España Fil: Maggio, Bruno. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Alonso, Alicia. Consejo Superior de Investigaciones Científicas; España. Universidad del País Vasco; España

Cells are complex systems whose interaction with nanocarriers, i.e., liposomes, are continuously under investigation to improve drug uptake. Model membranes can facilitate the understanding of the processes involved in fusion or endocytosis. In this work, we engineered two different lipid model membranes, vesicles and supported lipid bilayers (SLBs), mimicking the lipid composition of the HeLa cell plasma membrane. We characterized the model using atomic force microscopy (AFM) and fluorescence. We found that liposomes formed with four lipid components mimicking the HeLa cell bilayer show a liquid ordered fluid nature between 13 °C and 34 °C and yield featureless SLBs onto mica. We evaluated the fusion between the model and liposomes positively charged with and without cholesterol by AFM-based force spectroscopy and fluorescence techniques, such as Förster resonance energy transfer, fluorescence lifetime decay and fluorescence anisotropy. The results indicated a primary electrostatic interaction between the HeLa bilayer model and the liposomes. It was also confirmed the well-known fact that cholesterol enhances the fusion process with the engineered HeLa bilayer. All results support the usefulness of the engineered model in the rationale design of liposomes for drug delivery., This study was supported by the Spanish Ministry of Economy and Competitiveness (PID2019-110210GB-I00), the Catalan Government (Generalitat de Catalunya) (214SGR 1442), the Basque Government (grants No. IT1264-19 and IT1270-19), the Fundación Biofísica Bizkaia and the Basque Excellence Research Centre (BERC).

Toxicity evaluation for the proper use of graphene oxide (GO) in biomedical applications involving intravenous injections is crucial, but the GO circulation time and blood interactions are largely unknown. It is thought that GO may cause physical disruption (hemolysis) of red blood cells. The aim of this work is to characterize the interaction of GO with model and cell membranes and use this knowledge to improve GO hemocompatibility. We have found that GO interacts with both neutral and negatively charged lipid membranes; binding is decreased beyond a certain concentration of negatively charged lipids and favored in high-salt buffers. After this binding occurs, some of the vesicles remain intact, while others are disrupted and spread over the GO surface. Neutral membrane vesicles tend to break down and extend over the GO, while vesicles with negatively charged membranes are mainly bound to the GO without disruption. GO also interacts with red blood cells and causes hemolysis; hemolysis is decreased when GO is previously coated with lipid membranes, particularly with pure phosphatidylcholine vesicles.

The effects of increasing amounts of palmitoylceramide (pCer) on human red blood cell lipid membranes have been studied using atomic force microscopy of supported lipid bilayers, in both imaging (bilayer thickness) and force-spectroscopy (nanomechanical resistance) modes. Membranes appeared homogeneous with pCer concentrations up to 10 mol % because of the high concentration of cholesterol (Chol) present in the membrane (∼45 mol %). However, the presence of pCer at 30 mol % gave rise to a clearly distinguishable segregated phase with a nanomechanical resistance 7-fold higher than the continuous phase. These experiments were validated using differential scanning calorimetry. Furthermore, Chol depletion of the bilayers caused lipid domain generation in the originally homogeneous samples, and Chol-depleted domain stiffness significantly increased with higher amounts of pCer. These results point to the possibility of different kinds of transient and noncompositionally constant, complex gel-like phases present in RBC lipid membranes rich in both pCer and Chol, in contrast to the widespread opinion about the displacements between pCer-enriched "gel-like" domains and liquid-ordered "raft-like" Chol-enriched phases. Changes in the biophysical properties of these complex gel-like phases governed by local modulation of pCer:Chol ratios could be a cell mechanism for fine-tuning the properties of membranes as required.

Free volume voids in lipid bilayers can be measured by positron annihilation lifetime spectroscopy (PALS). This technique has been applied, together with differential scanning calorimetry and molecular dynamics (MD) simulations, to study the effects of cholesterol (Chol) and ceramide (Cer) on free volume voids in sphingomyelin (SM) or dipalmitoylphosphatidylcholine (DPPC) bilayers. Binary lipid samples with Chol were studied (DPPC:Chol 60:40, SM:Chol 60:40 mol ratio), and no phase transition was detected in the 20-60 °C range, in agreement with calorimetric data. Chol-driven liquid-ordered phase showed an intermediate free volume void size as compared to gel and fluid phases. For SM and SM:Cer (85:15 mol:mol) model membranes measured in the 20-60 °C range the gel-to-fluid phase transition could be observed with a related increase in free volume, which was more pronounced for the SM:Cer sample. MD simulations suggest a hitherto unsuspected lipid tilting in SM:Cer bilayers but not in pure SM. Ternary samples of DPPC:Cer:Chol (54:23:23) and SM:Cer:Chol (54:23:23) were measured, and a clear pattern of free volume increase was observed in the 20-60 °C because of the gel-to-fluid transition. Interestingly, MD simulations showed a tendency of Cer to change its distribution along the membrane to make room for Chol in ternary mixtures. The results suggest that the gel phase formed in these ternary mixtures is stabilized by Chol-Cer interactions.

Sphingolipids consist of a sphingoid base N-linked to a fatty acyl chain. Among them, sphingomyelins (SM) are major components of mammalian cells, while ceramide (Cer) plays an important role as a lipid second messenger. We have performed a quantitative lipidomic study of Cer and SM species in different mammalian tissues (adipose tissue, liver, brain and blood serum of human, mice, rat and dog), as well as in cell cultures of mammalian origin (primary hepatocytes, immortalized MDCK cells, mice melanoma b16 cells, and mice primary CD4 + T lymphocytes) using an ultra-high performance liquid chromatography coupled to time-of-flight mass spectrometry (UHPLC-ToF-MS)-based platform. The data have been compared with published, in general semi-quantitative, results from 20 other samples, with good agreement. The sphingoid base was predominantly d18-1 sphingosine (2-amino-4-octadecene-1,3-diol) in all cases. The fatty acid composition of SM was clearly different from that of Cer. In virtually all samples the most abundant Cer species were those containing C24:0 and C24:1 in their N-acyl chains, while the main species contained in SM was C16:0. Brain was the most divergent tissue, in which Cer and SM C18:0 were very abundant.

This work reports on the effects of two omega-3 fatty acids, namely docosahexaenoic (C22:64,7,10,13,16,19) acid (DHA), and eicosapentaenoic (C20:55,8,11,14,17) acid (EPA), with oleic (C18:19) acid (OA) as a control, on the gel-liquid crystalline phase transition of dipalmitoyl phosphatidylcholine (DPPC). Mainly differential scanning calorimetry has been used, together with Laurdan fluorescence, and confocal fluorescence microscopy. All three fatty acids DHA, EPA and OA exhibited fluidifying properties when added to the DPPC bilayers, decreasing the main transition temperature. DHA and EPA were somewhat more effective than OA in this respect, but the effects of all three were of the same order of magnitude, thus the long-chain omega-3 fatty acids failed to exhibit any peculiar fluidifying potency. The same was true when the omega-3 fatty acids were esterified in the sn-2 position of a phosphatidylcholine. Moreover the omega-3 fatty acids had very small or no effects on the fluidity of bilayers in the liquid-crystalline, or fluid disordered state (egg phosphatidylcholine and others), or in the fluid ordered state (phospholipid: cholesterol mixtures). The hypothesis that some physiological effects of long-chain omega-3 fatty acids could be related to their special fluidifying properties is not supported by these data.

The mechanisms of Pb(II) toxicity have been studied in human red blood cells using confocal microscopy, immunolabeling, fluorescence-activated cell sorting and atomic force microscopy. The process follows a sequence of events, starting with calcium entry, followed by potassium release, morphological change, generation of ceramide, lipid flip-flop and finally cell lysis. Clotrimazole blocks potassium channels and the whole process is inhibited. Immunolabeling reveals the generation of ceramide-enriched domains linked to a cell morphological change, while the use of a neutral sphingomyelinase inhibitor greatly delays the process after the morphological change, and lipid flip-flop is significantly reduced. These facts point to three major checkpoints in the process: first the upstream exchange of calcium and potassium, then ceramide domain formation, and finally the downstream scramblase activation necessary for cell lysis. In addition, partial non-cytotoxic cholesterol depletion of red blood cells accelerates the process as the morphological change occurs faster. Cholesterol could have a role in modulating the properties of the ceramide-enriched domains. This work is relevant in the context of cell death, heavy metal toxicity and sphingolipid signaling. AGA was a predoctoral student supported by the Basque Government and later by the University of the Basque Country (UPV/EHU). This work was also supported in part by grants from the Spanish Government (FEDER/MINECO BFU 2015-66306-P to F.M.G. and A.A.) and the Basque Government (IT849-13 to F.M.G. and IT838-13 to A.A.), and by the Swiss National Science Foundation.

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.

Type I phosphatidylinositol 4-phosphate 5-kinases (PIP5KIs; α, β, and γ) are a family of isoenzymes that produce phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] using phosphatidylinositol 4-phosphate as substrate. Their structural homology with the class II lipid kinases [type II phosphatidylinositol 5-phosphate 4-kinase (PIP4KII)] suggests that PIP5KI dimerizes, although this has not been formally demonstrated. Neither the hypothetical structural dimerization determinants nor the functional consequences of dimerization have been studied. Here, we used Förster resonance energy transfer, coprecipitation, and ELISA to show that PIP5KIβ forms homo- and heterodimers with PIP5KIγ_i2 in vitro and in live human cells. Dimerization appears to be a general phenomenon for PIP5KI isoenzymes because PIP5KIβ/PIP5KIα heterodimers were also detected by mass spectrometry. Dimerization was independent of actin cytoskeleton remodeling and was also observed using purified proteins. Mutagenesis studies of PIP5KIβ located the dimerization motif at the N terminus, in a region homologous to that implicated in PIP4KII dimerization. PIP5KIβ mutants whose dimerization was impaired showed a severe decrease in PI(4,5)P2 production and plasma membrane delocalization, although their association to lipid monolayers was unaltered. Our results identify dimerization as an integral feature of PIP5K proteins and a central determinant of their enzyme activity.—Lacalle, R. A., de Karam, J. C., Martínez-Muñoz, L., Artetxe, I., Peregil, R. M., Sot, J., Rojas, A. M., Goñi, F. M., Mellado, M., Mañes, S. Type I phosphatidylinositol 4-phosphate 5-kinase homo- and heterodimerization determines its membrane localization and activity., J.C.d.K. is a Ph.D. fellow of the La Caixa Foundation International Fellowship Programme. This work was supported in part by the Spanish Ministry of Science and Innovation (SAF2011-24453) and the Comunidad de Madrid (IMMUNOTHERCAN; S2010/BMD-2326).

The effects of C24:1 sphingolipids have been tested in phospholipid bilayers containing cholesterol. Confocal microscopy, differential scanning calorimetry, and atomic force microscopy imaging and force curves have been used. More precisely, the effects of C24:1 ceramide (nervonoyl ceramide, nCer) were evaluated and compared to those of C16:0 ceramide (palmitoyl ceramide, pCer) in bilayers composed basically of dioleoylphosphatidylcholine, sphingomyelin (either C24:1, nSM or C16:0, pSM) and cholesterol. Combination of equimolecular amounts of C24:1 and C16:0 sphingolipids were also studied under the same conditions. Results show that both pCer and nCer are capable of forming segregated gel domains. Force spectroscopy data point to nCer having a lower stiffening effect than pCer, while the presence of nSM reduces the stiffness. DSC reveals Tm reduction by nSM in every case. Furthermore, pSM seems to better accommodate both ceramides in a single phase of intermediate properties, while nSM partial accommodat...

Ceramides and dihydroceramides are N-acyl derivatives of sphingosine and sphinganine, respectively, which are the major sphingoid-base backbones of mammals. Recent studies have found that mammals, like certain other organisms, also produce 1-deoxy-(dihydro)ceramides (1-deoxyDHCers) that contain sphingoid bases lacking the 1-hydroxyl- or 1-hydroxymethyl- groups. The amounts of these compounds can be substantial—indeed, we have found comparable levels of 1-deoxyDHCers and ceramides in RAW 264.7 cells maintained in culture. The biophysical properties of 1-deoxyDHCers have not yet been reported, although these lipids might play important roles in normal cell regulation and in the pathology of diseases in which they are elevated, such as hereditary sensory autonomic neuropathies or diabetes. This study uses several approaches, including surface-pressure measurements, differential scanning calorimetry, and confocal microscopy, to study the behavior of 1-deoxyDHCers of different N-acyl-chain lengths and their interaction with sphingomyelin (SM). The thermotropic behaviors of 1-deoxyDHCers alone and in mixtures with SM are described, together with their interactions in monolayers and giant unilamellar vesicles. The gel-fluid transition temperatures of the pure compounds increase in the order 1-deoxyceramide < ceramide ≈ 1-deoxyDHCer < 1-(deoxymethyl)DHCer. In general, canonical ceramides are more miscible with SM in bilayers than are 1-deoxyceramides, and 1-(deoxymethyl)DHCers are the most hydrophobic among them, not even capable of forming monolayers at the air-water interface. Thus, these properties suggest that 1-deoxyDHCer can influence the properties of cellular membranes in ways that might affect biological function/malfunction.

Sphingosine [(2S, 3R, 4E)-2-amino-4-octadecen-1, 3-diol] is the most common sphingoid long chain base in sphingolipids. It is the precursor of important cell signaling molecules, such as ceramides. In the last decade it has been shown to act itself as a potent metabolic signaling molecule, by activating a number of protein kinases. Moreover, sphingosine has been found to permeabilize phospholipid bilayers, giving rise to vesicle leakage. The present contribution intends to analyze the mechanism by which this bioactive lipid induces vesicle contents release, and the effect of negatively charged bilayers in the release process. Fluorescence lifetime measurements and confocal fluorescence microscopy have been applied to observe the mechanism of sphingosine efflux from large and giant unilamellar vesicles; a graded-release efflux has been detected. Additionally, stopped-flow measurements have shown that the rate of vesicle permeabilization increases with sphingosine concentration. Because at the physiological pH sphingosine has a net positive charge, its interaction with negatively charged phospholipids (e.g., bilayers containing phosphatidic acid together with sphingomyelins, phosphatidylethanolamine, and cholesterol) gives rise to a release of vesicular contents, faster than with electrically neutral bilayers. Furthermore, phosphorous 31-NMR and x-ray data show the capacity of sphingosine to facilitate the formation of nonbilayer (cubic phase) intermediates in negatively charged membranes. The data might explain the pathogenesis of Niemann-Pick type C1 disease.

Lipid lateral segregation into specific domains in cellular membranes is associated with cell signaling and metabolic regulation. This phenomenon partially arises as a consequence of the very distinct bilayer-associated lipid physico-chemical properties that give rise to defined phase states at a given temperature. Until now lamellar gel (Lβ) phases have been described in detail in single or two-lipid systems. Using x-ray scattering, differential scanning calorimetry, confocal fluorescence microscopy, and atomic force microscopy, we have characterized phases of ternary lipid compositions in the presence of saturated phospholipids, cholesterol, and palmitoyl ceramide mixtures. These phases stabilized by direct cholesterol-ceramide interaction can exist either with palmitoyl sphingomyelin or with dipalmitoyl phosphatidylcholine and present intermediate properties between raft-associated phospholipid-cholesterol liquid-ordered and phospholipid-ceramide Lβ phases. The present data provide novel, to our knowledge, evidence of a chemically defined, multicomponent lipid system that could cooperate in building heterogeneous segregated platforms in cell membranes.

The thermotropic properties of aqueous dispersions of sphingomyelins (SM) and ceramides (Cer) with N-acyl chains varying from C6:0 to C24:1, either pure or in binary mixtures, have been examined by differential scanning calorimetry. Even in the pure state, Cer and particularly SM exhibited complex endotherms, and their thermal properties did not vary in a predictable way with changes in structure. In some cases, e.g. C18:0 SM, atomic force microscopy revealed coexisting lamellar domains made of a single lipid. Partial chain interdigitation and metastable crystalline states were deemed responsible for the complex behavior. SM:Cer mixtures (90:10 mol ratio) gave rise to bilayers containing separate SM-rich and Cer-rich domains. In vesicles made of more complex mixtures (SM:PE:Chol, 2:1:1), it is known that sphingomyelinase degradation of SM to Cer is accompanied by vesicle aggregation and release of aqueous contents. These vesicles did not reveal observable domain separation by confocal microscopy. Vesicle aggregation occurred at a faster rate for those bilayers that appeared to be more fluid according to differential scanning calorimetry. Content efflux rates measured by fluorescence spectroscopy were highest with C18:0 and C18:1 SM, and in general those rates did not vary regularly with other physical properties of SM or Cer. In general the individual SM and Cer appear to have particular thermotropic properties, often unrelated to the changes in N-acyl chain.

When giant unilamellar vesicles (GUVs) composed of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, and cholesterol are treated with PlcHR(2), a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa, the initial stages of lipid hydrolysis do not cause large changes in vesicle morphology (Ibarguren et al., 2011). However, when hydrolysis progresses confocal fluorescence microscopy reveals the formation of lipid aggregates, whose morphology is not compatible with that of bilayers. Smaller vesicles or droplets can also be seen inside the GUV. Our studies indicate that these aggregates or droplets are enriched in the non-lamellar lipid ceramide, an end-product of PlcHR(2) reaction. Moreover, the aggregates/droplets appear enriched in the hydrolytic enzyme PlcHR(2). At a final stage GUVs containing the enzyme-enriched droplets disintegrate and vanish from the microscope field. The observed non-lamellar enzyme-rich structures may be related to intermediates in the process of aggregation and fusion although the experimental design prevents vesicle free diffusion in the aqueous medium, thus actual aggregation or fusion cannot be observed.

The rational design and synthesis of a Trp-BODIPY cyclic peptide for the fluorescent labelling of apoptotic bodies is described. Affinity assays, confocal microscopy and flow cytometry analysis confirmed the binding of the peptide to negatively-chargedphospholipids associated with apoptosis, and its applicability for the detection and characterisation of subcellular structures released by apoptotic cells

Sphingomyelin and cholesterol can assemble into domains and segregate from other lipids in the membranes. These domains are reported to function as platforms for protein transport and signalling. Do similar domains exist in the Golgi membranes and are they required for protein secretion? We tested this hypothesis by using D-ceramide-C6 to manipulate lipid homeostasis of the Golgi membranes. Lipidomics of the Golgi membranes isolated from D-ceramide-C6-treated HeLa cells revealed an increase in the levels of C6-sphingomyelin, C6-glucosylceramide, and diacylglycerol. D-ceramide-C6 treatment in HeLa cells inhibited transport carrier formation at the Golgi membranes without affecting the fusion of incoming carriers. The defect in protein secretion as a result of D-ceramide-C6 treatment was alleviated by knockdown of the sphingomyelin synthases 1 and 2. C6-sphingomyelin prevented liquid-ordered domain formation in giant unilamellar vesicles and reduced the lipid order in the Golgi membranes of HeLa cells. These findings highlight the importance of a regulated production and organization of sphingomyelin in the biogenesis of transport carriers at the Golgi membranes.

Oleic acid vesicles have been used as model systems to study the properties of membranes that could be the evolutionary precursors of more complex, stable, and impermeable phospholipid biomembranes. Pure fatty acid vesicles in general show high sensitivity to ionic strength and pH variation, but there is growing evidence that this lack of stability can be counterbalanced through mixtures with other amphiphilic or surfactant compounds. Here, we present a systematic experimental analysis of the oleic acid system and explore the spontaneous formation of vesicles under different conditions, as well as the effects that alcohols and alkanes may have in the process. Our results support the hypothesis that alcohols (in particular 10- to 14-C-atom alcohols) contribute to the stability of oleic acid vesicles under a wider range of experimental conditions. Moreover, studies of mixed oleic-acid-alkane and oleic-acid-alcohol systems using infrared spectroscopy and Langmuir trough measurements indicate that precisely those alcohols that increased vesicle stability also decreased the mobility of oleic acid polar headgroups, as well as the area/molecule of lipid.

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 L(beta)/liquid-ordered (L(o)) phase coexistence within the range X(Chol) = 0-0.25 at 22 degrees C. At the latter compositional point and beyond, a single L(o) 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 L(o) 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 X(Chol) < 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 L(o) domains by ceramide upon sphingomyelinase activity.

The lateral organization of lipids in cell membranes is thought to regulate numerous cell processes. Most studies focus on the coexistence of two fluid phases, the liquid crystalline (ld) and the liquid-ordered (lo); the putative presence of gel domains (so) is not usually taken into account. We show that in phospholipid:sphingolipid:cholesterol mixtures, in which sphingomyelin (SM) promoted fluid lo domains, dihydrosphingomyelin (DHSM) tended to form rigid domains. Genetic and pharmacological blockade of the dihydroceramide desaturase (Des1), which replaced SM with DHSM in cultured cells, inhibited cell infection by replication-competent and -deficient HIV-1. Increased DHSM levels gave rise to more rigid membranes, resistant to the insertion of the gp41 fusion peptide, thus inhibiting viral-cell membrane fusion. These results clarify the function of dihydrosphingolipids in biological membranes and identify Des1 as a potential target in HIV-1 infection., We thank L. Martínez-Prat and E. Dalmau for technical help and C. Mark for editorial assistance. C.R.V. and J.M.M.-O. received predoctoral fellowships from the Portuguese Ministry of Science (MCTES) and the CSIC, respectively. This work was supported in part by the CSIC, the Foundation for Research and Prevention of AIDS in Spain (FIPSE), the Spanish Ministry of Science and Innovation (BFU2008-01637 (A.A.), BIO2008-00772 (J.L.N.), BFU2007-62062 (F.M.G.), SAF2008-00649 (S.M.), SAF2008-00706 (G.F.), SAF2007-64696 (J.M.-P.), the Spanish AIDS (RD006/0006) and RIER (RD08/0075/0007) Networks, the Generalitat of Catalunya (2009SRG-01072 to G.F.), and the European Union (CHAIN, FP7/2007-2013, 2233131; J.M.-P.), and FP6 (INNOCHEM, LSHB-CT-2005-518167) and e-rare (PI07/1314) programs to S.M.

Three ceramide analogues have been synthesized, with sphingosine-like chains containing five conjugated double bonds. Pentaene I has an N-palmitoyl acyl chain, while the other two pentaenes contain also a doxyl radical, respectively, at C5 (Penta5dox) and at C16 (Penta16dox) positions of the N-acyl chain. Pentaene I maximum excitation and emission wavelengths in a phospholipid bilayer are 353 and 478 nm, respectively. Pentaene I does not segregate from the other lipids in the way natural ceramide does, but rather mixes with them in a selective way according to the lipid phases involved. Fluorescence confocal microscopy studies show that when lipid domains in different physical states coexist, Pentaene I emission is higher in gel than in fluid domains, and in liquid-ordered than in liquid-disordered areas. Electron paramagnetic resonance of the pentaene doxyl probes confirms that these molecules are sensitive to the physical state of the bilayer. Calorimetric and fluorescence quenching experiments suggest that the lipids under study orient themselves in lipid bilayers with their polar moieties located at the lipid-water interface. The doxyl radical in the N-acyl chain quenches the fluorescence of the pentaene group when in close proximity. Because of this property, Penta16dox can detect gel-fluid transitions in phospholipids. The availability of probes for lipids in the gel phase is important in view of novel evidence for the existence of gel microdomains in cell membranes.

In a previous article, we demonstrated that histones (H1 or histone octamers) interact with negatively charged bilayers and induce extensive aggregation of vesicles containing phosphatidylinositol-4-phosphate (PIP) and, to a lesser extent, vesicles containing phosphatidylinositol (PI). Here, we found that vesicles containing PIP, but not those containing PI, can undergo fusion induced by histones. Fusion was demonstrated through the observation of intervesicular mixing of total lipids and inner monolayer lipids, and by ultrastructural and confocal microscopy studies. Moreover, in both PI- and PIP-containing vesicles, histones caused permeabilization and release of vesicular aqueous contents, but the leakage mechanism was different (all-or-none for PI and graded release for PIP vesicles). These results indicate that histones could play a role in the remodeling of the nuclear envelope that takes place during the mitotic cycle., This work was supported in part by grants from the Spanish Ministry of Economy (BFU 2011-28566 to A.A., BFU 2012-36241 to F.M.G., and BIO 2013-42978-P to M.M.) and the Basque Government (IT 838-13 and IT 849-13). M.G.L. was a predoctoral student supported by the Basque Government.

DAG-induced activation of PI-PLC has been studied using as substrates vesicles containing PI, either pure or in mixtures with DMPC, DSPC, sphingomyelin, or galactosylceramide. At 22°C DAG at 33 mol% increases PI-PLC activity in all the mixtures, but not in pure PI bilayers. DAG also causes an overall decrease in DPH polarization (decreased molecular order) in all samples, and increased overall enzyme binding. Confocal fluorescence microscopy examination of GUV of all the compositions under study, with or without DAG, and quantitative evaluation of the phase behaviour using LAURDAN generalized polarization, and of enzyme binding to the various domains, indicate 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:DMPC bilayers at 22°C DAG induced increased enzyme binding and activation, but no microscopic domain separation was observed, the presence of DAG-generated nanodomains is proposed instead for this system. In PI:galactosylceramide mixtures DAG may exert its activation role through the generation of small vesicles, that PI-PLC is known to degrade at higher rates. In general our results indicate that global measurements using fluorescent probes in vesicle suspensions in cuvette are not enough to understand DAG effects that take place at the domain level. The above data reinforce the idea of DAG as an important physical agent regulating membrane or cell properties.

Copyright © by Biophysical Society. Final full-text version of the paper available at: http://www.biophysj.org/cgi/content/abstract/90/11/4085, Sphingosine, at 5–15 mol % total lipids, remarkably increases the permeability to aqueous solutes of liposomal and erythrocyte ghostmembranes. The increased permeability cannot be interpreted in terms of leakage occurring at the early stages of a putative membrane solubilization by sphingosine, nor is it due to a sphingosine-induced generation of nonlamellar structures, or flipflop lipid movement. Instead, sphingosine stabilizes (rigidifies) gel domains in membranes, raising their melting temperatures and increasing the transition cooperativity. Structural defects originating during the lateral phase separation of the ‘‘more rigid’’ and ‘‘less rigid’’ domains are likely sites for the leakage of aqueous solutes to the extravesicularmedium. The presence of coexisting domains in the plasma membrane makes it a target for sphingosine permeabilization. The sphingosine-induced increase in rigidity and breakdown of the plasma membrane permeability barrier could be responsible for some of the physiological effects of sphingosine., This work was supported in part by grants from the Spanish Ministerio de Educación y Ciencia (BMC 2002-00784) and the University of the Basque Country (00042.310-13552/2001). F.X.C. and J.S. were predoctoral students supported, respectively, by the Ministerio de Educación y Ciencia and by the Basque government.

Alkanes (C6-C16) are often used as vehicles for hydrophobic reagents, e.g. long-chain ceramides, in cell biology studies. It is generally understood that they are inert solvents, particularly when added in small volumes. However, simple calculations show that, under standard experimental conditions in cell studies, alkane: phospholipid molar ratios in excess of 1000:1 may be found. Even at much smaller ratios (close to 1:1) our studies with liposomes show that alkanes induce vesicle aggregation. Differential scanning calorimetry shows marked changes in both the gel-fluid and the lamellar-hexagonal transitions. Alkanes inhibit bacterial sphingomyelinase when acting on large unilamellar vesicles, and activate bacterial phospholipase C under the same conditions. Thus, the use of alkanes in cell studies requires strict control experiments to avoid artefactual results.

The behaviour of N-hexadecanoylsphingosine (Cer16), N-hexanoylsphingosine (Cer6) and N-acetylsphingosine (Cer2) in aqueous media and in lipid-water systems, monolayers and bilayers has been comparatively examined using Langmuir balance and fluorescence techniques. Cer16 behaves as an insoluble non-swelling amphiphile, not partitioning into the air-water interface, thus not modifying the surface pressure of the aqueous solutions into which it is included. By contrast both Cer6 and Cer2 behave as soluble amphiphiles, up to approx. 100 microM. At low concentrations, they become oriented at the air-water interface, increasing surface pressure in a dose-dependent way up to ca. 5 microM bulk concentration. At higher concentrations, the excess ceramide forms micelles, critical micellar concentrations of both Cer6 and Cer2 being in the 5-6 microM range. When the air-water interface is occupied by a phospholipid, 6Cer2 and Cer6 become inserted in the phospholipid monolayer, causing a further increase in surface pressure. This increase is dose dependent, and reaches a plateau at ca. 2 microM ceramide bulk concentration. Both Cer2 and Cer6 become inserted in phospholipid monolayers with initial surface pressures of up to 43 and 46 mN m(-1), respectively, which ensures their capacity to become inserted into cell membranes whose monolayers are estimated to support a surface pressure of about 30 mN m(-1). Both Cer2 and Cer6, but not Cer16, had detergent-like properties, such as giving rise to phospholipid-ceramide mixed micelles, when added to phospholipid monolayers or bilayers. The short-chain ceramides form large aggregates and precipitate at concentrations above approx. 100 microM. These results are relevant in cell physiology studies in which short- and long-chain ceramides are sometimes used as equivalent molecules, in spite of their different biophysical behaviour.

Copyright © by Biophysical Society. Final full-text version of the paper available at: http://www.biophysj.org/cgi/content/abstract/88/5/3368, The effects on dielaidoylphosphatidylethanolamine (DEPE) bilayers of ceramides containing different N-acyl chains have been studied by differential scanning calorimetry small angle x-ray diffraction and 31P-NMR spectroscopy. N-palmitoyl (Cer16), N-hexanoyl (Cer6), and N-acetyl (Cer2) sphingosines have been used. Both the gel-fluid and the lamellarinverted hexagonal transitions of DEPE have been examined in the presence of the various ceramides in the 0-25 mol % concentration range. Pure hydrated ceramides exhibit cooperative endothermic order-disorder transitions at 93 C (Cer16), 60 C (Cer6), and 54 C (Cer2). In DEPE bilayers, Cer16 does not mix with the phospholipid in the gel phase, giving rise to highmelting ceramide-rich domains. Cer16 favors the lamellar-hexagonal transition of DEPE, decreasing the transition temperature. Cer2, on the other hand, is soluble in the gel phase of DEPE, decreasing the gel-fluid and increasing the lamellar-hexagonal transition temperatures, thus effectively stabilizing the lamellar fluid phase. In addition, Cer2 was peculiar in that no equilibrium could be reached for the Cer2-DEPE mixture above 60 C, the lamellar-hexagonal transition shifting with time to temperatures beyond the instrumental range. The properties of Cer6 are intermediate between those of the other two, this ceramide decreasing both the gel-fluid and lamellar-hexagonal transition temperatures. Temperature-composition diagrams have been constructed for the mixtures of DEPE with each of the three ceramides. The different behavior of the long- and short-chain ceramides can be rationalized in terms of their different molecular geometries, Cer16 favoring negative curvature in the monolayers, thus inverted phases, and the opposite being true of the micelle-forming Cer2. These differences may be at the origin of the different physiological effects that are sometimes observed for the long- and short-chain ceramides., This work was supported in part by grants from Ministerio de Educación y Ciencia (BCM 2002-00784) and Universidad del País Vasco (UPV 00042.310/13552). J. Sot was a predoctoral student supported by the Basque government.

In the past decade, the long-neglected ceramides (N-acylsphingosines) have become one of the most attractive lipid molecules in molecular cell biology, because of their involvement in essential structures (stratum corneum) and processes (cell signalling). Most natural ceramides have a long (16-24 C atoms) N-acyl chain, but short N-acyl chain ceramides (two to six C atoms) also exist in Nature, apart from being extensively used in experimentation, because they can be dispersed easily in water. Long-chain ceramides are among the most hydrophobic molecules in Nature, they are totally insoluble in water and they hardly mix with phospholipids in membranes, giving rise to ceramide-enriched domains. In situ enzymic generation, or external addition, of long-chain ceramides in membranes has at least three important effects: (i) the lipid monolayer tendency to adopt a negative curvature, e.g. through a transition to an inverted hexagonal structure, is increased, (ii) bilayer permeability to aqueous solutes is notoriously enhanced, and (iii) transbilayer (flip-flop) lipid motion is promoted. Short-chain ceramides mix much better with phospholipids, promote a positive curvature in lipid monolayers, and their capacities to increase bilayer permeability or transbilayer motion are very low or non-existent.

Bacillus cereus sphingomyelinase activity was assayed on large unilamellar vesicles composed of sphingomyelin (SM)/cholesterol (Ch) mixtures at varying proportions. Natural (egg) SM was used with a gel-fluid transition temperature at ca. 40 degrees C. When the enzyme was assayed at 37 degrees C, the activity on pure SM was exceedingly low, but a small increase was observed as soon as some Ch was added, and a large enhancement of activity occurred with Ch proportions above 25 mol%. The data were interpreted in terms of sphingomyelinase activity being higher in the cholesterol-induced liquid-ordered phase than in the gel phase. The abrupt increase in activity above 25 mol% Ch would occur as a result of a change in domain connectivity, when the Ch-rich liquid-ordered domains coalesced. In equimolar SM/Ch mixtures, that were in the liquid-ordered state in a wide range of temperatures, sphingomyelinase activity was virtually constant in the 30-70 degrees C range. The results demonstrate that at the mammalian and bird physiological temperatures Ch modulates sphingomyelinase activity, and that this can occur precisely because most SM have a gel-fluid transition temperature above the physiological temperature range. In addition, Ch activation of sphingomyelinase and the strong affinity of Ch for SM allow the rapid, localised and self-contained production of the metabolic signal ceramide in specific microdomains (rafts).

The molecular basis for the existence of the so-called “detergent-resistant membranes” has been explored. With that aim, vesicles composed of phosphatidylcholine, sphingolipid, and cholesterol were...

Some of the simplest sphingolipids, namely sphingosine, ceramide and their phosphorylated compounds [sphingosine 1-phosphate (Sph-1-P) and ceramide 1-phosphate (Cer-1-P)], are potent metabolic regulators. Each of these lipids modifies in marked and specific ways the physical properties of the cell membranes, in what can be the basis for some of their physiological actions. The present paper is an overview of the mechanisms by which these sphingolipid signals, sphingosine and ceramide, in particular, are able to modify the properties of cell membranes.

This study was conducted to explore how the nature of the acyl chains of sphingomyelin (SM) influence its lateral distribution in the ternary lipid mixture SM/cholesterol/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), focusing on the importance of the hydrophobic part of the SM molecule for domain formation. Atomic force microscopy (AFM) measurements showed that the presence of a double bond in the 24:1 SM molecule in mixtures with cholesterol (CHO) or in pure bilayers led to a decrease in the molecular packing. Confocal microscopy and AFM showed, at the meso- and nanoscales respectively, that unlike 16:0 and 24:0 SM, 24:1 SM does not induce phase segregation in ternary lipid mixtures with DOPC and CHO. This ternary lipid mixture had a nanomechanical stability intermediate between those displayed by liquid-ordered (Lo) and liquid-disordered (Ld) phases, as reported by AFM force spectroscopy measurements, demonstrating that 24:1 SM is able to accommodate both DOPC and CHO, forming a single phase. Confocal experiments on giant unilamellar vesicles made of human, sheep, and rabbit erythrocyte ghosts rich in 24:1 SM and CHO, showed no lateral domain segregation. This study provides insights into how the specific molecular structure of SM affects the lateral behavior and the physical properties of both model and natural membranes. Specifically, the data suggest that unsaturated SM may help to keep membrane lipids in a homogeneous mixture rather than in separate domains., Instituto de Investigaciones Bioquímicas de La Plata, Facultad de Ciencias Exactas

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.

The early stages of Triton X-100 solubilization of bilayers consisting of sphingomyelin/ceramide (SM/Cer) mixtures have been studied using a combination of calorimetric and spectroscopic techniques. Compositions based on sphingomyelin, containing up to 30 mol% Cer, at 4, 20 and 50 °C have been examined. The presence of Cer does not modify the affinity (in terms of ΔG of binding per mol total lipid) of the SM-based bilayers for Triton X-100, although it does increase the amount of detergent required for the onset of solubilization. At 50 °C more detergent was required to solubilize the SM/Cer bilayers than at 20 °C. The data can be rationalized in terms of lipid and detergent geometries and interactions ( Lichtenberg et al., 2013 ).

The binding and early stages of activity of a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa on giant unilamellar vesicles (GUV) have been monitored using fluorescence confocal microscopy. Both the lipids and the enzyme were labeled with specific fluorescent markers. GUV consisted of a mixture of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and cholesterol in equimolar ratios, to which 5–10 mol% of the enzyme end-product ceramide and/or diacylglycerol were occasionally added. Morphological examination of the GUV in the presence of enzyme reveals that, although the enzyme diffuses rapidly throughout the observation chamber, detectable enzyme binding appears to be a slow, random process, with new bound-enzyme-containing vesicles appearing for several minutes. Enzyme binding to the vesicles appears to be a cooperative process. After the initial cluster of bound enzyme is detected, further binding and catalytic activity follow rapidly. After the activity has started, the enzyme is not released by repeated washing, suggesting a “scooting” mechanism for the hydrolytic activity. The enzyme preferentially binds the more disordered domains, and, in most cases, the catalytic activity causes the disordering of the other domains. Simultaneously, peanut- or figure-eight-shaped vesicles containing two separate lipid domains become spherical. At a further stage of lipid hydrolysis, lipid aggregates are formed and vesicles disintegrate.

The present study deals with a combination of fluorescence spectroscopy, atomic force microscopy (AFM), differential scanning calorimetry (DSC) and confocal microscopy to study two different effects: i) lateral segregation in pSM/Chol binary mixtures, and ii) the effects of pCer incorporation into pSM/Chol mixtures. The data reveals the segregation of large cholesterol-enriched microdomains within the range XChol = 0-0.25 in the binary pSM/Chol mixtures. In comparison with the pSM/pCer mixture (Busto et al. 2009), sphingomyelin shows a higher preference for ceramide than for cholesterol. In ternary pSM/Chol/pCer mixtures, an immiscibility between cholesterol- (pSM/Chol) and ceramide-enriched (pSM/pCer) phases at high pSM/(Chol+pCer) ratio is observed, where no ceramide over cholesterol nor cholesterol over ceramide displacement is detected. Furthermore, the calorimetric and confocal microscopy data concur in showing an inability of pCer to displace cholesterol both at low and high cholesterol concentrations. Interestingly, an inverse cholesterol-mediated ceramide displacement from its tight packing with sphingomyelin is clearly observed. These observations in model membranes in the absence of the lipids commonly used to form a liquid-disordered (Lα) phase support the proposed role of raft-like domains (Silva et al, 2007) rather than ceramide, in regulating ceramide-induced platform formation within cell membranes.∗ Busto J.V. et al. (2009) Coexistence of immiscible mixtures of palmitoylsphingomyelin and palmitoylceramide in monolayers and bilayers. Biophys. J., in press.∗ Silva L.C. et al. (2007) Ceramide-domain formation and collapse in lipid rafts: membrane reorganization by an apoptotic lipid. Biophys. J., 92(2):502-516.

Giant unilamellar vesicles (GUVs) constitute a cell-sized model membrane system that allows direct visualization of particular membrane-related phenomena, such as domain formation, at the level of single vesicles using fluorescence microscopy-related techniques. Currently available protocols for the preparation of GUVs work only at very low salt concentrations, thus precluding experimentation under physiological conditions. In addition, the GUVs thus obtained lack membrane compositional asymmetry. Here we show how to prepare GUVs using a new protocol based on the electroformation method either from native membranes or organic lipid mixtures at physiological ionic strength. Additionally, we describe methods to test whether membrane proteins and glycosphingolipids preserve their natural orientation after electroformation of GUVs composed of native membranes.

Sphingosine-1-phosphate (S1P) is currently considered to be an important signaling molecule in cell metabolism. We studied a number of relevant biophysical properties of S1P, using mainly Langmuir balance, differential scanning calorimetry, (31)P-NMR, and infrared (IR) spectroscopy. We found that, at variance with other, structurally related sphingolipids that are very hydrophobic, S1P may occur in either an aqueous dispersion or a bilayer environment. S1P behaves in aqueous media as a soluble amphiphile, with a critical micelle concentration of approximately 12 muM. Micelles give rise to larger aggregates (in the micrometer size range) at and above a 1 mM concentration. The aggregates display a thermotropic transition at approximately 60 degrees C, presumably due to the formation of smaller structures at the higher temperatures. S1P can also be studied in mixtures with phospholipids. Studies with dielaidoylphosphatidylethanolamine (DEPE) or deuterated dipalmitoylphosphatidylcholine (DPPC) show that S1P modifies the gel-fluid transition of the glycerophospholipids, shifting it to lower temperatures and decreasing the transition enthalpy. Low (10 mol %) concentrations of S1P also have a clear effect on the lamellar-to-inverted hexagonal transition of DEPE, i.e., they increase the transition temperature and stabilize the lamellar versus the inverted hexagonal phase. IR spectroscopy of natural S1P mixed with deuterated DPPC allows the independent observation of transitions in each molecule, and demonstrates the existence of molecular interactions between S1P and the phospholipid at the polar headgroup level that lead to increased hydration of the carbonyl group. The combination of calorimetric, IR, and NMR data allowed the construction of a temperature-composition diagram ("partial phase diagram") to facilitate a comparative study of the properties of S1P and other related lipids (ceramide and sphingosine) in membranes. In conclusion, two important differences between S1P and ceramide are that S1P stabilizes the lipid bilayer structure, and physiologically relevant concentrations of S1P can exist dispersed in the cytosol.