Cholesterol and ceramide: An unlikely pair

Cholesterol (Chol) has been commonly considered as a structural component in lipid membranes, with the capacity to induce formation of liquid-ordered platforms that might in turn have a decisive effect on membrane trafficking and protein clustering. Meanwhile, sphingolipids have appeared as important pieces of the “membrane puzzle,” with many of them playing bioactive roles in a variety of cellular processes. Among sphingolipids, some of them (e.g., sphingomyelins, SM) appear to exhibit a preferential interaction with Chol, while others (such as ceramides, Cer) have been described to compete with Chol for the same interaction sites, mostly in the SM amide group, due to the high hydrophobicity of both molecules. However, publications in the last decade have demonstrated that Chol and Cer may not always displace each other; rather, they can interact in a mutually stabilizing fashion, particularly in the presence of SM. More importantly, experiments have shown that this interaction may also occur in predominantly fluid (but heterogeneous) membranes. Furthermore, while the early experiments to probe this interaction explored the effects of C16:0 sphingolipids, newer reports have shown that Chol-Cer interactions are also possible in the presence of C24:0 and/or C24:1 sphingolipids, which are (together with C16:0) among the most abundant sphingolipid species occurring in natural membranes. In this manuscript, we explore the latest reports on this issue, which are mainly centered on different scenarios where Chol-Cer interactions may appear, and the putative relevance of such interactions on membrane functionality.This work was supported in part by the Spanish Ministerio de Ciencia e Innovación (MCI), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) (grant No. PGC2018-099857-B-I00), by the Eusko Jaurlaritza (Grants No. IT1264-19, IT1196-19, and IT1270-19), by the Fundación Biofísica Bizkaia, and by the Basque Excellence Research Centre (BERC) program of the Basque Government.Peer reviewe

Sphingolipid interactions in model membrane systems (lipid bilayers) containing cholesterol

Póster presentado al 8th International Iberian Biophysics Congress celebrado en Bilbao los días 20 y 21 de junio de 2022.Sphingolipid molecular interactions are important because of their structural functions and of their role as second messengers in some cellular processes. Among them, sphingomyelin (SM) is the major sphingolipid component of mammalian cells, in whose plasma membrane it can constitute up to 10 mol% of the phospholipids. SM has attracted the attention of scientists due to its strong interaction with other lipids, such as cholesterol (Chol), which interacts with SM giving rise to cholesterol-enriched domains in a liquid ordered (L0) phase, and ceramide, which segregates into highly packed gel domains that could act as signaling platforms in several cellular processes as apoptosis, cell senescence and death. Recent lipidomic studies quantified the presence of different natural sphingolipids in several mammal tissues, showing that longer and saturated ceramides such as d18:1/18:0 and d18:1/24:0 predominate in brain tissue, while d18:1/16:0 are most abundant in other tissues. To further study the interactions between the different sphingolipids in the plasma membrane, and trying to elucidate how their distribution affects their biophysical properties, a liquid- disordered/liquid-ordered (LdL0) cell membrane environment is recreated in a membrane model system composed of DOPC:SM:Chol (2:1:1) + Cer. To characterize this model, a combination of techniques was used: atomic force microscopy (AFM), confocal microscopy, and differential scanning calorimetry (DSC)

β-Amyloid (1-42) peptide adsorbs but does not insert into ganglioside-containing phospholipid membranes in the liquid-disordered state: modelling and experimental studies

β-Amyloid (Aβ) is a 39-43 residue peptide involved in the pathogenesis of Alzheimer's disease. Aβ deposits onto the cells and gives rise to the plaques that are characteristic of the disease. In an effort to understand the molecular mechanism of plaque formation, we have examined the interaction of Aβ42, considered to be the most pathogenic of the peptides, with lipid bilayers consisting of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) to which small amounts of GM1 ganglioside (1-5 mol%) were incorporated. POPC bilayers exist in the fluid, or liquid-disordered state at room temperature, mimicking the fluidity of cell membranes. An Aβ42 preparation consisting essentially of peptide monomers was used. A combination of molecular dynamics (MD), isothermal calorimetry and Langmuir balance measurements was applied. Our results show that Aβ binds POPC bilayers, and that binding increases (ΔG of binding decreases) with GM1, but only up to 3 mol% of the ganglioside, larger concentrations appearing to have a lower effect. MD and Langmuir balance measurements concur in showing that the peptide adsorbs onto the bilayer surface, but does not become inserted into it at surface pressures compatible with the cell membrane conditions. Thioflavin T measurements agree with MD in revealing a very low degree of peptide oligomerization/aggregation under our conditions. This is in contrast with previous studies showing peptide aggregation and insertion when interacting with membranes in the liquid-ordered state. The present contribution underlines the importance of bilayer lipid composition and properties for Aβ plaque formation.This work was supported in part by grants from the Spanish Ministry of Economy (grant FEDER MINECO PGC2018-099857-B-I00) and the Basque Government (grants No. IT1264-19 and IT1270-19). ABGA was supported by the University of the Basque Country. This work was partially funded by FOCEM (MERCOSUR Structural Convergence Fund), COF 03/11.Peer reviewe

Membrane Permeabilization by <i>Bordetella</i> Adenylate Cyclase Toxin Involves Pores of Tunable Size

RTX (Repeats in ToXin) pore-forming toxins constitute an expanding family of exoproteins secreted by many Gram-negative bacteria and involved in infectious diseases caused by said pathogens. Despite the relevance in the host/pathogen interactions, the structure and characteristics of the lesions formed by these toxins remain enigmatic. Here, we capture the first direct nanoscale pictures of lytic pores formed by an RTX toxin, the Adenylate cyclase (ACT), secreted by the whooping cough bacterium Bordetella pertussis. We reveal that ACT associates into growing-size oligomers of variable stoichiometry and heterogeneous architecture (lines, arcs, and rings) that pierce the membrane, and that, depending on the incubation time and the toxin concentration, evolve into large enough &#8220;holes&#8222; so as to allow the flux of large molecular mass solutes, while vesicle integrity is preserved. We also resolve ACT assemblies of similar variable stoichiometry in the cell membrane of permeabilized target macrophages, proving that our model system recapitulates the process of ACT permeabilization in natural membranes. Based on our data we propose a non-concerted monomer insertion and sequential mechanism of toroidal pore formation by ACT. A size-tunable pore adds a new regulatory element to ACT-mediated cytotoxicity, with different pore sizes being putatively involved in different physiological scenarios or cell types

Patches and Blebs: A Comparative Study of the Composition and Biophysical Properties of Two Plasma Membrane Preparations from CHO Cells

This study was aimed at preparing and characterizing plasma membranes (PM) from Chinese Hamster Ovary (CHO) cells. Two methods of PM preparation were applied, one based on adhering cells to a poly-lysine-coated surface, followed by hypotonic lysis and removal of intracellular components, so that PM patches remain adhered to each other, and a second one consisting of bleb induction in cells, followed by separation of giant plasma membrane vesicles (GPMV). Both methods gave rise to PM in sufficient amounts to allow biophysical and biochemical characterization. Laurdan generalized polarization was used to measure molecular order in membranes, PM preparations were clearly more ordered than the average cell membranes (GP ≈0.450 vs. ≈0.20 respectively). Atomic force microscopy was used in the force spectroscopy mode to measure breakthrough forces of PM, both PM preparations provided values in the 4-6 nN range, while the corresponding value for whole cell lipid extracts was ≈2 nN. Lipidomic analysis of the PM preparations revealed that, as compared to the average cell membranes, PM were enriched in phospholipids containing 30-32 C atoms in their acyl chains but were relatively poor in those containing 34-40 C atoms. PM contained more saturated and less polyunsaturated fatty acids than the average cell membranes. Blebs (GPMV) and patches were very similar in their lipid composition, except that blebs contained four-fold the amount of cholesterol of patches (≈23 vs. ≈6 mol% total membrane lipids) while the average cell lipids contained 3 mol%. The differences in lipid composition are in agreement with the observed variations in physical properties between PM and whole cell membranes.This work was supported in part by grant PGC2018-099857-B-I00 (MCI/AEI/FEDER, UE) and grants IT1264-19 and IT1270-19 from the Basque Government.BGM was a predoctoral student supported by the University of the Basque Country. ABGA was a post-doctoral scientist supported by the University of the Basque Country. The technical assistance by A. Marcos was supported by Fundación Biofísica Bizkaia and the Basque Excellence Research Centre (BERC) program of the Basque Government.Peer reviewe

N-Nervonoylsphingomyelin (C24:1) Prevents Lateral Heterogeneity in Cholesterol-Containing 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.Fil: Maté, Sabina María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Busto, Jon V.. Universidad del País Vasco; España. Consejo Superior de Investigaciones Científicas; EspañaFil: García Arribas, Aritz B.. Universidad del País Vasco; España. Consejo Superior de Investigaciones Científicas; EspañaFil: Sot, Jesús. Consejo Superior de Investigaciones Científicas; España. Universidad del País Vasco; EspañaFil: Vazquez, Romina Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Herlax, Vanesa Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Wolf, Claude. Universite Pierre et Marie Curie; FranciaFil: Bakas, Laura Susana. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas; ArgentinaFil: Goñi, Felix M. Universidad del País Vasco; España. Consejo Superior de Investigaciones Científicas; Españ