Lipid Monolayers Research Articles

Three-dimensional architecture of ESCRT-III flat spirals on the membrane

The endosomal sorting complexes required for transport (ESCRTs) are responsible for membrane remodeling in many cellular processes, such as multivesicular body biogenesis, viral budding, and cytokinetic abscission. ESCRT-III, the most abundant ESCRT subunit, assembles into flat spirals as the primed state, essential to initiate membrane invagination. However, the three-dimensional architecture of ESCRT-III flat spirals remained vague for decades due to highly curved filaments with a small diameter and a single preferred orientation on the membrane. Here, we unveiled that yeast Snf7, a component of ESCRT-III, forms flat spirals on the lipid monolayers using cryogenic electron microscopy. We developed a geometry-constrained Euler angle-assigned reconstruction strategy and obtained moderate-resolution structures of Snf7 flat spirals with varying curvatures. Our analyses showed that Snf7 subunits recline on the membrane with N-terminal motifs α0 as anchors, adopt an open state with fused α2/3 helices, and bend α2/3 gradually from the outer to inner parts of flat spirals. In all, we provide the orientation and conformations of ESCRT-III flat spirals on the membrane and unveil the underlying assembly mechanism, which will serve as the initial step in understanding how ESCRTs drive membrane abscission.

The effect of nanobubbles on Langmuir-Blodgett films

Nanobubbles (NBs) are classified in two distinct categories: surface and bulk. Surface NBs are readily observed using atomic force microscopy (AFM), while the existence of bulk NBs has been a subject of debate, conflicting with the diffusion theory's predictions. Current methodologies for identifying bulk NBs yield inconclusive results. In this study, Langmuir Blodgett (LB) technique and AFM, are utilized to visualize NB imprints on anionic, cationic and zwitterionic lipid films deposited on glass-slide substrates. Our analysis of Langmuir monolayers compression isotherms reveals the impact of bulk NBs on lipid monolayer development. AFM scans of the deposited lipid films consistently show NB imprints. Notably, cationic and zwitterionic film depositions exhibit NB formations from the 1st layer, whereas in anionic films, these formations are observed only after the 3rd layer. These results suggest that the origin of these imprinted formations may be attributed to bulk NBs.

Fabrication of plasmonic probes for reproducible nanospectroscopic investigation of lipid monolayers – The electrochemical etching with DC-pulsed voltage

Tip-enhanced Raman spectroscopy (TERS) is a label-free analytical technique that characterizes molecular systems, potentially even with a nanometric resolution. In principle, the metallic plasmonic probe is illuminated with a laser beam generating the localized surface plasmons, which induce a strong local electric field enhancement in close proximity to the probe. Such field enhancement improves the Raman scattering cross-section from the sample volume localized near the probe apex. TERS provides a high spatial resolution and a great sensitivity, however, it is rather rarely used due to technical limitations causing unstable enhancement and the relative lack of data reproducibility. Despite many scientific efforts for the fabrication of effective TER probes providing robust TER enhancement still requires further investigations. In this work, we explore new possibilities based on preparation of scanning tunnelling microscopy (STM) plasmonic probes, since by nature of the tunnelling effect, they potentially could offer a very high spatial resolution in STM guided TERS experiments. Here we compare two methods of STM-TERS probe preparation for effective spectra acquisition. Our results strongly indicate that an application of square pulse voltage upon the etching procedure significantly improves the quality of the TER data over those obtained with a constant voltage one. To demonstrate the efficiency of our probes we present the results of hyperspectral TER mapping of the 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) monolayer deposited on an ultra-pure and atomically flat gold substrate.

Effects of three polyphenols with different numbers of phenolic hydroxyls on the structural and interfacial properties and lipid-protein co-oxidation of oil body emulsions

Oil bodies (OBs) are organelles for storing lipids, and they are composed of lipid cores and monolayer phospholipid protein membranes. During storage, membrane proteins anchored in the lipid core are often damaged by lipid oxidation products, leading to oxidative damage and a decreased quality of emulsified foods. Here, three natural antioxidants (gallic acid (GA), epigallocatechin gallate (EGCG), and tannic acid (TA)) were added to OB emulsions to study the effects of polyphenols on protein interfacial properties during oxidation and the lipid-protein co-oxidation behavior. Compared to GA, EGCG and TA with higher molecular weights and hydroxyl substitution showed better antioxidant activity, metal chelating activity, iron reduction ability, and affinity for OB. The polyphenols significantly reduced the contents of free amino and sulfhydryl groups in OB. Infrared spectroscopy analysis indicate that hydrogen bonding and hydrophobic interactions may exist between the polyphenols and OB. During storage, polyphenols inhibited >23% of the primary and secondary lipid oxidation products in the emulsions, whereas the formation of carbonyl groups and disappearance of sulfhydryl groups were only inhibited by 10.7% and 7.4%, respectively, indicating that polyphenols preferentially inhibited lipid oxidation over protein oxidation. Electrophoresis results showed that moderate oxidation (3 days of storage) disrupted the disulfide bonds of proteins. Note that moderate oxidation enhanced the interfacial properties of OB proteins, whereas excessive oxidation led to protein aggregation. This study offers new perspectives on using polyphenols to regulate the lipid-protein co-oxidation in OB, as well as the different properties of OB proteins after oxidation.

Depth-profiling alkyl chain order in unsaturated lipid monolayers on water.

Unsaturated lipids with C=C groups in their alkyl chains are widely present in the cell membrane and food. The C=C groups alter the lipid packing density, membrane stability, and persistence against lipid oxidation. Yet, molecular-level insights into the structure of the unsaturated lipids remain scarce. Here, we probe the molecular structure and organization of monolayers of unsaturated lipids on the water surface using heterodyne-detected sum-frequency generation (HD-SFG) spectroscopy. We vary the location of the C=C in the alkyl chain and find that at high lipid density, the location of the C=C group affects neither the interfacial water organization nor the tail of the alkyl chain. Based on this observation, we use the C=C stretch HD-SFG response to depth-profile the alkyl chain conformation of the unsaturated lipid. We find that the first 1/3 of carbon atoms from the headgroup are relatively rigid, oriented perpendicular to the surface. In contrast, the remaining carbon atoms can be approximated as free rotators, introducing the disordering of the alkyl chains.

A Sigma1 Receptor Agonist Alters Fluidity and Stability of Lipid Monolayers.

Interactions between the sigma1 receptor agonist PRE-084 and various lipid monolayers, including dipalmitoylphosphatidylcholine (DPPC), DPP-ethanolamine (DPPE), DPP-glycerol (DPPG), DPP-serine (DPPS), palmitoylsphingomyelin (PSM), and cholesterol (Ch), were investigated to elucidate the effects of PRE-084 on membrane fluidity and stability. Their interactions with sigma1 receptor agonists have potential implications for neuroprotection, antidepressant, analgesic, and cognitive enhancement effects. In this study, we observed that the presence of PRE-084 in the subphase led to increased fluidity in DPPC and DPPE monolayers, whereas decreasing fluidity was observed in DPPG, DPPS, and PSM monolayers. The interaction of PRE-084 with Ch monolayers was found to be distinct from its interaction with other lipids. Fluorescence microscopy images revealed changes in the size and shape of liquid-condensed domains in the presence of PRE-084, supporting the notion of altered membrane fluidity. Our findings provide new insights into the interaction of PRE-084 with lipid monolayers and its potential implications for biological and membrane science.

Cell-free translation system with artificial lipid-monolayer particles as a unique tool for characterizing lipid-monolayer binding proteins.

Methods for functional analysis of proteins specifically localizing to lipid monolayers such as rubber particles and lipid droplets are limited. We have succeeded in establishing a system in which artificially prepared lipid monolayer particles are added to a cell-free translation system to confirm the properties of proteins that specifically bind to lipid monolayers in a translation-coupled manner.

The mechanistic basis of the membrane-permeabilizing activities of the virulence-associated protein A (VapA) from Rhodococcus equi.

Pathogenic Rhodococcus equi release the virulence-associated protein A (VapA) within macrophage phagosomes. VapA permeabilizes phagosome and lysosome membranes and reduces acidification of both compartments. Using biophysical techniques, we found that VapA interacts with model membranes in four steps: (i) binding, change of mechanical properties, (ii) formation of specific membrane domains, (iii) permeabilization within the domains, and (iv) pH-specific transformation of domains. Biosensor data revealed that VapA binds to membranes in one step at pH 6.5 and in two steps at pH 4.5 and decreases membrane fluidity. The integration of VapA into lipid monolayers was only significant at lateral pressures <20 mN m-1 indicating preferential incorporation into membrane regions with reduced integrity. Atomic force microscopy of lipid mono- and bilayers showed that VapA increased the surface heterogeneity of liquid disordered domains. Furthermore, VapA led to the formation of a new microstructured domain type and, at pH 4.5, to the formation of 5 nm high domains. VapA binding, its integration and lipid domain formation depended on lipid composition, pH, protein concentration and lateral membrane pressure. VapA-mediated permeabilization is clearly distinct from that caused by classical microbial pore formers and is a key contribution to the multiplication of Rhodococcus equi in phagosomes.

Equilibrium behavior of branching curvature and comparison of enantiomer morphology in lipid monolayers

Equilibrium behavior of branching curvature and comparison of enantiomer morphology in lipid monolayers

Thermodynamic, rheological, and morphological properties of sakuranetin incorporated in langmuir monolayers of unsaturated lipids as a biomembrane model

Thermodynamic, rheological, and morphological properties of sakuranetin incorporated in langmuir monolayers of unsaturated lipids as a biomembrane model

Modeling shear banding lipid monolayer collapse with finite element analysis

Modeling shear banding lipid monolayer collapse with finite element analysis

Microbubble Biointerfacing by Regulation of the Platelet Membrane Surfactant Activity at the Gas‐Liquid Interface for Acute Thrombosis Targeting

AbstractBiointerfacing nanomaterials with cell membranes has been successful in the functionalization of nanoparticles or nanovesicles, but microbubble functionalization remains challenging due to the unique conformation of the lipid monolayer structure at the gas‐liquid interface that provides insufficient surfactant activity. Here, we describe a strategy to rationally regulate the surfactant activity of platelet membrane vesicles by adjusting the ratio of proteins to lipids through fusion with synthetic phospholipids (i.e., liposomes). A “platesome” with the optimized protein‐to‐lipid ratio can be assembled at the gas‐liquid interface in the same manner as pulmonary surfactants to stabilize a microsized gas bubble. Platesome microbubbles (PMBs) inherited 61.4 % of the platelet membrane vesicle proteins and maintained the active conformation of integrin αIIbβ3 without the talin 1 for fibrin binding. We demonstrated that the PMBs had good stability, long circulation, and superior functionality both in vitro and in vivo. Moreover, by molecular ultrasound imaging, the PMBs provide up to 11.8 dB of ultrasound signal‐to‐noise ratio enhancement for discriminating between acute and chronic thrombi. This surface tension regulating strategy may provide a paradigm for biointerfacing microbubbles with cell membranes, offering a potential new approach for the construction of molecular ultrasound contrast agents for the diagnosis of different diseases.

Microbubble Biointerfacing by Regulation of the Platelet Membrane Surfactant Activity at the Gas-Liquid Interface for Acute Thrombosis Targeting.

Biointerfacing nanomaterials with cell membranes has been successful in the functionalization of nanoparticles or nanovesicles, but microbubble functionalization remains challenging due to the unique conformation of the lipid monolayer structure at the gas-liquid interface that provides insufficient surfactant activity. Here, we describe a strategy to rationally regulate the surfactant activity of platelet membrane vesicles by adjusting the ratio of proteins to lipids through fusion with synthetic phospholipids (i.e., liposomes). A "platesome" with the optimized protein-to-lipid ratio can be assembled at the gas-liquid interface in the same manner as pulmonary surfactants to stabilize a microsized gas bubble. Platesome microbubbles (PMBs) inherited 61.4 % of the platelet membrane vesicle proteins and maintained the active conformation of integrin αIIbβ3 without the talin 1 for fibrin binding. We demonstrated that the PMBs had good stability, long circulation, and superior functionality both in vitro and in vivo. Moreover, by molecular ultrasound imaging, the PMBs provide up to 11.8 dB of ultrasound signal-to-noise ratio enhancement for discriminating between acute and chronic thrombi. This surface tension regulating strategy may provide a paradigm for biointerfacing microbubbles with cell membranes, offering a potential new approach for the construction of molecular ultrasound contrast agents for the diagnosis of different diseases.

Lipid Monolayer on Cell Surface Protein Templates Functional Extracellular Lipid Assembly.

When the ancestors of men moved from aquatic habitats to the drylands, their evolutionary strategy to restrict water loss is to seal the skin surface with lipids. It is unknown how these rigid ceramide-dominated lipids with densely packed chains squeeze through narrow extracellular spaces and how they assemble into their complex multilamellar architecture. Here it is shown that the human corneocyte lipid envelope, a monolayer of ultralong covalently bound lipids on the cell surface protein, templates the functional barrier assembly by partly fluidizing and rearranging the free extracellular lipids in its vicinity during the sculpting of a functional skin lipid barrier. The lipid envelope also maintains the fluidity of the extracellular lipids during mechanical stress. This local lipid fluidization does not compromise the permeability barrier. The results provide new testable hypotheses about epidermal homeostasis and the pathophysiology underlying diseases with impaired lipid binding to corneocytes, such as congenital ichthyosis. In a broader sense, this lipoprotein-mediated fluidization of rigid (sphingo)lipid patches may also be relevant to lipid rafts and cellular signaling events and inspire new functional materials.

Interaction of sakuranetin with unsaturated lipids forming Langmuir monolayers at the air-water interface: A biomembrane model

This study investigates the interaction between sakuranetin, a versatile pharmaceutical flavonoid, and monolayers composed of unsaturated phospholipids, serving as a surrogate for cell membranes. The phospholipids were 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE). We conducted a series of experiments to comprehensively investigate this interaction, including surface pressure assessments, Brewster angle microscopy (BAM), and polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS). Our findings unequivocally demonstrate that sakuranetin interacts with these phospholipids, expanding the monomolecular films. Notably, regarding POPC, the presence of sakuranetin led to a reduction in stability and a decline in surface elasticity, which can likely be attributed to intricate molecular rearrangements at the interface. The visual evidence of aggregations in BAM images reinforces the interactions substantiated by PM-IRRAS, highlighting sakuranetin's interaction with the polar and nonpolar regions of POPC. However, it is worth noting that these aggregations do not appear to contribute significantly to the viscosity of the mixed film, and our investigations did not reveal any substantial hysteresis. In contrast, when examining POPE, we observed a minor reduction in thermodynamic stability, indicative of fewer rearrangements within the monolayer. This notion was further reinforced by the limited presence of aggregations in the BAM images. Sakuranetin also increased the rigidity of the lipid monolayer; nevertheless, the monolayer remained predominantly elastic, facilitating easy re-spreading on the surface, especially for the first lipid. PM-IRRAS analysis unveiled interactions between sakuranetin and POPE's polar and nonpolar segments, compellingly explaining the observed monolayer expansion. Taken together, our data suggest that sakuranetin was more effectively incorporated into the monomolecular layer of POPE, indicating that membranes comprised of POPC might exhibit a greater degree of interaction in the presence of this pharmacologically active compound.

Nanoscale visualization of phase separation in binary supported lipid monolayer using tip-enhanced Raman spectroscopy.

Supported lipid membranes are an important model system to study the phase separation behavior at the nanoscale. However, the conventional nanoanalytical tools often fail to provide reliable chemical characterization of the phase separated domains in a non-destructive and label-free manner. This study demonstrates the application of scanning tunneling microscopy-based tip-enhanced Raman spectroscopy (TERS) to study the nanoscale phase separation in supported d62-DPPC : DOPC lipid monolayers. Hyperspectral TERS imaging successfully revealed a clear segregation of the d62-DPPC-rich and DOPC-rich domains. Interestingly, nanoscale deposits of d62-DPPC were observed inside the DOPC-rich domains and vice versa. High-resolution TERS imaging also revealed the presence of a 40-120 nm wide interfacial region between the d62-DPPC-rich and DOPC-rich domains signifying a smooth transition rather than a sharp boundary between them. The novel insights obtained in this study demonstrate the effectiveness of TERS in studying binary lipid monolayers at the nanoscale.

Selective assembly and insertion of ubiquicidin antimicrobial peptide in lipid monolayers.

Antimicrobial-resistant bacteria pose a significant threat to humans, prompting extensive research into developing new antimicrobial peptides (AMPs). The biomembrane is the first barrier of a biological cell, hence, comprehending the interaction and self-assembly of AMPs in and around such membranes is of great importance. In the present study, several biophysical techniques have been applied to explore the self-assembly of ubiquicidin (29-41), an archetypical AMP, in and around the phospholipid monolayers formed at air-water interface. Such a monolayer mimics one of the leaflets of a lipid bilayer. The surface pressure-area isotherm exhibits the strongest interaction with a negatively charged lipid, 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DPPG). The weakest affinity was towards the zwitterionic lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Another zwitterionic lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), shows an intermediate affinity. This affinity was quantified by analyzing alterations in the effective mean molecular area of the lipid, the in-plane compressional modulus of the assembly, and the electrostatic potential induced by the presence of peptides. The precise organization of the peptide around the lipid monolayer at a sub-nanometre length scale was revealed using synchrotron-based X-ray reflectivity measurements from the air-water interface. Information about the selective interaction of the peptide with lipids and their varied orientation at the lipid-water interface could be useful in understanding the selectivity of AMP in developing new antibiotics.

Structure and thermodynamics of supported lipid membranes on hydrophobic van der Waals surfaces.

Understanding the adsorption and physical characteristics of supported lipid membranes is crucial for their effective use as model cell membranes. Their morphological and thermodynamic properties at the nanoscale have traditionally been studied on hydrophilic substrates, such as mica and silicon oxide, which have proved to facilitate the reconstruction of biomembranes. However, in more recent years, with the advent of the van der Waals crystals technology, two-dimensional crystals such as graphene have been proposed as potential substrates in biosensing devices. Membranes formed on these crystals are expected to behave differently owing to their intrinsic hydrophobicity, however thus far knowledge of their morphological and thermodynamic properties is lacking. Here we present a comprehensive nanoscale analysis of the adsorption of phosphatidylcholine lipid monolayers on two of the most commonly used van der Waals crystals, graphite and hexagonal boron nitride. Both morphological and thermodynamic properties of the lipid membranes were investigated using temperature-controlled atomic force microscopy. Our experiments show that the lipids adsorb onto the crystals, forming monolayers with their orientation dependent upon their concentration. Furthermore, we found that the hydrophobicity of van der Waals crystals determines a strong increase in the transition temperature of the lipid monolayer compared to that observed on hydrophilic substrates. These results are important for understanding the properties of lipid membranes at solid surfaces and extending their use to novel drug delivery and biosensing devices made of van der Waals crystals.

Biological lipid hydration: distinct mechanisms of interfacial water alignment and charge screening for model lipid membranes.

Studying lipid monolayers as model biological membranes, we demonstrate that water molecules interfacing with different model membranes can display preferential orientation for two distinct reasons: due to charges on the membrane, and due to large dipole fields resulting from zwitterionic headgroups. This preferential water orientation caused by the charge or the dipolar field can be effectively neutralized to net-zero water orientation by introducing monolayer counter-charges (i.e. lipids with oppositely charged headgroups). Following the Gouy-Chapman model, the effect of monolayer surface charge on water orientation is furthermore strongly dependent on the electrolyte concentration and thus on the counterions in solution. In contrast, the effect of ions in the subphase on the dipolar alignment of water is zero. As a result, the capability of monolayer counter-charges to null the effect of dipolar orientation is strongly electrolyte-dependent. Notably, the different effects are additive for mixed charged/zwitterionic lipid systems occurring in nature. Specifically, for an E. coli lipid membrane extract consisting of both zwitterionic and negatively charged lipids, the water orientation can be explained by the sum of the constituents. Our results can be quantitatively reproduced using Gouy-Chapman theory, revealing the relatively straightforward electrostatic effects on the hydration of complex membrane interfaces.

Membrane fluidity properties of lipid-coated polylactic acid nanoparticles.

Lipid coating is considered a versatile strategy to equip nanoparticles (NPs) with a biomimetic surface coating, but the membrane properties of these nanoassemblies remain in many cases insufficiently understood. In this work, we apply C-Laurdan generalized polarization (GP) measurements to probe the temperature-dependent polarity of hybrid membranes consisting of a lipid monolayer adsorbed onto a polylactic acid (PLA) polymer core as function of lipid composition and compare the behavior of the lipid coated NPs (LNPs) with that of liposomes assembled from identical lipid mixtures. The LNPs were generated by nanoprecipitation of the polymer in aqueous solutions containing two types of lipid mixtures: (i) cholesterol, dipalmitoylphosphatidylcholine (DPPC), and the ganglioside GM3, as well as (ii) dioleoylphosphatidylcholine (DOPC), DPPC and GM3. LNPs were found to exhibit more distinct and narrower phase transitions than corresponding liposomes and to retain detectable phase transitions even for cholesterol or DOPC concentrations that yielded no detectable transitions in liposomes. These findings together with higher GP values in the case of the LNPs for temperatures above the phase transition temperature indicate a stabilization of the membrane through the polymer core. LNP binding studies to GM3-recognizing cells indicate that differences in the membrane fluidity affect binding avidity in the investigated model system.