Model Cell Membranes Research Articles

Substrate-induced electrostatic potential varies composition of supported lipid bilayer containing anionic lipid

Supported lipid bilayers (SLBs) are artificial lipid bilayers at solid–liquid interfaces applied as cell membrane model systems. An advantage of the artificial system is that the lipid composition can be controlled arbitrarily. On the other hand, the SLB formation process and its efficiency are affected by the properties of the solid substrate surface. In this study, we investigated the effect of the electrostatic interaction between the negatively charged SiO2/Si substrate surface and the lipid bilayer membrane on the composition of binary SLBs comprising anionic and neutral lipids. The phase transition temperature and the area fraction of lipid domains of SLB were evaluated by fluorescence microscopy and the fluorescence recovery after photobleaching. The neutral lipid was preferably included in SLB, but the anionic lipid ratio increased with Ca2+ concentration during the SLB formation. The lipid composition in SLB can be controlled by modulating the substrate-induced electrostatic potential.

The Past and the Future of Langmuir and Langmuir-Blodgett Films.

The Langmuir-Blodgett (LB) technique, through which monolayers are transferred from the air/water interface onto a solid substrate, was the first method to allow for the controlled assembly of organic molecules. With its almost 100 year history, it has been the inspiration for most methods to functionalize surfaces and produce nanocoatings, in addition to serving to explore concepts in molecular electronics and nanoarchitectonics. This paper provides an overview of the history of Langmuir monolayers and LB films, including the potential use in devices and a discussion on why LB films are seldom considered for practical applications today. Emphasis is then given to two areas where these films offer unique opportunities, namely, in mimicking cell membrane models and exploiting nanoarchitectonics concepts to produce sensors, investigate molecular recognitions, and assemble molecular machines. The most promising topics for the short- and long-term prospects of the LB technique are also highlighted.

Structure and hydrodynamic factors in lipid vesicle solutions

Lipid vesicles are widely used as models for cell membranes and extracellular vesicles, hosts for membrane protein studies, and containers for hydrophilic biotherapeutic molecules. Vesicle solutions are usually prepared at a specific lipid concentration; however, because vesicles are solvent-filled structures, the corresponding volume fraction of vesicles is at least a factor or 3 times higher than the corresponding lipid volume fraction and critically depends on the vesicle radii. Seemingly low lipid concentrations correspond to significantly higher vesicle volume fractions and closer face-to-face distances between their surfaces than may be expected.

Effect of functionalized polystyrene nanoparticles on model cell membranes

To understand applications including drug delivery, MRI contrast agents, and consumer products involving nanoparticle (NP) interactions with cell membranes, the interfacial contact between NPs and the lipid membrane and subsequent effects were characterized. To measure these properties, phospholipid model cell membranes in the form of small unilamellar vesicles (SUVs) and giant unilamellar vesicles (GUVs) were exposed to carboxyl modified, amine modified, or sulfate modified NPs to determine their effect on vesicle size, stability, and morphology.

Interaction of the huntingtin N-terminal sequence with model cell membranes

Huntington’s disease, a fatal neurodegenerative disorder, is caused by the expansion of the polyglutamine (polyQ) tract near the N-terminus of huntingtin protein (htt). Expanded polyQ domains are correlated with a tendency to aggregate into oligomers and fibrils, species that are likely neurotoxic. The number of polyQ repeats is directly associated with the severity and age of onset of Huntington’s disease. The htt protein has several proposed functions, many of which are related to interactions with lipid membranes.

Effects of charge on the interaction of the huntingtin N-terminal sequence with model cell membranes

Huntington’s Disease (HD) is a genetic neurodegenerative disorder caused by an expansion in the polyglutamine (poly-Q) region near the N-terminus of the huntingtin (htt) protein. When the poly-Q expansion surpasses a threshold of 35-40 glutamine repeats, the protein misfolds into a beta sheet rich structure that forms insoluble neuronal aggregates. The poly-Q region of htt is flanked by a 17 amino acid sequence (Nt17) that facilitates insertion of htt into lipid membranes via an amphipathic alpha helical structure, a function that is proposed to anchor the protein and may affect the aggregation process.

Structural determination of model phospholipid membranes by Raman spectroscopy: Laboratory experiment.

In an upper-division interdisciplinary laboratory experiment, students use Raman spectroscopy to highlight how the overall structure and conformational order of lipid bilayers can be influenced by their individual phospholipid composition. Students prepare a supported lipid bilayer, as a model cell membrane, by spreading liposomes made of various phospholipids on a solid support. The characterization of phospholipid bilayers, a major component of cellular membranes, can advance our fundamental understanding of important biological phenomena, with significant implications in various fields including drug delivery and development. We use Raman spectroscopy as an analytical tool to investigate the structural and packing properties of model cell membranes. The spectral frequency, intensity, and line-width of lipid Raman bands are extremely sensitive to structural alterations. This experimental module effectively exposes students to the fundamentals of Raman spectroscopy and teaches students the importance of interdisciplinary education as they integrate concepts from chemical structure, molecular interactions, and analytical spectroscopic techniques to gain a more holistic understanding of biological membrane properties.

Focal opening of the neuronal plasma membrane by shock-induced bubble collapse for drug delivery: a coarse-grained molecular dynamics simulation.

Cell permeabilization using shock-induced bubble collapse provides an attractive choice for drug delivery systems. In this work, based on a realistically human brain plasma membrane (PM) model, we investigated the focal opening of this complex model by the jet from cavitation, focusing on the effect of characteristic membrane components, particle velocity (up) and bubble diameters (D). Both high levels of cholesterol and specific cerebrosides in the PM model limit the pore opening of cavitation jets. Sphingomyelin is the opposite, but has little effect due to its low content. Two adjustable parameters of up and D can be coupled to control the opening size. The relationship between them and the maximum pore area was provided for the first time. The maximum pore area increases with the up (or the impulse that is positively related to up) in the low-speed range, which agrees with the experimentally observed impulse determinism. However, the maximum area drops in the high-speed range. Combined with D, we proposed that the jet size determines the pore size, not the impulse. Larger bubbles that can create a larger pore in the membrane have a larger jet size, but their impulse is relatively small. Finally, the recovery simulation shows that the membrane with a small pore can be quickly recovered within 300 ps, while that with a larger pore did not recover until 2 μs. These rules from this work may be helpful to optimize the choice of shock waves for the delivery of different drugs across membranes.

Insight into carbon quantum dot-vesicles interactions: role of functional groups.

Understanding carbon quantum dot–cell membrane interaction is essential for designing an effective nanoparticle-based drug delivery system. In this study, an attempt has been made to study the interaction involving phosphatidylcholine vesicles (PHOS VES, as model cell membrane) and four different carbon quantum dots bearing different functional groups (–COOH, –NH2, –OH, and protein bovine serum albumin coated) using various tools such as PL behavior, surface charge on vesicles, QCM, ITC, TEM, LSV, and FTIR. From the above studies, it was observed that the –NH2 terminating carbon dots were capable of binding strongly with the vesicles whereas other functional groups bearing carbon dots were not significantly interacting. This observation was also supported by direct visual evidence as shown by transmission electron microscopy, which shows that the polyethyleneimine carbon dot (PEICD) bearing –NH2 functionality has greater affinity towards PHOS VES. The mechanistic insight presented in the paper indicates greater possibility of higher H-bonding, signifying better interaction between –NH2 functionalized carbon dots and PHOS VES supported by FTIR, QCM, ITC and TEM. Moreover, the transport of neurotransmitters (which are generally amine compound) in neurons for cellular communication through synapse is only possible through vesicular platforms, showing that in our body, such interactions are already present. Such studies on the nano–bio interface will help biomedical researchers design efficient carbon-based nanomaterial as drug/gene delivery vehicles.

Membrane model as key tool in the study of glutathione-s-transferase mediated anticancer drug resistance

Glutathione-s-transferase is believed to be involved in the resistance to chemotherapeutic drugs, which depends on the interaction with the cell membranes. In this study, we employed Langmuir monolayers of a mixture of phospholipids and cholesterol (MIX) as models for tumor cell membranes and investigated their interaction with the anticancer drugs cisplatin (CDDP) and doxorubicin (DOX). We found that both DOX and CDDP expand and affect the elasticity of MIX monolayers, but these effects are hindered when glutathione-s-transferase (GST) and its cofactor glutathione (GSH) are incorporated. Changes are induced by DOX or CDDP on the polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) data for MIX/GST/GSH monolayers, thus denoting some degree of interaction that is not sufficient to alter the monolayer mechanical properties. Overall, the results presented here give support to the hypothesis of the inactivation of DOX and CDDP by GST and point to possible directions to detect and fight drug resistance.

Predicting the conformational variability of oncogenic GTP-bound G12D mutated KRas-4B proteins at zwitterionic model cell membranes.

KRas proteins are the largest family of mutated Ras isoforms, participating in a wide variety of cancers. Due to their importance, large effort is being carried out on drug development by small-molecule inhibitors. However, understanding protein conformational variability remains a challenge in drug discovery. In the case of the Ras family, their multiple conformational states can affect the binding of potential drug inhibitors. To overcome this challenge, we propose a computational framework based on combined all-atom Molecular Dynamics and Metadynamics simulations in order to accurately access conformational variants of the target protein. We tested the methodology using a G12D mutated GTP bound oncogenic KRas-4B protein located at the interface of a DOPC/DOPS/cholesterol model anionic cell membrane. Two main orientations of KRas-4B at the anionic membrane have been determined. The corresponding torsional angles are taken as reliable reaction coordinates so that free-energy landscapes are obtained by well-tempered metadynamics simulations, revealing local and global minima of the free-energy hypersurface and unveiling reactive paths of the system between the two preferential orientations. We have observed that GTP-binding to KRas-4B has huge influence on the stabilisation of the protein and it can potentially help to open Switch I/II druggable pockets, lowering energy barriers between stable states and resulting in cumulative conformers of KRas-4B. This may highlight new opportunities for targeting the unique meta-stable states through the design of new efficient drugs.

Ascorbate-and iron-driven redox activity of Dp44mT and Emodin facilitates peroxidation of micelles and bicelles

BackgroundIron (Fe)-induced oxidative stress leads to reactive oxygen species that damage biomembranes, with this mechanism being involved in the activity of some anti-cancer chemotherapeutics. MethodsHerein, we compared the effect of the ligand, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), or the potential ligand, Emodin, on Fe-catalyzed lipid peroxidation in cell membrane models (micelles and bicelles). These studies were performed in the presence of hydrogen peroxide (H2O2) and the absence or presence of ascorbate. ResultsIn the absence of ascorbate, Fe(II)/Emodin mixtures incubated with H2O2 demonstrated slight pro-oxidant properties on micelles versus Fe(II) alone, while the Fe(III)-Dp44mT complex exhibited marked antioxidant properties. Examining more physiologically relevant phospholipid-containing bicelles, the Fe(II)- and Fe(III)-Dp44mT complexes demonstrated antioxidant activity without ascorbate. Upon adding ascorbate, there was a significant increase in the peroxidation of micelles and bicelles in the presence of unchelated Fe(II) and H2O2. The addition of ascorbate to Fe(III)-Dp44mT substantially increased the peroxidation of micelles and bicelles, with the Fe(III)-Dp44mT complex being reduced by ascorbate to the Fe(II) state, explaining the increased reactivity. Electron paramagnetic resonance spectroscopy demonstrated ascorbyl radical anion generation after mixing ascorbate and Emodin, with signal intensity being enhanced by H2O2. This finding suggested Emodin semiquinone radical formation that could play a role in its reactivity via ascorbate-driven redox cycling. Examining cultured melanoma cells in vitro, ascorbate at pharmacological levels enhanced the anti-proliferative activity of Dp44mT and Emodin. Conclusions and general significanceAscorbate-driven redox cycling of Dp44mT and Emodin promotes their anti-proliferative activity.

Mechanisms of Interaction of Small Hydroxylated Cryosolvents with Dehydrated Model Cell Membranes: Stabilization vs Destruction.

The mechanism by which cryosolvents such as alcohols modify and penetrate cell membranes as a function of their concentration and hydration state remains poorly understood. We conducted molecular dynamics simulations of 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayers in the presence of aqueous solutions of four common penetrating hydroxylated cryosolvents (methanol, ethylene glycol, propylene glycol, and glycerol) at varying concentration ranges and across three different hydration states. All cryosolvents were found to preferentially replace water at the bilayer interface, and a reduction in hydration state correlates with a higher proportion of cryosolvent at the interface for relative concentrations. Minor differences in chemical structure had a profound effect on cryosolvent-membrane interactions, as the lone methyl groups of methanol and propylene glycol enhanced their membrane localization and penetration, but with increasing concentrations acted to destabilize the membrane structure in a process heightened at higher hydration states. By contrast, ethylene glycol and glycerol promoted and retained membrane structural integrity by forming hydrogen-bonded lipid bridges via distally located hydroxyl groups. Glycerol exhibited the highest capacity to cross-link lipids at relative concentrations, as well as promoted a bilayer structure consistent with a fully hydrated bilayer in the absence of cryosolvent for all hydration states investigated.

Membrane Potentials Trigger Molecular-Scale Rearrangements in the Outer Membrane of Gram-Negative Bacteria.

The structural complexity of the cell envelope of Gram-negative bacteria limits the fabrication of realistic models of bacterial cell membranes. A vertical Langmuir-Blodgett withdrawing was used to deposit a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) monolayer on the Au(111) surface. The second leaflet composed of di[3-deoxy-D-manno-octulosonyl]-lipid A (KLA) was deposited using Langmuir-Schaefer transfer. The use of an electrode material as a support for the POPE-KLA bilayer allowed electrochemical control of the membrane's stability, compactness, and structure. Capacitance-potential curves showed a typical pattern for the supported lipid bilayers electrochemical characteristic. The minimum membrane capacitance was ∼4 μF cm-2 and did not change in the following desorption-adsorption cycles, indicating the presence of a stable bilayer structure with an asymmetric composition of both leaflets. However, at a molecular scale, as elucidated in spectroelectrochemical experiments, large differences in the response of both leaflets to electric potentials were observed. The acyl chains in POPE and KLA existed in a liquid state. The quantitative analysis of the CH stretching modes indicated potential-driven reorientations in the hydrophobic fragment of the bilayer, already in the adsorbed state. To assign observed rearrangements to POPE and KLA lipids in both leaflets, per-deuterated d31-POPE was transferred into the inner leaflet. Since no potential-dependent changes of the CD2 stretching modes in the d31-POPE-KLA bilayer were observed, reorientations in the acyl chain region were assigned to the KLA molecules. Mg2+ ions were bound to the polar head groups of KLA. The strength of electrostatic interactions in the polar head group region of KLA was dependent on the direction of the electric field. At negative electric potentials, the binding of divalent cations weakened, which gave the KLA molecules increased orientational flexibility. This behavior in electric fields is peculiar for the outer membrane and indicates that the microbial cell membranes have different electrochemical properties than phospholipid bilayers.

Lipid Membrane State Change by Catalytic Protonation and the Implications for Synaptic Transmission.

In cholinergic synapses, the neurotransmitter acetylcholine (ACh) is rapidly hydrolyzed by esterases to choline and acetic acid (AH). It is believed that this reaction serves the purpose of deactivating ACh once it has exerted its effect on a receptor protein (AChR). The protons liberated in this reaction, however, may by themselves excite the postsynaptic membrane. Herein, we investigated the response of cell membrane models made from phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidic acid (PA) to ACh in the presence and absence of acetylcholinesterase (AChE). Without a catalyst, there were no significant effects of ACh on the membrane state (lateral pressure change mN/m). In contrast, strong responses were observed in membranes made from PS and PA when ACh was applied in presence of AChE (>5 mN/m). Control experiments demonstrated that this effect was due to the protonation of lipid headgroups, which is maximal at the pK (for PS: ; for PA: ). These findings are physiologically relevant, because both of these lipids are present in postsynaptic membranes. Furthermore, we discussed evidence which suggests that AChR assembles a lipid-protein interface that is proton-sensitive in the vicinity of pH . Such a membrane could be excited by hydrolysis of micromolar amounts of ACh. Based on these results, we proposed that cholinergic transmission is due to postsynaptic membrane protonation. Our model will be falsified if cholinergic membranes do not respond to acidification.

Influence of lipophilicity of anthracyclines on the interactions with cholesterol in the model cell membranes – Langmuir monolayer and SEIRAS studies

The interactions of anthracyclines with biological membranes strongly depend on the drug lipophilicity, which might also determine the specific affinity to cholesterol molecules. Therefore, in this work we show the studies concerning the effect of two selected anthracyclines, daunorubicin (DNR) and idarubicin (IDA) on simple models of healthy (DMPC:Chol 7:3) and cancer cells membranes with increased level of cholesterol (DMPC:Chol 3:7) as well as pure cholesterol monolayers prepared at the air-water interface and supported on gold surface. It has been shown that more lipophilic IDA is able to penetrate cholesterol monolayers more effectively than DNR due to the formation of IDA-cholesterol arrangements at the interface, as proved by the thermodynamic analysis of compression-expansion cycles. The increased interactions of IDA were also confirmed by the time measurements of pre-compressed monolayers exposed to drug solutions as well as grazing incidence X-ray diffraction studies demonstrating differences in the 2D organization of cholesterol monolayers. Langmuir studies of mixed DMPC:Chol membranes revealed the reorganization of molecules in the cancer cell models at the air-water interface at higher surface pressures due to the removal of DNR, while increased affinity of IDA towards cholesterol allowed this drug to penetrate the layer more efficiently without its removal. The SEIRAS spectra obtained for supported DMPC:Chol bilayers proved that IDA locates both in the ester group and in the acyl chain region of the bilayer, while DNR does not penetrate the membranes as deeply as IDA. The increased penetration of the mixed phospholipid layers by idarubicin might be attributed to the higher lipophilicity caused by the lack of methoxy group and resulting in a specific affinity towards cholesterol.

Hybrid polymer/lipid vesicles: Influence of polymer architecture and molar mass on line tension

Hybrid polymer/lipid vesicles are self-assembled structures that have been the subject of an increasing number of studies in recent years. They are particularly promising tools in the development of cell membrane models because they offer the possibility to fine-tune their membrane structure by adjusting the distribution of components (presence or absence of “raft-like” lipid domains), which is of prime importance to control their membrane properties. Line tension in multiphase membranes is known to be a key parameter on membrane structuration, but remains unexplored, either experimentally or by computer modeling for hybrid polymer/lipid vesicles. In this study, we were able to measure the line tension on different budded hybrid vesicles, using a micropipette aspiration technique, and show the influence of the molar mass and the architecture of block copolymers on line tension and its consequences for membrane structuration.

Purification, molecular characterization and ligand binding properties of the major donkey seminal plasma protein DSP-1

Fibronectin type-II (FnII) family proteins are the major proteins in many mammalian species including bull, horse and pig. In the present study, a major FnII protein has been identified and isolated from donkey (Equus hemionus) seminal plasma, which we refer to as Donkey Seminal Plasma protein-1 (DSP-1). The amino acid sequence determined by mass spectrometry and computational modeling studies revealed that DSP-1 is homologous to other mammalian seminal plasma proteins, including bovine PDC-109 (also known as BSP-A1/A2) and equine HSP-1/2. High-resolution LC-MS analysis indicated that the protein is heterogeneously glycosylated and also contains multiple acetylations, occurring in the attached glycans. Structural and thermal stability studies on DSP-1 employing CD spectroscopy and differential scanning calorimetry showed that the protein unfolds at ~43 °C and binding to phosphorylcholine (PrC) – the head group moiety of choline phospholipids – increases its thermal stability. Intrinsic fluorescence titrations revealed that DSP-1 recognizes lyso-phosphatidylcholine with over 100-fold higher affinity than PrC. Further, interaction of DSP-1 with erythrocytes, a model cell membrane, revealed that DSP-1 binding is mediated by a specific interaction with choline phospholipids and results in membrane perturbation, suggesting that binding of this protein to sperm plasma membrane could be physiologically significant.

A Brief Introduction to Some Aspects of the Fluid-Mosaic Model of Cell Membrane Structure and Its Importance in Membrane Lipid Replacement.

Early cell membrane models placed most proteins external to lipid bilayers in trimolecular structures or as modular lipoprotein units. These thermodynamically untenable structures did not allow lipid lateral movements independent of membrane proteins. The Fluid–Mosaic Membrane Model accounted for these and other properties, such as membrane asymmetry, variable lateral mobilities of membrane components and their associations with dynamic complexes. Integral membrane proteins can transform into globular structures that are intercalated to various degrees into a heterogeneous lipid bilayer matrix. This simplified version of cell membrane structure was never proposed as the ultimate biomembrane description, but it provided a basic nanometer scale framework for membrane organization. Subsequently, the structures associated with membranes were considered, including peripheral membrane proteins, and cytoskeletal and extracellular matrix components that restricted lateral mobility. In addition, lipid–lipid and lipid–protein membrane domains, essential for cellular signaling, were proposed and eventually discovered. The presence of specialized membrane domains significantly reduced the extent of the fluid lipid matrix, so membranes have become more mosaic with some fluid areas over time. However, the fluid regions of membranes are very important in lipid transport and exchange. Various lipid globules, droplets, vesicles and other membranes can fuse to incorporate new lipids or expel damaged lipids from membranes, or they can be internalized in endosomes that eventually fuse with other internal vesicles and membranes. They can also be externalized in a reverse process and released as extracellular vesicles and exosomes. In this Special Issue, the use of membrane phospholipids to modify cellular membranes in order to modulate clinically relevant host properties is considered.

Altering model cell membranes by means of photoactivated organic functionalized gold nanorods

A possible non-invasive photothermal therapy employs photothermal agents with high light-to-heat conversion efficiency. Gold nanorods (Au-NRs) can be applied as photothermal agents because of their optical properties and specific tumor-targeting capability. The potential therapeutic applications and toxicological effects of photothermal agents depend on the interaction of the Au-NRs with the cell membrane, the understanding of which is of great importance. Although studies in this area have been performed, the interactions between model cell membranes and photoactivated Au-NRs have not yet been described. In this study, we explain how local excitation of the Au-NRs caused a local temperature increase in their vicinity that affected the properties of a model membrane composed of dipalmitoylphosphatidylcholine (DPPC). Our results showed that the illumination of the Au-NRs changed the DPPC’s organization in the Langmuir monolayer. Upon photoactivation, the mutual distances between the DPPC molecules increased; however, the conformation of the lipid tails remained unchanged. Moreover, the illumination of the membrane at a surface pressure corresponding to that in a native cellular membrane caused a more stable and elastic behavior of the Langmuir monolayer. The results obtained corroborate the theory that photoactivation of Au-NRs influences the packing and phase behavior of mimetic cell membranes. In the long term, it may allow us to understand how the photoactivation of nanoparticles facilitates the transport of medicinal substances.