Validity and applicability of membrane model systems for studying interactions of peripheral membrane proteins with lipids

The cell membrane serves, at the same time, both as a barrier that segregates as well as a functional layer that facilitates selective communication. It is characterized as much by the complexity of its components as by the myriad of signaling process that it supports. And, herein lays the problems in its study and understanding of its behavior — it has a complex and dynamic nature that is further entangled by the fact that many events are both temporal and transient in their nature. Model membrane systems that bypass cellular complexity and compositional diversity have tremendously accelerated our understanding of the mechanisms and biological consequences of lipid–lipid and protein–lipid interactions. Concurrently, in some cases, the validity and applicability of model membrane systems are tarnished by inherent methodical limitations as well as undefined quality criteria. In this review we introduce membrane model systems widely used to study protein–lipid interactions in the context of key parameters of the membrane that govern lipid availability for peripheral membrane proteins. This article is part of a Special Issue entitled Tools to study lipid functions.     

Detection of lipid domains in model and cell membranes by fluorescence lifetime imaging microscopy

The discovery that the lipids constituting the plasma membrane are not randomly distributed, but instead are able to form laterally segregated lipid domains with different properties has given hints how the formation of such lipid domains influences and regulates many processes occurring at the plasma membrane. While in model systems these lipid domains can be easily accessed and their properties studied, it is still challenging to determine the properties of cholesterol rich lipid domains, the so called “Rafts”, in the plasma membrane of living cells due to their small size and transient nature. One promising technique to address such issues is fluorescence lifetime imaging (FLIM) microscopy, as spatially resolved images make the visualization of the lateral lipid distribution possible, while at the same time the fluorescence lifetime of a membrane probe yields information about the bilayer structure and organization of the lipids in lipid domains and various properties like preferential protein-protein interactions or the enrichment of membrane probes. This review aims to give an overview of the techniques underlying FLIM probes which can be applied to investigate the formation of lipid domains and their respective properties in model membrane and biological systems. Also a short technical introduction into the techniques of a FLIM microscope is given.     

Nanostructure of cationic lipid-oligonucleotide complexes

Complexes (lipoplexes) between cationic liposomes and single-strand oligodeoxynucleotides (ODN) are potential delivery systems for antisense therapy. The nanometer-scale morphology of these assemblies is relevant to their transfection efficiency. In this work the monocationic lipid dioleoyloxytrimethylammoniumpropane, the neutral "helper" lipid cholesterol, and an 18-mer anti-bcl2 ODN were combined at different ratios. The lipoplexes formed were characterized for the quantity of ODN bound, for the degree of lipid mixing, and for their size. The nanostructure of the system was examined by cryogenic-temperature transmission electron microscopy, augmented by small-angle x-ray scattering. Addition of ODN to cationic liposomes induced both liposome aggregation and the formation of a novel condensed lamellar phase. This phase is proposed to be stabilized by anionic single-strand ODN molecules intercalated between cationic bilayers. The proportion of cholesterol present apparently did not affect the nature of lipoplex microstructure, but changed the interlamellar spacing.     

Membrane lipid domains and rafts: current applications of fluorescence lifetime spectroscopy and imaging

Membrane microdomains and their involvement in cellular processes are part of the current paradigm of biomembranes. However, a better characterization of domains, namely lipid rafts, is needed. In this review, it is shown how the use of time-resolved fluorescence, with the adequate parameters and probes, helps elucidating the type, number, fraction, composition and size of lipid phases and domains in multicomponent model systems. The determination of phase diagrams for lipid mixtures containing sphingolipids and/or cholesterol is exemplified. The use of fluorescence quenching and Förster resonance energy transfer (FRET) are also illustrated. Strategies for studying protein-induced domains are presented. The advantages of using single point microscopic decays and fluorescence lifetime imaging microscopy (FLIM) in systems with three-phase coexistence are explained. Finally, the introduction of FLIM allows studies in live cell membranes, and the nature of the microdomains observed is readily elucidated due to the information retrieved from fluorescence lifetimes.     

Lipid peroxidation in membranes and low-density lipoproteins: similarities and differences

Lipid peroxidation has been a central aspect of studies of the nature of free radical species and their origin in biological systems. Moreover, there has been a growing interest in lipid peroxidation based on evidence that biologically active products are formed that influence cell function and the course of major human diseases. A review of the work in this area is contributed by Lars Ernster is presented with an emphasis on the mechanisms by which lipid peroxidation is initiated in biological lipid systems. Based on what was described for metal catalyzed oxidation of cell membranes, and the seminal studies on cytochrome P-450-mediated lipid peroxidation, several parallel and distinct aspects of lipid peroxidation are described. A key distinction between lipid peroxidation in cell membranes and lipoproteins reveals aspects of free radical initiated reactions involving proteins and lipids that determine pro- vs. anti-oxidant outcomes, and the role of lipid structure and order in delineating the progress of oxidation.     

Applications of electron spin resonance spectroscopy to the identification of radicals produced during lipid peroxidation

Electron spin resonance (ESR) spectroscopy, which is the only commonly available method for directly detecting free radicals in biological systems, has now been quite extensively used to study radicals produced by metabolism of xenobiotic chemicals and the interaction of such species with lipid molecules. This review examines a variety of different xenobiotic systems and tissues and summarises the information obtained from these studies, with particular reference to the elucidation of the nature of the radicals involved in the initiation and propagation of lipid peroxidation.     

Characteristics of interaction of adenylate cyclase modulators and phosphoinositide cell signaling systems with lipid langmuir monolayers

Interaction of two groups of bioregulators, which oppositely affect activity of adenylate cyclase and phosphoinositide cellular signaling systems, with the Langmuir monolayer films made of natural lecithin was studied. Most significant influence on the structural and energy characteristics of lipid monolayers was revealed for the group of bioregulators, which inhibit polyphosphoinositide signaling system or/and activate adenylate cyclase signaling system. It is shown, that using the cluster analysis the bioregulators can be divided into two groups according to general orientation of their action on the considered systems of transduction of a signal.     

Minimal cells: relevance and interplay of physical and biochemical factors

Research on the construction of minimal cell-like systems is continuously progressing. The aim is to assemble a synthetic or semi-synthetic cell by encapsulating the minimal set of different macromolecules into a lipid vesicle (liposome). Synthetic cells have their relevance as new biotechnological tool for use in synthetic biology and in research into the origin of life. In recent years, several technical advances have been reported and reviewed, but most deal with the biochemical and molecular biology of protein synthesis inside liposomes, whereas a discussion on the importance and the interplay of some physical factors has not been discussed. In this short review, we comment on physical aspects, such as compartment formation and solute entrapment, and on the nature of lipid membrane. Emphasis is given to their relevance for the technology of construction of synthetic cells, and for new aspects of vesicle population studies.     

Investigation of the interaction between modified ISCOMs and stratum corneum lipid model systems

The modified ISCOMs, so-called Posintro™ nanoparticles, provide an opportunity for altering the surface charge of the particles, which influences their affinity for the negatively charged antigen sites, cell membranes and lipids in the skin. Hypothetically, this increases the passage of the ISCOMs (or their components) and their load through the stratum corneum. The subsequent increase in the uptake by the antigen-presenting cells results in enhanced transcutaneous immunization. To understand the nature of penetration of Posintro™ nanoparticles into the intercorneocyte space of the stratum corneum, the interaction between the nanoparticles and lipid model systems in form of liposomes and/or supported lipid bilayer was studied. As a lipid model we used Stratum Corneum Lipid (SCL), a mixture similar in composition to the lipids of the intercorneocyte space. By Förster Resonance Energy Transfer (FRET), Atomic Force Microscopy (AFM), Electrochemical Impedance Spectroscopy (EIS) and cryo-Transmission Electron Microscopy (cryo-TEM) it was shown that application of nanoparticles to the SCL bilayers results in lipid disturbance. Investigation of this interaction by means of Isothermal Titration Calorimetry (ITC) confirmed existence of an enthalpically unfavorable reaction. All these methods demonstrated that the strength of electrostatic repulsion between the negatively charged SCL and the nanoparticles affected their interaction, as decreasing the negative charge of the Posintro™ nanoparticles leads to enhanced disruption of lipid organization.     

Fluidity enhancement: a critical factor for performance of liposomal transdermal drug delivery system

Liposomes are well known lipid carriers for drug delivery of bioactive molecules encapsulated inside their membrane. Liposomes as skin drug delivery systems were initially promoted primarily for localized effects with minimal systemic delivery. Subsequently, a novel vesicular system, transferosomes was reported for transdermal delivery with efficiency similar to subcutaneous injection. The multiple bilayered organizations of lipids applied in these vesicles structure are somewhat similar to complex nature of stratum corneal intercellular lipids domains. The incorporation of novel agents into these lipid vesicles results in the loss of entrapped markers but it is similar to fluidization of stratum corneum lipids on treatment with a penetration enhancer. This approach generated the utility of penetration enhancers/fluidizing agents in lipids vesicular systems for skin delivery. For the transdermal and topical applications of liposomes, fluidity of bilayer lipid membrane is rate limiting which governs the permeation. This article critically reviews the relevance of using different types of vesicles as a model for skin in permeation enhancement studies. This study has also been designed to encompass all enhancement measurements and analytical tools for characterization of permeability in liposomal vesicular system.     

The electrical interplay between proteins and lipids in membranes

All molecular interactions that are relevant to cellular and molecular structures are electrical in nature but manifest in a rich variety of forms that each has its own range and influences on the net effect of how molecular species interact. This article outlines how electrical interactions between the protein and lipid membrane components underlie many of the activities of membrane function. Particular emphasis is placed on spatially localised behaviour in membranes involving modulation of protein activity and microdomain structure.The interactions between membrane lipids and membrane proteins together with their role within cell biology represent an enormous body of work. Broad conclusions are not easy given the complexities of the various systems and even consensus with model membrane systems containing two or three lipid types is difficult. By defining two types of broad lipid–protein interaction, respectively Type I as specific and Type II as more non-specific and focussing on the electrical interactions mostly in the extra-membrane regions it is possible to assemble broad rules or a consensus of the dominant features of the interplay between these two fundamentally important classes of membrane component. This article is part of a special issue entitled: Lipid–protein interactions.     

Fluorescent lipid probes: properties and application

This review is devoted to fluorescent lipid probes: the characteristics of their fluorophores; the main methods of their synthesis; and the potentialities, scope, and limitations of their use in studies of biological systems (cells, membranes and their models, enzymes of lipid metabolism, etc.). Particular attention is paid to the lipid specificity of the probes, i.e., the correspondence of their physicochemical characteristics and behavior in biological systems to those of natural lipids.     

Injectable Lipid Emulsions—Advancements,Opportunities and Challenges

Injectable lipid emulsions, for decades, have been clinically used as an energy source for hospitalized patients by providing essential fatty acids and vitamins. Recent interest in utilizing lipid emulsions for delivering lipid soluble therapeutic agents, intravenously, has been continuously growing due to the biocompatible nature of the lipid-based delivery systems. Advancements in the area of novel lipids (olive oil and fish oil) have opened a new area for future clinical application of lipid-based injectable delivery systems that may provide a better safety profile over traditionally used long- and medium-chain triglycerides to critically ill patients. Formulation components and process parameters play critical role in the success of lipid injectable emulsions as drug delivery vehicles and hence need to be well integrated in the formulation development strategies. Physico-chemical properties of active therapeutic agents significantly impact pharmacokinetics and tissue disposition following intravenous administration of drug-containing lipid emulsion and hence need special attention while selecting such delivery vehicles. In summary, this review provides a broad overview of recent advancements in the field of novel lipids, opportunities for intravenous drug delivery, and challenges associated with injectable lipid emulsions.     

Kinetics and stability of alamethicin conducting channels in lipid bilayers

It is already well-established that conduction in lipid bilayers containing alamethicin arises from the presence of complexes in which there are several molecules of the polypeptide. It is with the nature of these complexes that this paper is primarily concerned. While it is clear that increasing alamethicin concentration and increasing potential across the membrane favour their formation, the nature of the reactions involved has not yet been elucidated. Attempts have therefore been made to clarify the sequence of events leading to the establishment of a complex in its conducting state. It has been concluded that the most likely mechanism involves, initially, a non-field-dependent aggregation of the alamethicin, in the plane of the membrane, into non-conducting oligomers. These then appear to undergo movement normal to the membrane (which is field dependent) to form the conducting species. Temperature studies have shown that the various conducting states of the oligomer have effectively equal enthalpies, and that the activation energies for transitions between these states are all approx. 1.2 kcal/mol. The corresponding rate constants are very sensitive to the lipid composition of the membrane and a variety of different systems has been examined in order to clarify the origins of this effect. The only conclusion from this part of the work is that lipid fluidity might be involved.     

Lipid composition regulates the conformation and insertion of the antimicrobial peptide maculatin 1.1

Antimicrobial peptides interact with cell membranes and their selectivity is contingent on the nature of the constituent lipids. Eukaryotic and bacterial membranes are comprised of different proportions of a range of lipid species with different physical properties. Hence, characterisation of antimicrobial peptides with respect to the magnitude of their interactions with model membranes of different lipid types is needed. Maculatin 1.1 is a short antimicrobial peptide secreted from the skin of several Australian tree-frog species. Circular dichroism spectroscopy (CD) was used to explore the interaction of maculatin 1.1 with a wide range of model membrane systems of different head group and acyl chain characteristics. For neutral phosphatidylcholine (PC), unlike anionic phospholipids, the magnitude of the peptide interactions was dependent on the length and degree of saturation of the constituent acyl chains. Oriented circular dichroism (OCD) data indicated that helical structure was likely promoted by peptide insertion into the hydrophobic core of PC bilayers. The addition of cholesterol (30% mol/mol) tended to decrease the membrane interaction of maculatin 1.1. Anionic lipids locked maculatin 1.1 via electrostatic interactions onto the surface of oriented bilayers as seen in OCD spectra. Furthermore, increasing the membrane curvature by reducing the vesicle radii only slightly reduced the proportion of helical structure in all systems by approximately 10%. The peptide-lipid interaction was strongly dependent on both the lipid chain length and head group, which highlights the importance of the lipid composition used to mimic different cell types. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Interaction of nonionic PEO-PPO diblock copolymers with lipid bilayers

The relationship between the molecular architecture of a series of poly(ethylene oxide)-b-poly(propylene oxide) (PEO-PPO) diblock copolymers and the nature of their interactions with lipid bilayers has been studied using small- and wide-angle X-ray scattering (SAXS and WAXS) and differential scanning calorimetry (DSC). The number of molecular repeat units in the hydrophobic PPO block has been found to be a critical determinant of the nature of diblock copolymer-lipid bilayer association. For dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-based biomembrane structures, polymers whose PPO chain length approximates that of the acyl chains of the lipid bilayer yield highly ordered, expanded lamellar structures consistent with well-integrated (into the lipid bilayer) PPO blocks. Shorter diblock copolymers produce mixed lamellar and nonlamellar mesophases. The thermotropic phase behavior of the polymer-doped membrane systems is highly influenced by the presence and molecular architecture of the diblock copolymer, as evidenced by shifting of the main phase transition to higher temperatures, broadening of the main transition, and the appearance of other features. Studies of temperature-induced changes in the mesophase structure for compositions prepared with well-integrated PEO-PPO polymers indicate that they undergo reversible changes to a nonlamellar structure as the temperature is lowered. Increasing either the number of repeat units in the PEO block or the polymer concentration promotes a greater degree of structural ordering.     

Organization of the Brain Systems of Aim-Directed Behavior: New Data

Currently, the dominating approach to studying functional brain organization is based on the so-called activation studies, in the frameworks of which the functional specializations of different brain structures in the context of studied nature of activity are specified according to their energy states. The concept of organization of brain systems is formed largely thanks to such activation research. However, our studies devoted to the analysis of functional relations between different nodes of the brain systems show that they are much more complicated than they are presented in the activation studies. The structure of brain systems is not limited to those elements that are involved in its work by changing their local neuronal activity. This fact dramatically changes our views on how the brain systems are organized.     

Defects in ordered aggregates of cardiolipin visualized by atomic force microscopy

The formation and the nature of defects in ordered aggregates of cardiolipin (tetra acyl diphosphatidylglycerol) supported on solid substrates have been investigated by atomic force microscopy (AFM). The experiments were performed on two model systems, i.e. three-dimensional liquid crystals dispersed in water and partially de-hydrated on a hydrophilic surface, and two-dimensional films of molecules self-assembled onto an isotropic hydrophobic surface. Defects were induced both by varying the preparation temperature and by treatment with specific chemicals known to modify the order parameters in natural and artificial membranes, specifically: 2,4-dinitro-phenol (DNP) and pentachloro-phenol (PCP). The effect of lipid oxidation on the nanocrystalline order was also investigated. The images obtained by AFM allow to characterize the type of defects and their local density at nanoscale level. They also provide additional information to differentiate the specific role of acyl chains and polar heads in the process of lipid self-organization.     

Principles of classification and the strategy of immunomodulators used in medicine

The review deals with problems of the subdivision of the presently known immunomodulators in accordance with the mechanisms of their action on individual elements and systems of immunity. The necessity of immunomodulators systematization according to their purpose with a view to the prophylaxis or therapy of definite diseases caused by the pathology of the immune system or accompanied by its disturbances is emphasized. The approximate scheme and principles of the immunomodulators classification are proposed with due regard to the nature, character and mechanisms of action of the preparations.     

Electron density mapping of triblock copolymers associated with model biomembranes: insights into conformational states and effect on bilayer structure

One-dimensional electron-density profiles derived from synchrotron small-angle X-ray scattering (SAXS) have been constructed and used to determine the conformational state of selected poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers and the region of their association with a lipid bilayer. The number of molecular repeat units in the hydrophobic PPO block has been found to determine both the nature of triblock polymer-membrane association and the conformational state of the symmetric, flanking hydrophilic PEO units. For DMPC-based biomembranes, polymers whose PPO chain length is less than that of the bilayer thickness insert weakly into the membrane with the PEO blocks on the same side of the bilayer, leading to delocalization of the PEO at the membrane-water interface. This polymer architecture has been found not to alter the membrane fluidity and roughness. Conversely, polymers whose chain length is sufficient to span the lipid bilayer are tightly integrated, projecting their PEO chains into the water channels on opposing sides of the bilayer. The coiled conformational state of the PEO chains produces steric pressure on the bilayer, causing a thinning of the membrane and leading to a rigid, less-mobile bilayer than systems where the polymer is introduced as the lipid conjugate.