Effect of detergents on the ATPase activity and barrier properties of the plasma membrane of smooth muscle cells

The effect of detergents on enzymic and barrier properties of membrane structures is studied in the plasma membrane fraction, postsynaptic membranes of smooth muscle cells and sterine bilipid membranes. The formation of hydrophylic pores in the membrane, as well as changes in the phase state of the lipid matrix and fluidity of lipid microenvironment of membrane enzymes are supposed.     

Enantiomers of phospholipids and cholesterol: A key to decipher lipid-lipid interplay in membrane

Most phospholipids constituting biological membranes are chiral molecules with a hydrophilic head group and hydrophobic alkyl chains, rendering biphasic property characteristic of membrane lipids. Some lipids assemble into small domains via chirality-dependent homophilic and heterophilic interactions, the latter of which sometimes include cholesterol to form lipid rafts and other microdomains. On the other hand, lipid mediators and hormones derived from chiral lipids are recognized by specific membrane or nuclear receptors to induce downstream signaling. It is crucial to clarify the physicochemical properties of the lipid self-assembly for the study of the functions and behavior of biological membranes, which often become elusive due to effects of membrane proteins and other biological events. Three major lipids with different skeletal structures were discussed: sphingolipids including ceramides, phosphoglycerolipids, and cholesterol. The physicochemical properties of membranes and physiological functions of lipid enantiomers and diastereomers were described in comparison to natural lipids. When each enantiomer formed a self-assembly or interacted with achiral lipids, both lipid enantiomers exhibited identical membrane physicochemical properties, while when the enantiomer interacted with chiral lipids or with the opposite enantiomer, mixed membranes exhibited different properties. For example, racemic membranes comprising native sphingomyelin and its antipode exhibited phase segregation due to their strong homophilic interactions. Therefore, lipid enantiomers and diastereomers can be good probes to investigate stereospecific lipid-lipid and lipid-protein interactions occurring in biological membranes.     

Hybrid materials from amphiphilic block copolymers and membrane proteins

Self-assembly of reactive amphiphilic block copolymers is used to prepare nanostructured hydrogels with exceptional permeability properties, vesicular structures and planar, freestanding membranes in aqueous solution. Although the underlying block copolymer membranes are two-three-fold thicker than conventional lipid bilayers, they can be regarded as mimetic of biological membranes and can be used as a matrix for membrane-spanning proteins. Surprisingly, the proteins remain functional, despite the extreme thickness of the membranes and even after polymerization of the reactive block copolymers. The unique combination of block copolymers with membrane proteins allows the preparation of mechanically stable, defect-free membranes and nanocapsules that have highly selective permeability and/or specific recognition sites. This is documented by some representative examples.     

Excimer-forming lipids in membrane research

Pyrenedecanoic acid and pyrene lecithin are optical probes well suited to investigate lipid bilayer membranes. The method is based on the determination of the formation of excited dimers or excimers. The rate of excimer formation yields information on the dynamic molecular properties of artificial as well as of natural membranes. This article will review applications of the excimer-forming probes.Pyrene lipid probes are used to determine the coefficient of the lateral diffusion in fluid lipid membranes. Results in artificial membranes are comparable to the values obtained in erythrocyte membranes.Moreover, the excimer formation rate is a very sensitive measure of changes in membrane fluidity. Membrane fluidity is an important regulator of membrane functional proteins. For example, there is a correlation between membrane fluidity and enzyme activities of the adenylate cyclase system.The excimer formation technique is not restricted to the measurement of lateral mobility in membranes. It can also be used to determine the transversal mobility, that is, the lipid exchange between the lipid layers of one bilayer or between bilayers of different vesicles. Again, artificial as well as natural membranes can be investigated by this technique.Another important area of investigation in membrane research is the interaction between lipids and proteins. Lipids, in the presence of a protein, show a different dynamic behavior from free lipids. Because of changes in fluidity and a modified solubility of the pyrene probes within different membrane regions, our methods could also be applied to the examination of phase separation phenomena and to lipid-protein interactions.     

Optical techniques for imaging membrane lipid microdomains in living cells

Lateral organisation of cellular membranes, particularly the plasma membrane, is of benefit to the cell as it allows complicated cellular processes to be regulated and efficient. For example, trafficking and secretion of molecules can be targeted and directed, cells polarised and signalling events modulated and propagated. The fluid mosaic model allows for significant heterogeneity on the part of the lipids themselves and of membrane associated proteins. By exploiting the tendency of complex lipid bilayers to undergo spontaneous or induced phase-separation into non-miscible domains, the cell could achieve this desired spatial organisation. While phase-separation is readily observed in simple, artificial bilayers, its occurrence in physiological membranes remains controversial. This stems mainly from our inability to image lipid microdomains directly - possibly due to their small size, short lifespan and/or morphological similarity to the bulk membrane. In this review, we seek to examine the techniques used to try to image membrane lipid microdomains, concentrating mainly on optical microscopy techniques that are applicable to live cells. We also look at novel emerging instruments and methods that promise to overcome our current technological limitations and shed new light on these important structures.     

Biomimetic design of nanopatterned membranes

The biomimetic approach copying the supramolecular building principle of many archaeal cell envelopes (i.e., a plasma membrane with associated S-layer proteins) has resulted in stable lipid membranes with excellent reconstitution properties for transmembrane proteins. This is a particular challenge as one-third of all proteins in an organism are membrane proteins like pores, ion channels, or receptors. At S-layer supported lipid membranes, spatial well-defined domains on the S-layer protein interact noncovalently with lipid head groups within the lipid membrane resulting in a nanopatterning of a few anchored and scores of diffusional free-lipid molecules. In addition, no impact on the hydrophobic core region and on the function of reconstituted integral proteins has been determined. Among others, particularly S-layer stabilized membranes can be used for structure-function studies on reconstituted integral proteins and also in the membrane protein-based molecular nanotechnology, e.g., in the design of biosensing devices (e.g., lipid chip or lab-on-a-chip), or for receptor or ion channel-based high-throughput screening.     

The myeloid expressed EF-hand proteins display a diverse pattern of lipid raft association

EF-hand proteins are known to translocate to membranes, suggesting that they are involved in signaling events located in the cell membrane. Many proteins involved in signaling events associate cholesterol rich membrane domains, so called lipid rafts, which serve as platforms for controlled protein-protein interaction. Here, we demonstrate that the myeloid expressed EF-hand proteins can be distinguished into three classes with respect to their membrane association. Grancalcin, a myeloid expressed penta EF-hand protein, is constitutively located in lipid rafts. S100A9 (MRP14) and S100A8 (MRP8) are translocated into detergent resistant lipid structures only after calcium activation of the neutrophils. However, the S100A9/A8 membrane association is cholesterol and sphingolipid independent. On the other hand, the association of S100A12 (EN-RAGE) and S100A6 (calcyclin) with membranes is detergent sensitive. These diverse affinities to lipid structures of the myeloid expressed EF-hand proteins most likely reflect their different functions in neutrophils.     

Folding and assembly of beta-barrel membrane proteins

Beta-barrel membrane proteins occur in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. The membrane-spanning sequences of beta-barrel membrane proteins are less hydrophobic than those of alpha-helical membrane proteins, which is probably the main reason why completely different folding and membrane assembly pathways have evolved for these two classes of membrane proteins. Some beta-barrel membrane proteins can be spontaneously refolded into lipid bilayer model membranes in vitro. They may also have this ability in vivo although lipid and protein chaperones likely assist with their assembly in appropriate target membranes. This review summarizes recent work on the thermodynamic stability and the mechanism of membrane insertion of beta-barrel membrane proteins in lipid model and biological membranes. How lipid compositions affect folding and assembly of beta-barrel membrane proteins is also reviewed. The stability of these proteins in membranes is not as large as previously thought (<10 kcal/mol) and is modulated by elastic forces of the lipid bilayer. Detailed kinetic studies indicate that beta-barrel membrane proteins fold in distinct steps with several intermediates that can be characterized in vitro. Formation of the barrel is synchronized with membrane insertion and all beta-hairpins insert simultaneously in a concerted pathway.     

Aspirin inhibits formation of cholesterol rafts in fluid lipid membranes

Aspirin and other non-steroidal anti-inflammatory drugs have a high affinity for phospholipid membranes, altering their structure and biophysical properties. Aspirin has been shown to partition into the lipid head groups, thereby increasing membrane fluidity. Cholesterol is another well known mediator of membrane fluidity, in turn increasing membrane stiffness. As well, cholesterol is believed to distribute unevenly within lipid membranes leading to the formation of lipid rafts or plaques. In many studies, aspirin has increased positive outcomes for patients with high cholesterol. We are interested if these effects may be, at least partially, the result of a non-specific interaction between aspirin and cholesterol in lipid membranes.We have studied the effect of aspirin on the organization of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) membranes containing cholesterol. Through Langmuir–Blodgett experiments we show that aspirin increases the area per lipid and decreases compressibility at 32.5 mol% cholesterol, leading to a significant increase of fluidity of the membranes. Differential scanning calorimetry provides evidence for the formation of meta-stable structures in the presence of aspirin. The molecular organization of lipids, cholesterol and aspirin was studied using neutron diffraction. While the formation of rafts has been reported in binary DPPC/cholesterol membranes, aspirin was found to locally disrupt membrane organization and lead to the frustration of raft formation. Our results suggest that aspirin is able to directly oppose the formation of cholesterol structures through non-specific interactions with lipid membranes.     

Artificial biomembrane morphology: a dissipative particle dynamics study

Artificial membranes mimicking biological structures are rapidly breaking new ground in the areas of medicine and soft-matter physics. In this endeavor, we use dissipative particle dynamics simulation to investigate the morphology and behavior of lipid-based biomembranes under conditions of varied lipid density and self-interaction. Our results show that a less-than-normal initial lipid density does not create the traditional membrane; but instead results in the formation of a ‘net’, or at very low densities, a series of disparate ‘clumps’ similar to the micelles formed by lipids in nature. When the initial lipid density is high, a membrane forms, but due to the large number of lipids, the naturally formed membrane would be larger than the simulation box, leading to ‘rippling’ behavior as the excess repulsive force of the membrane interior overcomes the bending energy of the membrane. Once the density reaches a certain point however, ‘bubbles’ appear inside the membrane, reducing the rippling behavior and eventually generating a relatively flat, but thick, structure with micelles of water inside the membrane itself. Our simulations also demonstrate that the interaction parameter between individual lipids plays a significant role in the formation and behavior of lipid membrane assemblies, creating similar structures as the initial lipid density distribution. This work provides a comprehensive approach to the intricacies of lipid membranes, and offers a guideline to design biological or polymeric membranes through self-assembly processes as well as develop novel cellular manipulation and destruction techniques.     

The hydrophobic insertion mechanism of membrane curvature generation by proteins

A wide spectrum of intracellular processes is dependent on the ability of cells to dynamically regulate membrane shape. Membrane bending by proteins is necessary for the generation of intracellular transport carriers and for the maintenance of otherwise intrinsically unstable regions of high membrane curvature in cell organelles. Understanding the mechanisms by which proteins curve membranes is therefore of primary importance. Here we suggest, for the first time to our knowledge, a quantitative mechanism of lipid membrane bending by hydrophobic or amphipathic rodlike inclusions which simulate amphipathic α-helices—structures shown to sculpt membranes. Considering the lipid monolayer matrix as an anisotropic elastic material, we compute the intramembrane stresses and strains generated by the embedded inclusions, determine the resulting membrane shapes, and the accumulated elastic energy. We characterize the ability of an inclusion to bend membranes by an effective spontaneous curvature, and show that shallow rodlike inclusions are more effective in membrane shaping than are lipids having a high propensity for curvature. Our computations provide experimentally testable predictions on the protein amounts needed to generate intracellular membrane shapes for various insertion depths and membrane thicknesses. We also predict that the ability of N-BAR domains to produce membrane tubules in vivo can be ascribed solely to insertion of their amphipathic helices.     

Effect of cholesterol on the interaction of the HIV GP41 fusion peptide with model membranes. Importance of the membrane dipole potential

Fusion of viral and cell membranes is a key event in the process by which the human immunodeficiency virus (HIV) enters the target cell. Membrane fusion is facilitated by the interaction of the viral gp41 fusion peptide with the cell membrane. Using synthetic peptides and model membrane systems, it has been established that the sequence of events implies the binding of the peptide to the membrane, followed by a conformational change (transformation of unordered and helical structures into beta-aggregates) which precedes lipid mixing. It is known that this process can be influenced by the membrane lipid composition. In the present work we have undertaken a systematic study in order to determine the influence of cholesterol (abundant in the viral membrane) in the sequence of events leading to lipid mixing. Besides its effect on membrane fluidity, cholesterol can affect a less known physical parameter, the membrane dipole potential. Using the dipole potential fluorescent sensor di-8-ANEPPS together with other biophysical techniques, we show that cholesterol increases the affinity of the fusion peptide for the model membranes, and although it lowers the extent of lipid mixing, it increases the mixing rate. The influence of cholesterol on the peptide affinity and the lipid mixing rate are shown to be mainly due to its influence of the membrane dipole potential, whereas the lipid mixing extent and peptide conformational changes seem to be more dependent on other membrane parameters such as membrane fluidity and hydration.     

S-layer-supported lipid membranes

Many prokaryotic organisms (archaea and bacteria) are covered by a regularly ordered surface layer (S-layer) as the outermost cell wall component. S-layers are built up of a single protein or glycoprotein species and represent the simplest biological membrane developed during evolution. Pores in S-layers are of regular size and morphology, and functional groups on the protein lattice are aligned in well-defined positions and orientations. Due to the high degree of structural regularity S-layers represent unique systems for studying the structure, morphogenesis, and function of layered supramolecular assemblies. Isolated S-layer subunits of numerous organisms are able to assemble into monomolecular arrays either in suspension, at air/water interfaces, on planar mono- and bilayer lipid films, on liposomes and on solid supports (e.g. silicon wafers). Detailed studies on composite S-layer/lipid structures have been performed with Langmuir films, freestanding bilayer lipid membranes, solid supported lipid membranes, and liposomes. Lipid molecules in planar films and liposomes interact via their head groups with defined domains on the S-layer lattice. Electrostatic interactions are the most prevalent forces. The hydrophobic chains of the lipid monolayers are almost unaffected by the attachment of the S-layer and no impact on the hydrophobic thickness of the membranes has been observed. Upon crystallization of a coherent S-layer lattice on planar and vesicular lipid membranes, an increase in molecular order is observed, which is reflected in a decrease of the membrane tension and an enhanced mobility of probe molecules within an S-layer-supported bilayer. Thus, the terminology 'semifluid membrane' has been introduced for describing S-layer-supported lipid membranes. The most important feature of composite S-layer/lipid membranes is an enhanced stability in comparison to unsupported membranes.     

Do sterols reduce proton and sodium leaks through lipid bilayers?

Proton and/or sodium electrochemical gradients are critical to energy handling at the plasma membranes of all living cells. Sodium gradients are used for animal plasma membranes, all other living organisms use proton gradients. These chemical and electrical gradients are either created by a cation pumping ATPase or are created by photons or redox, used to make ATP. It has been established that both hydrogen and sodium ions leak through lipid bilayers at approximately the same rate at the concentration they occur in living organisms. Although the gradients are achieved by pumping the cations out of the cell, the plasma membrane potential enhances the leakage rate of these cations into the cell because of the orientation of the potential. This review proposes that cells use certain lipids to inhibit cation leakage through the membrane bilayers. It assumes that Na(+) leaks through the bilayer by a defect mechanism. For Na(+) leakage in animal plasma membranes, the evidence suggests that cholesterol is a key inhibitor of Na(+) leakage. Here I put forth a novel mechanism for proton leakage through lipid bilayers. The mechanism assumes water forms protonated and deprotonated clusters in the lipid bilayer. The model suggests how two features of lipid structures may inhibit H(+) leakage. One feature is the fused ring structure of sterols, hopanoids and tetrahymenol which extrude water and therefore clusters from the bilayer. The second feature is lipid structures that crowd the center of the bilayer with hydrocarbon. This can be accomplished either by separating the two monolayers with hydrocarbons such as isoprenes or isopranes in the bilayer's cleavage plane or by branching the lipid chains in the center of the bilayers with hydrocarbon. The natural distribution of lipids that contain these features are examined. Data in the literature shows that plasma membranes exposed to extreme concentrations of cations are particularly rich in the lipids containing the predicted qualities. Prokaryote plasma membranes that reside in extreme acids (acidophiles) contain both hopanoids and iso/anteiso- terminal lipid branching. Plasma membranes that reside in extreme base (alkaliphiles) contain both squalene and iso/anteiso- lipids. The mole fraction of squalene in alkaliphile bilayers increases, as they are cultured at higher pH. In eukaryotes, cation leak inhibition is here attributed to sterols and certain isoprenes, dolichol for lysosomes and peroxysomes, ubiquinone for these in addition to mitochondrion, and plastoquinone for the chloroplast. Phytosterols differ from cholesterol because they contain methyl and ethyl branches on the side chain. The proposal provides a structure-function rationale for distinguishing the structures of the phytosterols as inhibitors of proton leaks from that of cholesterol which is proposed to inhibit leaks of Na(+). The most extensively studied of sterols, cholesterol, occurs only in animal cells where there is a sodium gradient across the plasma membrane. In mammals, nearly 100 proteins participate in cholesterol's biosynthetic and degradation pathway, its regulatory mechanisms and cell-delivery system. Although a fat, cholesterol yields no energy on degradation. Experiments have shown that it reduces Na(+) and K(+) leakage through lipid bilayers to approximately one third of bilayers that lack the sterol. If sterols significantly inhibit cation leakage through the lipids of the plasma membrane, then the general role of all sterols is to save metabolic ATP energy, which is the penalty for cation leaks into the cytosol. The regulation of cholesterol's appearance in the plasma membrane and the evolution of sterols is discussed in light of this proposed role.     

Modulation of membrane surface curvature by peptide-lipid interactions

Recent reports on the interaction of cardiotoxin and melittin with phospholipid model membranes are reviewed and analyzed. These types of peptide toxins are able to modulate lipid surface curvature and polymorphism in a highly lipid-specific way. It is demonstrated that the remarkable variety of effects of melittin on the organization of different membrane phospholipids can be understood in a relatively simple model, based on the shape-structure concept of lipid polymorphism and taking into account the position of the peptide molecule with respect to the lipids. Based on the strong preference of the peptides for negatively charged lipids and the structural consequences thereof, and on preliminary studies of signal peptide-lipid interaction, a role of inverted or concave lipid structures in the process of protein translocation across membranes is suggested.     

Induction of non-bilayer lipid structures by functional signal peptides.

Using 31P NMR and freeze-fracture electron microscopy we investigated the effect of several synthetic signal peptides on lipid structure in model membranes mimicking the lipid composition of the Escherichia coli inner membrane. It is demonstrated that the signal peptide of the E. coli outer membrane protein PhoE, as well as that of the M13 phage coat protein, strongly promote the formation of non-bilayer lipid structures. This effect appears to be correlated to in vivo translocation efficiency, since a less functional analogue of the PhoE signal peptide was found to be less active in destabilizing the bilayer. It is proposed that signal sequences can induce local changes in lipid structure that are involved in protein translocation across the membrane.     

Membrane lipid therapy: Modulation of the cell membrane composition and structure as a molecular base for drug discovery and new disease treatment

Nowadays we understand cell membranes not as a simple double lipid layer but as a collection of complex and dynamic protein–lipid structures and microdomains that serve as functional platforms for interacting signaling lipids and proteins. Membrane lipids and lipid structures participate directly as messengers or regulators of signal transduction. In addition, protein–lipid interactions participate in the localization of signaling protein partners to specific membrane microdomains. Thus, lipid alterations change cell signaling that are associated with a variety of diseases including cancer, obesity, neurodegenerative disorders, cardiovascular pathologies, etc. This article reviews the newly emerging field of membrane lipid therapy which involves the pharmacological regulation of membrane lipid composition and structure for the treatment of diseases. Membrane lipid therapy proposes the use of new molecules specifically designed to modify membrane lipid structures and microdomains as pharmaceutical disease-modifying agents by reversing the malfunction or altering the expression of disease-specific protein or lipid signal cascades. Here, we provide an in-depth analysis of this emerging field, especially its molecular bases and its relevance to the development of innovative therapeutic approaches.     

The Ca2+- and phospholipid-binding protein Annexin A2 is able to increase and decrease plasma membrane order

Annexin A2 (AnxA2) is a calcium- and phospholipid-binding protein that plays roles in cellular processes involving membrane and cytoskeleton dynamics and is able to associate to several partner proteins. However, the principal molecular partners of AnxA2 are negatively charged phospholipids such as phosphatidylserine and phosphatidyl-inositol-(4,5)-phosphate. Herein we have studied different aspects of membrane lipid rearrangements induced by AnxA2 membrane binding. X-ray diffraction data revealed that AnxA2 has the property to stabilize lamellar structures and to block the formation of highly curved lipid phases (inverted hexagonal phase, H_II). By using pyrene-labelled cholesterol and the environmental probe di-4-ANEPPDHQ, we observed that in model membranes, AnxA2 is able to modify both, cholesterol distribution and lipid compaction. In epithelial cells, we observed that AnxA2 localizes to membranes of different lipid order. The protein binding to membranes resulted in both, increases and/or decreases in membrane order depending on the cellular membrane regions. Overall, AnxA2 showed the capacity to modulate plasma membrane properties by inducing lipid redistribution that may lead to an increase in order or disorder of the membranes.     

Addition of lipid to the photosynthetic membrane: effects on membrane structure and energy transfer

We have carried out a series of experiments in which the lipid composition of the photosynthetic membrane has been altered by the addition of lipid from a defined source under experimental conditions. Liposomes prepared by sonication are mixed with purified photosynthetic membranes obtained from spinach chloroplasts and are taken through cycles of freezing and thawing. Several lines of evidence, including gel electrophoresis and freeze-fracture electron microscopy, indicate that an actual addition of lipid has taken place. Structural analysis by freeze-fracture shows that intramembrane particles are widely separated after the addition of large amounts of lipid, with one exception: large hexagonal lattices of particles appear in some regions of the membrane. These lattices are identical in appearance with lattices formed from a single purified component of the membrane known as chlorophyll-protein complex II. The suggestion that the presence of such lattices in lipid-enriched membranes reflects a profound rearrangement of photosynthetic structures has been confirmed by analysis of the fluorescence emission spectra of natural and lipid- enriched membranes. Specifically, lipid addition in each of the cases we have studied results in the apparent detachment of chlorophyll- protein complex II from photosynthetic reaction centers. It is concluded that specific arrangements of components in the photosynthetic membrane, necessary for the normal functioning of the membrane in the light reaction of photosynthesis, can be regulated to a large extent by the lipid content of the membrane.