Asymmetric glycerophospholipids impart distinctive biophysical properties to lipid bilayers

Phospholipids are a diverse group of biomolecules consisting of a hydrophilic headgroup and two hydrophobic acyl tails. The nature of the head and length and saturation of the acyl tails are important for defining the biophysical properties of lipid bilayers. It has recently been shown that the membranes of certain yeast species contain high levels of unusual asymmetric phospholipids consisting of one long and one medium-chain acyl moiety, a configuration not common in mammalian cells or other well-studied model yeast species. This raises the possibility that structurally asymmetric glycerophospholipids impart distinctive biophysical properties to the yeast membranes. Previously, it has been shown that lipids with asymmetric length tails form a mixed interdigitated gel phase and exhibit unusual endotherm behavior upon heating and cooling. Here, however, we address physiologically relevant temperature conditions and, using atomistic molecular dynamics simulations and environmentally sensitive fluorescent membrane probes, characterize key biophysical parameters (such as lipid packing, diffusion coefficient, membrane thickness, and area per lipid) in membranes composed of both length-asymmetric glycerophospholipids and ergosterol. Interestingly, we show that saturated but asymmetric glycerophospholipids maintain membrane lipid order across a wide range of temperatures. We also show that these asymmetric lipids can substiture of unsaturated symmetric lipids in the phase behaviour of ternary lipid bilayers. This may allow cells to maintain membrane fluidity, even in environments that lack oxygen, which is required for the synthesis of unsaturated lipids and sterols.     

The lipid network

Natural cell membranes are composed of a remarkable variety of lipids, which provide specific biophysical properties to support membrane protein function. An improved understanding of this complexity of membrane composition may also allow the design of membrane active drugs. Crafting a relevant model of a cell membrane with controlled composition is becoming an art, with the ability to reveal the molecular mechanisms of biological processes and lead to better treatment of pathologies. By matching physiological observations from in vivo experiments to high-resolution information, more easily obtained from in vitro studies, complex interactions at the lipid interface are determined. The role of the lipid network in biological membranes is, therefore, the subject of increasing attention.     

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.     

Tocopherols and tocotrienols in membranes: a critical review

The familiar role of tocols (tocopherols and tocotrienols) as lipid-soluble chain-terminating inhibitors of lipid peroxidation is currently in the midst of a reinterpretation. New biological activities have been described for tocols that apparently are not dependent on their well-established antioxidant behaviour. These activities could well be real, but there remain large gaps in our understanding of the behaviour of tocols in membranes, especially when it comes to the alpha-, beta-, gamma-, delta-chroman methylation patterns and the seemingly special nature of tocotrienols. It is inappropriate to make conclusions and develop models based on in vivo (or cell culture) results with reference to in vitro measurements of antioxidant activity. When present in biological membranes, tocols will experience a large variation in the local composition of phospholipids and the presence of neutral lipids such as cholesterol, both of which would be expected to change the efficiency of antioxidant action. It is likely that tocols are not homogeneously dispersed in a membrane, but it is still not known whether any specific combination of lipid head group and acyl chains are conferred special protection from peroxidation, nor do we currently appreciate the structural role that tocols play in membranes. Tocols may enhance curvature stress or counteract similar stresses generated by other lipids such as lysolipids. This review will outline what is known about the location and behaviour of tocols in phospholipid bilayers. We will draw mainly from the biophysical literature, but will attempt to extend the discussion to biologically relevant phenomena when appropriate. We hope that it will assist researchers when designing new experiments and when critically assessing the results, in turn providing a more thorough understanding of the biochemistry of tocols.     

Role of membrane organization and membrane domains in endocytic lipid trafficking

Lipid compositions vary greatly among organelles, and specific sorting mechanisms are required to establish and maintain these distinct compositions. In this review, we discuss how the biophysical properties of the membrane bilayer and the chemistry of individual lipid molecules play a role in the intracellular trafficking of the lipids themselves, as well as influencing the trafficking of transmembrane proteins. The large diversity of lipid head groups and acyl chains lead to a variety of weak interactions, such as ionic and hydrogen bonding at the lipid/water interfacial region, hydrophobic interactions, and van-der-Waals interactions based on packing density. In simple model bilayers, these weak interactions can lead to large-scale phase separations, but in more complex mixtures, which mimic cell membranes, such phase separations are not observed. Nevertheless, there is growing evidence that domains (i.e., localized regions with non-random lipid compositions) exist in biological membranes, and it is likely that the formation of these domains are based on interactions similar to those that lead to phase separations in model systems. Sorting of lipids appears to be based in part on the inclusion or exclusion of certain types of lipids in vesicles or tubules as they bud from membrane organelles.     

Biomimetic lipid/polymer colorimetric membranes: molecular and cooperative properties

Characterization of membranes and of biological processes occurring within membranes is essential for understanding fundamental cellular behavior. Here we present a detailed biophysical study of a recently developed colorimetric biomimetic membrane assembly constructed from physiological lipid molecules and conjugated polydiacetylene. Various analytical techniques have been applied to characterize the organization of the lipid components in the chromatic vesicles and their contributions to the observed blue-to-red color transitions. Experiments reveal that both the polymerized units as well as the lipids exhibit microscopic phases and form domains whose properties and bilayer organization are interdependent. These domains are interspersed within mixed lipid/polymer vesicles that have a size distribution different from those of aggregates of the individual molecular constituents. The finding that fluidity changes induced within the lipid domains are correlated with the chromatic transitions demonstrates that the colorimetric platform can be used to evaluate the effects of individual molecular components, such as negatively charged lipids and cholesterol, upon membrane fluidity and thermal stability.     

Domain-specific lipid distribution in macrophage plasma membranes

Lipid rafts, defined as cholesterol- and sphingolipid-rich domains, provide specialized lipid environments understood to regulate the organization and function of many plasma membrane proteins. Growing evidence of their existence, protein cargo, and regulation is based largely on the study of isolated lipid rafts; however, the consistency and validity of common isolation methods is controversial. Here, we provide a detailed and direct comparison of the lipid and protein composition of plasma membrane "rafts" prepared from human macrophages by different methods, including several detergent-based isolations and a detergent-free method. We find that detergent-based and detergent-free methods can generate raft fractions with similar lipid contents and a biophysical structure close to that previously found on living cells, even in cells not expressing caveolin-1, such as primary human macrophages. However, important differences between isolation methods are demonstrated. Triton X-100-resistant rafts are less sensitive to cholesterol or sphingomyelin depletion than those prepared by detergent-free methods. Moreover, we show that detergent-based methods can scramble membrane lipids during the isolation process, reorganizing lipids previously in sonication-derived nonraft domains to generate new detergent-resistant rafts. The role of rafts in regulating the biological activities of macrophage plasma membrane proteins may require careful reevaluation using multiple isolation procedures, analyses of lipids, and microscopic techniques.     

Lipid nanoparticle technologies for the study of G protein-coupled receptors in lipid environments

G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins which conduct a wide range of biological roles and represent significant drug targets. Most biophysical and structural studies of GPCRs have been conducted on detergent-solubilised receptors, and it is clear that detergents can have detrimental effects on GPCR function. Simultaneously, there is increasing appreciation of roles for specific lipids in modulation of GPCR function. Lipid nanoparticles such as nanodiscs and styrene maleic acid lipid particles (SMALPs) offer opportunities to study integral membrane proteins in lipid environments, in a form that is soluble and amenable to structural and biophysical experiments. Here, we review the application of lipid nanoparticle technologies to the study of GPCRs, assessing the relative merits and limitations of each system. We highlight how these technologies can provide superior platforms to detergents for structural and biophysical studies of GPCRs and inform on roles for protein-lipid interactions in GPCR function.     

Lipid domains in bacterial membranes and the action of antimicrobial agents

There has been increasing interest in recent years in describing the lateral organization of membranes and the formation of membrane domains. Much of the focus in this area has been on the formation of cholesterol-rich domains in mammalian membranes. However, it is likely that there are domains in all biological membranes. One of the challenges has been to define the chemical composition, lifetime and size of these domains. There is evidence that bacteria have domains that are enriched in cardiolipin. In addition, the formation of lipid domains can be induced in bacteria by clustering negatively charged lipids with polycationic substances. Many antimicrobial compounds have multiple positive charges. Such polycationic compounds can sequester anionic lipids to induce lipid phase separation. The molecular interactions among lipids and their lateral packing density will be different in a domain from its environment. This will lead to phase boundary defects that will lower the permeability barrier between the cell and its surroundings. The formation of these clusters of anionic lipids may also alter the stability or composition of existing membrane domains that may affect bacterial function. Interestingly many antimicrobial agents are polycationic and therefore likely have some effect in promoting lipid phase segregation between anionic and zwitterionic lipids. However, this mechanism is expected to be most important for substances with sequential positive charges contained within a flexible molecule that can adapt to the arrangement of charged groups on the surface of the bacterial cell. When this mechanism is dominant it can allow the prediction of the bacterial species that will be most affected by the agent as a consequence of the nature of the lipid composition of the bacterial membrane.     

Lipid domains in bacterial membranes and the action of antimicrobial agents

There has been increasing interest in recent years in describing the lateral organization of membranes and the formation of membrane domains. Much of the focus in this area has been on the formation of cholesterol-rich domains in mammalian membranes. However, it is likely that there are domains in all biological membranes. One of the challenges has been to define the chemical composition, lifetime and size of these domains. There is evidence that bacteria have domains that are enriched in cardiolipin. In addition, the formation of lipid domains can be induced in bacteria by clustering negatively charged lipids with polycationic substances. Many antimicrobial compounds have multiple positive charges. Such polycationic compounds can sequester anionic lipids to induce lipid phase separation. The molecular interactions among lipids and their lateral packing density will be different in a domain from its environment. This will lead to phase boundary defects that will lower the permeability barrier between the cell and its surroundings. The formation of these clusters of anionic lipids may also alter the stability or composition of existing membrane domains that may affect bacterial function. Interestingly many antimicrobial agents are polycationic and therefore likely have some effect in promoting lipid phase segregation between anionic and zwitterionic lipids. However, this mechanism is expected to be most important for substances with sequential positive charges contained within a flexible molecule that can adapt to the arrangement of charged groups on the surface of the bacterial cell. When this mechanism is dominant it can allow the prediction of the bacterial species that will be most affected by the agent as a consequence of the nature of the lipid composition of the bacterial membrane.     

Chemistry and biology of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids: fluorescent probes of biological and model membranes

Lipids that are covalently labeled with the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group are widely used as fluorescent analogues of native lipids in model and biological membranes to study a variety of processes. The fluorescent NBD group may be attached either to the polar or the apolar regions of a wide variety of lipid molecules. Synthetic routes for preparing the lipids, and spectroscopic and ionization properties of these probes are reviewed in this report. The orientation of various NBD-labeled lipids in membranes, as indicated by the location of the NBD group, is also discussed. The NBD group is uncharged at neutral pH in membranes, but loops up to the surface if attached to acyl chains of phospholipids. These lipids find applications in a variety of membrane-related studies which include membrane fusion, lipid motion and dynamics, organization of lipids and proteins in membranes, intracellular lipid transfer, and bilayer to hexagonal phase transition in liposomes. Use of NBD-labeled lipids as analogues of natural lipids is critically evaluated.     

Investigation on lipid asymmetry using lipid probes: Comparison between spin-labeled lipids and fluorescent lipids

Synthetic lipids with a nitroxide or a fluorescent probe have been extensively used during the last 30 years to determine the transmembrane diffusion of phospholipids in artificial or biological membranes. However, the relevance of data obtained with these modified lipids has sometimes been questioned. Beside possible artefacts introduced by the reporter probe, synthetic lipids used in cells often contain a short fatty acid chain in the sn-2 position, which gives them higher water solubility than naturally occurring lipids. In the present review, we have attempted to give a critical appraisal. Main strategies are recalled and important discoveries obtained with lipid probes on transmembrane lipid traffic in eukaryotic cells are briefly summarized. Examples of artefacts caused by lipid probes are given. Comparisons between data obtained by different techniques such as ESR and fluorescence allow us to emphasize the complementary character of the two approaches and more generally show the necessity to use several probes before drawing conclusions concerning endogenous lipids. In spite of these pitfalls, overall, lipid probes have provided a wealth of useful information that, to date, cannot be obtained with unlabeled lipids.     

Molecular Model for the Solubilization of Membranes into Nanodisks by Styrene Maleic Acid Copolymers

A recent discovery in membrane research is the ability of styrene-maleic acid (SMA) copolymers to solubilize membranes in the form of nanodisks allowing extraction and purification of membrane proteins from their native environment in a single detergent-free step. This has important implications for membrane research because it allows isolation as well as characterization of proteins and lipids in a near-native environment. Here, we aimed to unravel the molecular mode of action of SMA copolymers by performing systematic studies using model membranes of varying compositions and employing complementary biophysical approaches. We found that the SMA copolymer is a highly efficient membrane-solubilizing agent and that lipid bilayer properties such as fluidity, thickness, lateral pressure profile, and charge density all play distinct roles in the kinetics of solubilization. More specifically, relatively thin membranes, decreased lateral chain pressure, low charge density at the membrane surface, and increased salt concentration promote the speed and yield of vesicle solubilization. Experiments using a native membrane lipid extract showed that the SMA copolymer does not discriminate between different lipids and thus retains the native lipid composition in the solubilized particles. A model is proposed for the mode of action of SMA copolymers in which membrane solubilization is mainly driven by the hydrophobic effect and is further favored by physical properties of the polymer such as its relatively small cross-sectional area and rigid pendant groups. These results may be helpful for development of novel applications for this new type of solubilizing agent, and for optimization of the SMA technology for solubilization of the wide variety of cell membranes found in nature.     

Lipid rafts: at a crossroad between cell biology and physics

Membrane lateral heterogeneity is accepted as a requirement for the function of biological membranes and the notion of lipid rafts gives specificity to this broad concept. However, the lipid raft field is now at a technical impasse because the physical tools to study biological membranes as a liquid that is ordered in space and time are still being developed. This has lead to a disconnection between the concept of lipid rafts as derived from biochemical and biophysical assays and their existence in the cell. Here, we compare the concept of lipid rafts as it has emerged from the study of synthetic membranes with the reality of lateral heterogeneity in biological membranes. Further application of existing tools and the development of new tools are needed to understand the dynamic heterogeneity of biological membranes.     

Cytotoxicity of commonly used nitroxide radical spin probes

Since nitroxide radical spin probes are used frequently to test biophysical properties of cells, their use should be restricted to conditions that do not perturb normal cell growth and viability. Eight commonly used nitroxide radical spin probes have been tested for their effects on the survival of CHO cells. These include water-soluble spin probes Tempol, Tempamine, CTPO, CTPC and 4-maleimido-Tempo, and lipid soluble spin probes 5-Doxyl-, 12-Doxyl-, and 16-Doxylstearates. With the exception of 4-maleimido-Tempo, none of the water soluble spin labels inhibited cell survival at concentrations as high as 1 mM. At concentrations of 75 microM and higher, 4-maleimido-Tempo inhibited cell survival in a dose dependent manner. At concentrations commonly used for spin labeling of cells (30-50 microM) none of the lipid soluble spin probes tested was cytotoxic. At 100 microM only 5-Doxylstearate inhibited cell survival, whereas 12-Doxylstearate and 16-Doxylstearate had no effect.     

Fluorescence resonance energy transfer between lipid probes detects nanoscopic heterogeneity in the plasma membrane of live cells

Fluorescence resonance energy transfer (FRET) between matched carbocyanine lipid analogs in the plasma membrane outer leaflet of RBL mast cells was used to investigate lateral distributions of lipids and to develop a general method for quantitative measurements of lipid heterogeneity in live cell membranes. FRET measured as fluorescence quenching of long-chain donor probes such as DiO-C18 is greater with long-chain, saturated acceptor probes such as DiI-C16 than with unsaturated or shorter-chain acceptors with the same chromophoric headgroup compared at identical concentrations. FRET measurements between these lipid probes in model membranes support the conclusion that differential donor quenching is not caused by nonideal mixing or spectroscopic differences. Sucrose gradient analysis of plasma membrane-labeled, Triton X-100-lysed cells shows that proximity measured by FRET correlates with the extent of lipid probe partitioning into detergent-resistant membranes. FRET between DiO-C16 and DiI-C16 is sensitive to cholesterol depletion and disruption of liquid order (Lo) by short-chain ceramides, and it is enhanced by cross linking of Lo-associated proteins. Consistent results are obtained when homo-FRET is measured by decreased fluorescence anisotropy of DiI-C16. These results support the existence of nanometer-scale Lo/liquid disorder heterogeneity of lipids in the outer leaflet of the plasma membrane in live cells.     

How calcium may cause exocytosis in sea urchin eggs

The process of secretory granule-plasma membrane fusion can be studied in sea urchin eggs. Micromolar calcium concentrations are all that is required to bring about exocytosisin vitro. I discuss recent experiments with sea urchin eggs that concentrate on the biophysical aspects of granule-membrane fusion. The backbone of biological membranes is the lipid bilayer. Sea urchin egg membrane lipids have negatively charged head groups that give rise to an electrical potential at the bilayer-water interface. We have found that this surface potential can affect the calcium required for exocytosis. Effects on the surface potential may also explain why drugs like trifluoperazine and tetracaine inhibit exocytosis: they absorb to the bilayer and reduce the surface potential. The membrane lipids may also be crucial to the formation of the exocytotic pore through which the secretory granule contents are released. We have measured calcium-induced production of the lipid, diacylglycerol. This lipid can induce a phase transition that will promote fusion of apposed lipid bilayers. The process of exocytosis involves the secretory granule core as well as the lipids of the membrane. The osmotic properties of the granule contents lead to swelling of the granule during exocytosis. Swelling promotes the dispersal of the contents as they are extruded through the exocytotic pore. The movements of water and ions during exocytosis may also stabilize the transient fusion intermediate and consolidate the exocytotic pore as fusion occurs.     

Understanding covalent modifications of proteins by lipids: where cell biology and biophysics mingle

Much effort has been expended on the in vitro characterization of enzymes that covalently attach lipids to proteins. Less information is available about properties conferred on modified proteins by their attached lipid groups, but biophysical studies of simple model systems have begun to shed light on this issue. Recent evidence suggests that the specificity of lipid modifications may be dependent upon the intracellular compartmentalization of the lipid and protein substrates of lipidating enzymes. The function and targeting of their lipidated products appear to be regulated dynamically through addition or subtraction of lipid moieties, other covalent or noncovalent modifications, as well as several devices that at this point can only be inferred. This field of research illustrates the necessity of integrating cell-biological and biophysical perspectives.     

Insertion of lipid domains into plasma membranes by fusion with erythrocytes

After prelabeling the plasma membrane with several lipid-specific fluorescent probes, erythrocytes with symmetric lipid bilayers were fused with culture cells using either poly(ethylene glycol) or Sendai virus as fusogen. Several nonspecific probes were transferred to, and became uniformly distributed within, the culture cell membrane upon fusion. In contrast, when merocyanine 540, which displays preferential binding to bilayers in which the lipids are loosely packed, was used to prelabel erythrocytes, fluorescence remained localized within a small confined area of the membrane, even 24 h after fusion. These results suggest that insertion of the lipids of the erythrocyte membrane into the plasma membrane of the culture cell can produce discrete domains which persist as such for long periods following fusion. Because the inserted proteins of the erythrocyte membrane similarly do not freely diffuse throughout the culture cell membrane, interactions between membrane proteins and lipids may be involved in this singular compartmentalization.     

Nuclear lipids: New functions for old molecules?

It is becoming increasingly evident that stimulation of nuclear lipid metabolism plays a central role in many signal transduction pathways that ultimately result in various cell responses including proliferation and differentiation. Nuclear lipid metabolism seems to be at least as complex as that existing at the plasma membrane. However, a distinctive feature of nuclear lipid biochemical pathways is their operational independence from their cell periphery counterparts. Although initially it was thought that nuclear lipids would serve as a source for second messengers, recent evidence points to the likelihood that lipids present in the nucleus also fulfil other roles. The aim of this review is to highlight the most intriguing advances made in the field over the last year, such as the production of new probes for the in situ mapping of nuclear phosphoinositides, the identification of two sources for nuclear diacylglycerol production, the emerging details about the peculiar regulation of nuclear phosphoinositide synthesizing enzymes, and the distinct possibility that nuclear lipids are involved in processes such as chromatin organization and pre-mRNA splicing.