Stability of lipid domains

Membranes with simple lipid composition exhibit complex phase behavior. Ordered and disordered liquid phases can coexist in cholesterol-containing membranes with lipid compositions resembling biological membranes and at physiological temperatures. Research during the last years suggests that these lipid domains play a role in the organization of biological membranes. Understanding the principles that govern the formation and stability of lipid domains is of great importance to build a model that properly describes membrane structure and function. In this review, we describe the current knowledge of the chemical and physical basis of lipid domains and its application to biological membranes.     

Cardiolipin mediates curcumin interactions with mitochondrial membranes

Curcumin, the main molecular ingredient of the turmeric spice, has been reported to exhibit therapeutic properties for varied diseases and pathological conditions. While curcumin appears to trigger multiple signaling pathways, the precise mechanisms accounting for its therapeutic activity have not been deciphered. Here we show that curcumin exhibits significant interactions with cardiolipin (CL), a lipid exclusively residing in the mitochondrial membrane. Specifically, we found that curcumin affected the structures and dynamics of CL-containing biomimetic and biological mitochondrial membranes. Application of several biophysical techniques reveals the CL-promoted association and internalization of curcumin into lipid bilayers. In parallel, curcumin association with CL containing bilayers increased their fluidity and reduced lipid ordering. These findings suggest that membrane modifications mediated by CL interactions may play a role in the therapeutic functions of curcumin, and that the inner mitochondrial membrane in general might constitute a potential drug target.     

Thallium(III)-mediated changes in membrane physical properties and lipid oxidation affect cardiolipin-cytochrome c interactions

Trivalent thallium (Tl(III)) is a highly toxic heavy metal through not completely understood mechanisms. Previously, we demonstrated that Tl(III) causes mitochondrial depolarization in PC12 cells leading to a decrease in cell viability. Given the role of the phospholipid cardiolipin (CL) in mitochondrial events, we evaluated in vitro the short- (2 min) and long- (60 min) time effects of Tl(III) (1-75 microM) on CL-containing membranes physical properties, and the consequences on cytochrome c binding to CL. After 2 min of incubation, Tl(III) significantly decreased liposome surface potential, lipid packing, and hydration of phosphatidylcholine:CL liposomes, while CL pK2 decreased from 9.8 to 8.2. The magnitude of these changes was even higher after 60 min of incubation. While no Tl(III) was found bound to membranes, Tl(I) was present in the samples. Accordingly, significant oxidative damage to both CL fatty acids and polar headgroup was observed. Cytochrome c binding to CL was decreased in Tl(III)-treated liposomes. The present results indicate that Tl(III) interaction with CL-containing membranes affected their physical properties, caused lipid oxidation and CL hydrolysis, and resulted in a decrease of cytochrome c binding. If occurring in vivo, these effects of Tl(III) could partially account for mitochondrial dysfunction in cells exposed to this metal.     

Thallium(III)-mediated changes in membrane physical properties and lipid oxidation affect cardiolipin-cytochrome c interactions

Trivalent thallium (Tl(III)) is a highly toxic heavy metal through not completely understood mechanisms. Previously, we demonstrated that Tl(III) causes mitochondrial depolarization in PC12 cells leading to a decrease in cell viability. Given the role of the phospholipid cardiolipin (CL) in mitochondrial events, we evaluated in vitro the short- (2 min) and long- (60 min) time effects of Tl(III) (1-75 μM) on CL-containing membranes physical properties, and the consequences on cytochrome c binding to CL. After 2 min of incubation, Tl(III) significantly decreased liposome surface potential, lipid packing, and hydration of phosphatidylcholine:CL liposomes, while CL pK₂ decreased from 9.8 to 8.2. The magnitude of these changes was even higher after 60 min of incubation. While no Tl(III) was found bound to membranes, Tl(I) was present in the samples. Accordingly, significant oxidative damage to both CL fatty acids and polar headgroup was observed. Cytochrome c binding to CL was decreased in Tl(III)-treated liposomes. The present results indicate that Tl(III) interaction with CL-containing membranes affected their physical properties, caused lipid oxidation and CL hydrolysis, and resulted in a decrease of cytochrome c binding. If occurring in vivo, these effects of Tl(III) could partially account for mitochondrial dysfunction in cells exposed to this metal.     

Membrane lipids: where they are and how they behave

Throughout the biological world, a 30 A hydrophobic film typically delimits the environments that serve as the margin between life and death for individual cells. Biochemical and biophysical findings have provided a detailed model of the composition and structure of membranes, which includes levels of dynamic organization both across the lipid bilayer (lipid asymmetry) and in the lateral dimension (lipid domains) of membranes. How do cells apply anabolic and catabolic enzymes, translocases and transporters, plus the intrinsic physical phase behaviour of lipids and their interactions with membrane proteins, to create the unique compositions and multiple functionalities of their individual membranes?     

Effect of ring-substituted oxysterols on the phase behavior of dipalmitoylphosphatidylcholine membranes

Oxysterols are oxygenated derivatives of cholesterol that form a class of potent regulatory molecules with diverse biological activity. Given the implications of oxysterols in several physiological/pathophysiological pathways of human diseases, it is important to identify how their presence affects the biophysical properties of cell membranes. In this article we first describe the structure, formation, and biological functions of oxysterols, and previous work on the effect of these molecules on the structure and phase behavior of lipid membranes. We then present results of our X-ray diffraction experiments on aligned multilayers of dipalmitoylphosphatidylcholine (DPPC) membranes containing ring-substituted oxysterols. The effect of these molecules on the phase behavior of DPPC membranes is found to be very similar to that of cholesterol. All the oxysterols studied induce a modulated phase in DPPC membranes, similar to that reported in DPPC–cholesterol membranes. However, some differences are observed in the ability of these molecules to suppress the main transition of the lipid and to induce chain ordering, which might be related to differences in their orientation in the bilayer.     

Structural Changes and Proapoptotic Peroxidase Activity of Cardiolipin-Bound Mitochondrial Cytochrome c

The cellular process of intrinsic apoptosis relies on the peroxidation of mitochondrial lipids as a critical molecular signal. Lipid peroxidation is connected to increases in mitochondrial reactive oxygen species, but there is also a required role for mitochondrial cytochrome c (cyt-c). In apoptotic mitochondria, cyt-c gains a new function as a lipid peroxidase that catalyzes the reactive oxygen species-mediated chemical modification of the mitochondrial lipid cardiolipin (CL). This peroxidase activity is caused by a conformational change in the protein, resulting from interactions between cyt-c and CL. The nature of the conformational change and how it causes this gain-of-function remain uncertain. Via a combination of functional, structural, and biophysical experiments we investigate the structure and peroxidase activity of cyt-c in its membrane-bound state. We reconstituted cyt-c with CL-containing lipid vesicles, and determined the increase in peroxidase activity resulting from membrane binding. We combined these assays of CL-induced proapoptotic activity with structural and dynamic studies of the membrane-bound protein via solid-state NMR and optical spectroscopy. Multidimensional magic angle spinning (MAS) solid-state NMR of uniformly ¹³C,¹⁵N-labeled protein was used to detect site-specific conformational changes in oxidized and reduced horse heart cyt-c bound to CL-containing lipid bilayers. MAS NMR and Fourier transform infrared measurements show that the peripherally membrane-bound cyt-c experiences significant dynamics, but also retains most or all of its secondary structure. Moreover, in two-dimensional and three-dimensional MAS NMR spectra the CL-bound cyt-c displays a spectral resolution, and thus structural homogeneity, that is inconsistent with extensive membrane-induced unfolding. Cyt-c is found to interact primarily with the membrane interface, without significantly disrupting the lipid bilayer. Thus, membrane binding results in cyt-c gaining the increased peroxidase activity that represents its pivotal proapoptotic function, but we do not observe evidence for large-scale unfolding or penetration into the membrane core.     

Regulatory role of cardiolipin in the activity of an ATP-dependent protease, Lon, from Escherichia coli

Lon is an ATP-dependent serine protease that plays a significant role in the quality control of proteins in cells, degrading misfolded proteins and certain short-lived regulatory proteins under stresses as such heat-shock and UV irradiation. It is known that some polymers containing phosphate groups regulate enzymatic activity by binding with Lon. We focused on the phospholipids of biological membrane components such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and cardiolipin (CL), and examined whether or not liposomes containing these phospholipids regulate the enzymatic activity of Lon. CL-containing liposomes specifically inhibited both the proteolytic and ATPase activities of Lon in a dose-dependent manner. In addition, on pull-down assay, we found that CL-containing liposomes selectively bound to Lon. The interaction between CL-containing liposomes and Lon changed with the order of addition of Mg(2+)/ATP. When CL-containing liposomes were added after the addition of Mg(2+)/ATP to Lon, the binding of CL-containing liposomes to Lon was significantly decreased as compared with the reversed order. In fact, we found that CL-containing liposomes bound to Lon, resulting in inhibition of the enzymatic activity of Lon. These results suggest that Lon interacts with CL in biological membranes, which may regulate the functions of Lon as a protein-degrading centre in accordance with environmental changes inside cells.     

Melatonin Alters Fluid Phase Coexistence in POPC/DPPC/Cholesterol Membranes

The structure and biophysical properties of lipid membranes are important for cellular functions in health and disease. In Alzheimer’s disease, the neuronal membrane is a target for toxic amyloid-β (Aβ). Melatonin is an important pineal gland hormone that has been shown to protect against Aβ toxicity in cellular and animal studies, but the molecular mechanism of this protection is not fully understood. Melatonin is a small membrane-active molecule that has been shown to interact with model lipid membranes and alter the membrane biophysical properties, such as membrane molecular order and dynamics. This effect of melatonin has been previously studied in simple model bilayers with one or two lipid components. To make it more relevant to neuronal membranes, we used a more complex ternary lipid mixture as our membrane model. In this study, we used ²H-NMR to investigate the effect of melatonin on the phase behavior of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and cholesterol lipid membranes. We used deuterium-labeled POPC-d₃₁ and DPPC-d₆₂,separately to probe the changes in hydrocarbon chain order as a function of temperature and melatonin concentration. We find that POPC/DPPC/cholesterol at molar proportions of 3:3:2 is close to liquid-disordered/liquid-ordered phase separation and that melatonin can induce phase separation in these ternary mixtures by preferentially incorporating into the disordered phase and increasing its level of disorder. At 5 mol% melatonin, we observed phase separation in samples with POPC-d₃₁, but not with DPPC-d₆₂, whereas at 10 mol% melatonin, phase separation was observed in both samples with either POPC-d₃₁ or DPPC-d₆₂. These results indicate that melatonin can have a strong effect on membrane structure and physical properties, which may provide some clues to understanding how melatonin protects against Aβ, and that choice of chain perdeuteration is an important consideration from a technical point of view.     

The effect of variations in phospholipid and sterol structure on the nature of lipid–sterol interactions in lipid bilayer model membranes

This review deals with the effect of variations in phospholipid and sterol structure on the nature and magnitude of lipid-sterol interactions in lipid bilayer model membranes. The first portion of the review covers the effect of Chol itself on the thermotropic phase behavior and organization of a variety of different glycero- and sphingolipid membrane lipid classes, varying in the structure and charge of their polar headgroups and in the length and structure of their fatty acyl chains. The second part of this review deals with the effect of variations in sterol structure on the thermotropic phase behavior and organization primarily of the well studied DPPC model membrane system. In the third section, we focus on some of the contributions of sterol functional group chemistry, molecular conformation and dynamics, to sterol-lipid interactions. Using those studies, we re-examine the results of recently published experimental and computer-modeling studies to provide a new more dynamic molecular interpretation of sterol-lipid interactions. We suggest that the established view of the rigid sterol ring system and extended alkyl side-chain obtained from physical studies of cholesterol-phospholipid mixtures may not apply in lipid mixtures differing in their sterol chemical structure.     

Influence of cholesterol and ergosterol on membrane dynamics: a fluorescence approach

Sterols are essential membrane components of eukaryotic cells and are important for membrane organization and function. Cholesterol is the most representative sterol present in higher eukaryotes. It is often found distributed non-randomly in domains or pools in biological and model membranes. Cholesterol-rich functional microdomains (lipid rafts) are often implicated in cell signaling and membrane traffic. Interestingly, lipid rafts have also recently been isolated from organisms such as yeast and Drosophila, which have ergosterol as their major sterol component. Although detailed biophysical characterization of the effect of cholesterol on membranes is well documented, the effect of ergosterol on the organization and dynamics of membranes is not very clear. We have monitored the effect of cholesterol and ergosterol on the dynamic properties of both fluid (POPC) and gel (DPPC) phase membranes utilizing the environment-sensitive fluorescent membrane probe DPH. Our results from steady state and time-resolved fluorescence measurements show, for the first time, differential effects of ergosterol and cholesterol toward membrane organization. These novel results are relevant in the context of lipid rafts in ergosterol-containing organisms such as Drosophila which maintain a low level of sterol compared to higher eukaryotes.     

Engineering the bilayer: Emerging genetic tool kits for mechanistic lipid biology

The structural diversity of lipids underpins the biophysical properties of cellular membranes, which vary across all scales of biological organization. Because lipid composition results from complex metabolic and transport pathways, its experimental control has been a major goal of mechanistic membrane biology. Here, we argue that in the wake of synthetic biology, similar metabolic engineering strategies can be applied to control the composition, physicochemical properties, and function of cell membranes. In one emerging area, titratable expression platforms allow for specific and genome-wide alterations in lipid biosynthetic genes, providing analog control over lipidome stoichiometry in membranes. Simultaneously, heterologous expression of biosynthetic genes and pathways has allowed for gain-of-function experiments with diverse lipids in non-native systems. Finally, we highlight future directions for tool development, including recently discovered lipid transport pathways to intracellular lipid pools. Further tool development providing synthetic control of membrane properties can allow biologists to untangle membrane lipid structure-associated functions.     

Phospholipid phase transitions in model and biological membranes as studied by infrared spectroscopy

Fourier transform infrared (FT-IR) spectroscopy is an extremely powerful yet non-perturbing physical technique for monitoring the conformation and dynamics of all portions of the phospholipid molecule. In this brief review we summarize some recent FT-IR spectroscopic studies of phospholipid phase transitions in model lipid bilayer and in biological membranes which illustrate the great utility of this technique. We show that FT-IR spectroscopy can accurately monitor the gel to liquid-crystalline phase transition and can provide a large amount of detailed information about phospholipid structure and organization in both the gel and liquid-crystalline states of lipid bilayers.     

Formation of inverted hexagonal phase in SDPE as observed by solid-state (31)P NMR

Docosahexaenoic acid (DHA), the longest and most unsaturated fatty acid commonly found in biological membranes, is known to affect various membrane properties. In a variety of cell membranes, DHA is primarily incorporated in phosphatidylethanolamines, where its function remains poorly understood. In order to understand the role of DHA in influencing membrane structure, we utilize (31)P NMR spectroscopy to study the phase behavior of 1-stearoyl-2-docosahexaenoyl-sn-glycerophosphoethanolamine (SDPE) in comparison to 1-palmitoyl-2-oleoyl-sn-glycerophosphoethanolamine (POPE) from 20 to 50 degrees C. Spectra of SDPE phospholipids show the formation of inverted hexagonal phase (H(II)) from 20 to 50 degrees C; in contrast, POPE mutilamellar dispersions exist in a lamellar liquid-crystalline phase (L(alpha)) at the same temperatures. The ability of SDPE to adopt nonbilayer phases at a physiological temperature may indicate its role in imparting negative curvature stress upon the membrane and may affect local molecular organization including the formation of lipid microdomains within biological membranes.     

The effects of temperature, pressure and peptide incorporation on ternary model raft mixtures--a Laurdan fluorescence spectroscopy study

Recently, an increasing evidence accumulated for the existence of lipid microdomains, called lipid rafts, in cell membranes, which may play an important role in many important membrane-associated biological processes. Suitable model systems for studying biophysical properties of lipid rafts are lipid vesicles composed of three-component lipid mixtures, such as POPC/SM/cholesterol, which exhibit a rich phase diagram, including raft-like liquid-ordered/liquid-disordered phase coexistence regions. We explored the temperature, pressure and concentration-dependent phase behavior of such canonical model raft mixtures using the Laurdan fluorescence spectroscopic technique. Hydrostatic pressure has not only been used as a physical parameter for studying the stability and energetics of these systems, but also because high pressure is an important feature of certain natural membrane environments. We show that the liquid-disordered/liquid-ordered phase coexistence regions of POPC/SM/cholesterol model raft mixtures extends over a very wide temperature range of about 50 degrees C. Upon pressurization, an overall ordered membrane state is reached at pressures of approximately 1,000 bar at 20 degrees C, and of approximately 2,000 bar at 40 degrees C. Incorporation of 5 mol% gramicidin as a model ion channel slightly increases the overall order parameter profile in the l(o)+l(d) two-phase coexistence region, probably by selectively partitioning into l(d) domains, does not change the overall phase behavior, however. This behavior is in contrast to the effect of the peptide incorporation into simple, one-component phospholipid bilayer systems.     

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.     

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.     

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.     

Influence of squalene on lipid particle/droplet and membrane organization in the yeast Saccharomyces cerevisiae

In a previous study (Spanova et al., 2010, J. Biol. Chem., 285, 6127-6133) we demonstrated that squalene, an intermediate of sterol biosynthesis, accumulates in yeast strains bearing a deletion of the HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets together with triacylglycerols and steryl esters. In mutants lacking the ability to form lipid particles, however, substantial amounts of squalene accumulate in organelle membranes. In the present study, we investigated the effect of squalene on biophysical properties of lipid particles and biological membranes and compared these results to artificial membranes. Our experiments showed that squalene together with triacylglycerols forms the fluid core of lipid particles surrounded by only a few steryl ester shells which transform into a fluid phase below growth temperature. In the hem1? deletion mutant a slight disordering effect on steryl esters was observed indicated by loss of the high temperature transition. Also in biological membranes from the hem1? mutant strain the effect of squalene per se is difficult to pinpoint because multiple effects such as levels of sterols and unsaturated fatty acids contribute to physical membrane properties. Fluorescence spectroscopic studies using endoplasmic reticulum, plasma membrane and artificial membranes revealed that it is not the absolute squalene level in membranes but rather the squalene to sterol ratio which mainly affects membrane fluidity/rigidity. In a fluid membrane environment squalene induces rigidity of the membrane, whereas in rigid membranes there is almost no additive effect of squalene. In summary, our results demonstrate that squalene (i) can be well accommodated in yeast lipid particles and organelle membranes without causing deleterious effects; and (ii) although not being a typical membrane lipid may be regarded as a mild modulator of biophysical membrane properties.     

Cardiolipin switch in mitochondria: shutting off the reduction of cytochrome c and turning on the peroxidase activity

Upon interaction with anionic phospholipids, particularly mitochondria-specific cardiolipin (CL), cytochrome c (cyt c) loses its tertiary structure and its peroxidase activity dramatically increases. CL-induced peroxidase activity of cyt c has been found to be important for selective CL oxidation in cells undergoing programmed death. During apoptosis, the peroxidase activity and the fraction of CL-bound cyt c markedly increase, suggesting that CL may act as a switch to regulate cyt c's mitochondrial functions. Using cyclic voltammetry and equilibrium redox titrations, we show that the redox potential of cyt c shifts negatively by 350-400 mV upon binding to CL-containing membranes. Consequently, functions of cyt c as an electron transporter and cyt c reduction by Complex III are strongly inhibited. Further, CL/cyt c complexes are not effective in scavenging superoxide anions and are not effectively reduced by ascorbate. Thus, both redox properties and functions of cyt c change upon interaction with CL in the mitochondrial membrane, diminishing cyt c's electron donor/acceptor role and stimulating its peroxidase activity.