Lateral Organization Of Biological Membranes Research Articles

Multidimensional Spectral Phasors of LAURDAN's Excitation-Emission Matrices: The Ultimate Sensor for Lipid Phases?

The impact of lipid diversity on the lateral organization of biological membranes remains a topic of debate. While the existence of domains in lamellar membranes is well-established, the nonlamellar phases occurring in biological systems are less explored due to technical constraints. Here, we present the measurement of the excitation-emission matrices (EEM) of LAURDAN in several lipid structures. LAURDAN is a fluorescence probe widely used for characterizing lipid assemblies. The EEMs were analyzed by multidimensional spectral phasors (MdSP), an approach that seizes information from both the excitation and emission spectra. We developed a computer algorithm to construct EEM data based on a model for LAURDAN's photophysics. The MdSP calculated from the simulated EEMs reveals that all feasible possibilities lie inside a universal triangle in the phasor's plot. We use this triangle to propose a ternary representation for the phasors, allowing a better assessment of LAURDAN's surroundings in terms of hydration, water mobility, and local electronic environment. Building upon this foundation, we constructed a theoretical "phase map" that can assess both lamellar and nonlamellar membranes. We thoroughly validated this theory using well-known lipid mixtures under different phase-state conditions and enzymatically generated systems. Our results confirm that the use of MdSP is a powerful tool for obtaining quantitative information on both lamellar and nonlamellar structures. This study not only advances our understanding of the impact of lipid diversity on membrane organization but also provides a robust and general framework for the assessment of fluorescence properties that can be further extended to other probes.

The Role of Growth Temperature and Lipid Composition in Phase Separation of Yeast Vacuole Membranes

Membranes of vacuoles, the lysosomal organelle in yeast, phase separate into two coexisting liquid phases. This phenomenon provides a physical mechanism for lateral organization of biological membranes. Recent studies show that these domains play a key role in a central eukaryotic signaling pathway. Howthe cell adapts and tunes its vacuole membrane to invoke phase separation is still largely unknown. Here we examine how membrane composition changes due to two different environmental stimuli: growth temperature and nutrient depletion. If it is important for cells to regulate phase separation of their membranes, then we expect yeast cells to acclimate to external conditions. We test this hypothesis by growing yeast at 25°C and 30°C and find that the yeast adapts to this change by dramatically shifting the demixing temperature of vacuole membranes (to 29.7°C and 42.7°C respectively). This stark change in vacuole miscibility temperatures scales with growth temperature both in live cells and in isolated vacuoles, showing that yeast alter their membrane compositions to maintain phase separation. We also directly investigate the role of ergosterol, the main sterol in yeast, in domain formation in vacuoles. The yeast growth cycle starts with a logarithmic phase of rapid growth followed by a stationary phase in which glucose becomes scarce and vacuole membranes exhibit coexisting liquid domains. We isolate vacuoles from yeast in the logarithmic phase of growth, in which the membranes do not natively exhibit domains. Using fluorescence microscopy, we observe changes in vacuole phase separation as a function of ergosterol content. We find the surprising result that domains appear in vacuole membranes with reduced ergosterol.

Complex Phase Behavior of GUVs Containing Different Sphingomyelins

Understanding the lateral organization of biological membranes plays a key role on the road to fully appreciate the physiological functions of this fundamental barrier between the inside and outside regions of a cell. Ternary lipid bilayers composed of a high and a low melting temperature lipid and cholesterol represent a model system that mimics some of the important thermodynamical features of much more complex lipid mixtures such as those found in mammal membranes. The phase diagram of these ternary mixtures can be studied exploiting fluorescence microscopy in giant unilamellar vesicles, and it is typically expected to give rise, for specific combinations of composition and temperature, to regions of two-phase coexistence and a region with three-phase coexistence, namely, the liquid-ordered, liquid-disordered, and solid phases. Whereas the observation of two-phase coexistence is routinely possible using fluorescence microscopy, the three-phase region is more elusive to study. In this article, we show that particular lipid mixtures containing diphytanoyl-phosphatidylcholine and cholesterol plus different types of sphingomyelin (SM) are prone to produce bilayer regions with more than two levels of fluorescence intensity. We found that these intensity levels occur at low temperature and are linked to the copresence of long and asymmetric chains in SMs and diphytanoyl-phosphatidylcholine in the lipid mixtures. We discuss the possible interpretations for this observation in terms of bilayer phase organization in the presence of sphingolipids. Additionally, we also show that in some cases, liposomes in the three-phase coexistence state exhibit extreme sensitivity to lateral tension. We hypothesize that the appearance of the different phases is related to the asymmetric structure of SMs and to interdigitation effects.

Capturing suboptical dynamic structures in lipid bilayer patches formed from free-standing giant unilamellar vesicles.

There is accumulating evidence that the small-scale lateral organization of biological membranes has a crucial role in signaling and trafficking in cells. However, it has been difficult to characterize these features with existing methods for preparing and analyzing freestanding membranes, because the dynamics occurs below the optical resolution possible with these protocols. We have developed a protocol that permits the imaging of lipid nanodomains and lateral protein organization in membranes of giant unilamellar vesicles (GUVs). Freestanding GUVs are transferred onto a mica support, and after treatment with magnesium chloride, they collapse to form planar lipid bilayer (PLB) patches. Rapid GUV collapse onto the mica preserves the lateral organization of freestanding membranes and thus makes it possible to image 'snapshots' of GUVs up to nanometer resolution by high-resolution microscopy. The method has been applied to classical lipid raft mixtures in which suboptical domain fluctuations have been imaged in both the liquid-ordered and liquid-disordered membrane phases. High-resolution scanning by atomic force microscopy (AFM) of membranes composed of binary and ternary lipid mixtures reconstituted with Na+/K+-ATPase (NKA) has revealed the spatial distribution and orientations of individual proteins, as well as details of membrane lateral structure. Immunolabeling followed by confocal microscopy can also provide information about the spatial distribution of proteins. The protocol opens up a new avenue for quantitative biophysical studies of suboptical dynamic structures in biomembranes, which are local and short-lived. Preparation of GUVs, PLB patches and their imaging takes <24 h.

Nanometer-Scale Lipid Clusters in Model Membranes Revealed by Atomic Recombination in Nanosims

Much of what is known about the lateral organization of biological membranes is inferred from the analogy between phase diagrams of ternary mixtures of lipids and the plasma membrane of mammalian cells. However, the relevance of phase diagrams of simple lipid mixtures to the compositionally complex and dynamic plasma membrane has not yet been established. Additionally, the fluorescence and spin labels used to build phase diagrams often interact with lipid bilayers, and detecting nanometer-scale phases remains challenging. We have previously introduced atomic recombination in dynamic SIMS as a technique for detecting lateral heterogeneities that occur on the nanometer length scale, below the resolution of both fluorescence microscopy and conventional NanoSIMS imaging. In this method, the formation of 13C15N- secondary ions from 13C and 15N atoms installed on different lipids only occurs if the isotopically-labeled lipids are within approximately 3 nm of each other. Isotope labels are desirable because they avoid problems caused by fluorescent dyes and because they directly report on the organization of the lipids of interest. Facile isotope-labeling chemistry allows the labels to be placed on any lipid of interest, allowing us to determine which lipids are clustering in complex mixtures. Here, we expand this technique to study the formation and composition of nanodomains of lipids in more complex mixtures for which phase diagrams have not been determined and would be too laborious to fully solve. Furthermore, we explore the formation of lipid clusters outside of phase boundaries, where lipids may still be inhomogenously distributed but display no first order phase transition. These more complex model membranes may be better models for lipid rafts given that lipids in the plasma membrane show no micrometer scale phase separation and no first order phase transition.

Atomic Recombination in Dynamic Secondary Ion Mass Spectrometry Probes Distance in Lipid Assemblies: A Nanometer Chemical Ruler

The lateral organization of biological membranes is thought to take place on the nanometer length scale. However, this length scale and the dynamic nature of small lipid and protein domains have made characterization of such organization in biological membranes and model systems difficult. Here we introduce a new method for measuring the colocalization of lipids in monolayers and bilayers using stable isotope labeling. We take advantage of a process that occurs in dynamic SIMS called atomic recombination, in which atoms on different molecules combine to form diatomic ions that are detected with a NanoSIMS instrument. This process is highly sensitive to the distance between molecules. By measuring the efficiency of the formation of 13C15N- ions from 13C and 15N atoms on different lipid molecules, we measure variations in the lateral organization of bilayers even though these heterogeneities occur on a length scale of only a few nm, well below the diameter of the primary ion beam of the NanoSIMS instrument or even the best super-resolution fluorescence methods. Using this technique, we provide direct evidence for nanoscale phase separation in a model membrane, which may provide a better model for the organization of biological membranes than lipid mixtures with microscale phase separation. We expect this technique to be broadly applicable to any assembly where very short scale proximity is of interest or unknown, both in chemical and biological systems.

Influence of the sterol aliphatic side chain on membrane properties: a molecular dynamics study.

Following a recent experimental investigation of the effect of the length of the alkyl side chain in a series of cholesterol analogues (Angew. Chem., Int. Ed., 2013, 52, 12848-12851), we report here an atomistic molecular dynamics characterization of the behaviour of methyl-branched side chain sterols (iso series) in POPC bilayers. The studied sterols included androstenol (i-C0-sterol) and cholesterol (i-C8-sterol), as well as four other derivatives (i-C5, i-C10, i-C12 and i-C14-sterol). For each sterol, both subtle local effects and more substantial differential alterations of membrane properties along the iso series were investigated. The location and orientation of the tetracyclic ring system is almost identical in all compounds. Among all the studied sterols, cholesterol is the sterol that presents the best matching with the hydrophobic length of POPC acyl chains, whereas longer-chained sterols interdigitate into the opposing membrane leaflet. In accordance with the experimental observations, a maximal ordering effect is observed for intermediate sterol chain length (i-C5, cholesterol, i-C10). Only for these sterols a preferential interaction with the saturated sn-1 chain of POPC (compared to the unsaturated sn-2 chain) was observed, but not for either shorter or longer-chained derivatives. This work highlights the importance of the sterol alkyl chain in the modulation of membrane properties and lateral organization in biological membranes.

The Mechanism of Collapse of Heterogeneous Lipid Monolayers

Collapse of homogeneous lipid monolayers is known to proceed via wrinkling/buckling, followed by folding into bilayers in water. For heterogeneous monolayers with phase coexistence, the mechanism of collapse remains unclear. Here, we investigated collapse of lipid monolayers with coexisting liquid-liquid and liquid-solid domains using molecular dynamics simulations. The MARTINI coarse-grained model was employed to simulate monolayers of ∼80 nm in lateral dimension for 10–25 μs. The monolayer minimum surface tension decreased in the presence of solid domains, especially if they percolated. Liquid-ordered domains facilitated monolayer collapse due to the spontaneous curvature induced at a high cholesterol concentration. Upon collapse, bilayer folds formed in the liquid (disordered) phase; curved domains shifted the nucleation sites toward the phase boundary. The liquid (disordered) phase was preferentially transferred into bilayers, in agreement with the squeeze-out hypothesis. As a result, the composition and phase distribution were altered in the monolayer in equilibrium with bilayers compared to a flat monolayer at the same surface tension. The composition and phase behavior of the bilayers depended on the degree of monolayer compression. The monolayer-bilayer connection region was enriched in unsaturated lipids. Percolation of solid domains slowed down monolayer collapse by several orders of magnitude. These results are important for understanding the mechanism of two-to-three-dimensional transformations in heterogeneous thin films and the role of lateral organization in biological membranes. The study is directly relevant for the function of lung surfactant, and can explain the role of nanodomains in its surface activity and inhibition by an increased cholesterol concentration.

Asymmetry Determines the Effect of Ceramides on Model Membranes. In Natural Membranes Too?

Ceramides can dramatically influence the lateral organization of biological membranes. In particular, ceramide-induced alterations of protein-lipid domains can be involved in several cellular processes, ranging from senescence to immune response. In this context, an important role is played by the length of the fatty acid bound to the sphingosine moiety. Asymmetric, heterogeneous ceramides, with more than 20 or less than 16 carbon atoms in the fatty acyl chain, in fact exert diverging effects in vivo if compared to their symmetric counterparts. In this work, we investigated the role of ceramide asymmetry and heterogeneity in model membranes showing raft-like phase separation, using a combination of fluorescence imaging, atomic force microscopy, fluorescence correlation spectroscopy and differential scanning calorimetry. We show that ceramide produced enzymatically from natural mixtures of sphingomyelin can dramatically alter the mixing behaviour of proteins and lipids in the membrane, inducing a homogenization of the bilayer. Furthermore, we characterized the physical properties of coexisting lipid phases at equilibrium in membranes with varying ceramide content, emphasizing the differences between symmetric-homogeneous and asymmetric-heterogeneous ceramides. While symmetric ceramides always produce enhanced order, asymmetric ceramides display a more complex behavior similar to that of cholesterol. Our results might help contribute to a more precise understanding of the rearrangements induced by different kinds of ceramide generation in cellular membranes.

Lateral organization of biological membranes

The lateral organization of biological membranes is of great importance in many biological processes, both for the formation of specific structures such as super-complexes and for function as observed in signal transduction systems. Over the last years, AFM studies, particularly of bacterial photosynthetic membranes, have revealed that certain proteins are able to segregate into functional domains with a specific organization. Furthermore, the extended non-random nature of the organization has been suggested to be important for the energy and redox transport properties of these specialized membranes. In the work reported here, using a coarse-grained Monte Carlo approach, we have investigated the nature of interaction potentials able to drive the formation and segregation of specialized membrane domains from the rest of the membrane and furthermore how the internal organization of the segregated domains can be modulated by the interaction potentials. These simulations show that long-range interactions are necessary to allow formation of membrane domains of realistic structure. We suggest that such possibly non-specific interactions may be of great importance in the lateral organization of biological membranes in general and in photosynthetic systems in particular. Finally, we consider the possible molecular origins of such interactions and suggest a fundamental role for lipid-mediated interactions in driving the formation of specialized photosynthetic membrane domains. We call these lipid-mediated interactions a 'lipophobic effect.'

Computer simulations of the phase separation in model membranes

We used computer simulations to investigate the properties of model lipid membranes with coexisting phases. This is relevant for understanding lipid-lipid interactions underlying lateral organization in biological membranes. Molecular dynamics simulations with the MARTINI coarse-grained force field were employed to study lipid bilayers -40 nm in lateral dimension on a 20 micros time scale. The simulations retain near atomic-level detail and lipid chemical specificity, and allow formation of multiple domains of tens of nanometers in size. Using ternary lipid mixtures of saturated and unsaturated lipids and cholesterol, we reproduced the coexistence of the Lalpha/gel phases and the Lo/Ld phases. Phase transformation proceeded by either nucleation or spinodal decomposition. The properties of coexisting phases were characterized in detail, including partial lipid areas, composition, phase boundary and domain registry, based on Voronoi tessellation. We investigated variations of these properties with temperature and surface tension, and compared them to our recent simulations of lipid monolayers of the same size and composition. We found substantial overlap in bilayer and monolayer properties. Increasing the temperature in bilayers produced similar effects as increasing the surface tension in monolayers. This information can be used for interpreting experimental data on model membranes.

Molecular View of Phase Coexistence in Model Membranes

We used computer simulations to investigate phase transformations in lipid monolayers. This is important for understanding lipid-lipid interactions underlying lateral organization in biological membranes, and the role of phase coexistence in the regulation of surface tension by lung surfactant. Molecular dynamics simulations with the coarse-grained force field MARTINI were employed to achieve large length (∼80 nm in lateral dimension) and time (tens of microseconds) scales. Lipid mixtures containing saturated and unsaturated lipids and cholesterol were investigated under varying surface tension and temperature. We reproduced compositional lipid de-mixing and transformation into liquid-expanded (LE) and liquid-condensed (LC) phases, and into liquid-ordered (Lo) and liquid-disordered (Ld) phases. Transformation proceeded via either nucleation and growth, or spinodal decomposition, with distinct coarsening kinetics. Nucleation rate and growth exponents were calculated. Partial lipid areas and phase composition showed a different dependence on surface tension. The domain boundary length increased and the line tension decreased with reducing surface tension. Domains of Lo phase manifested spontaneous curvature at low surface tensions. The surface viscosity of monolayers with phase coexistence increased due to domain re-organization under shear. In the Lo/Ld mixture, strong compositional fluctuations were observed at higher temperatures. Monolayer collapse occurred in the disordered phase (LE or Ld), which then transferred into bilayer folds and monolayer-bilayer connection. Domains of coexisting phased either increased or reduced monolayer stability. We also investigated lipid bilayers of the same composition. Decreasing surface tension in monolayers and temperature in bilayers had similar effects on the properties of coexisting phases.

Imaging lipid lateral organization in membranes with C-laurdan in a confocal microscope

Lateral organization of biological membranes is frequently studied using fluorescence microscopy. One of the most widely used probes for these studies is 2-dimethylamino-6-lauroylnaphthalene (laurdan). The fluorescence of this probe is sensitive to the environment polarity, and thus laurdan reports the local penetration of water when inserted in membranes. Unfortunately, this probe can only be used under two-photon excitation due to its low photostability. This is a very important limitation, because there are not too many laboratories with capability for two-photon microscopy. In this work, we explored the performance of 6-dodecanoyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene (C-laurdan), a carboxyl-modified version of laurdan, for imaging biological membranes using a conventional confocal microscopy setup. We acquired generalized polarization (GP) images of C-laurdan inserted in giant unillamelar vesicles composed of binary mixtures of lipids and verified that the probe allows observing the coexistence of different phases. We also tested the performance of the probe for measurement with living cells and registered GP images of melanophore cells labeled with C-laurdan in which we could observe highly ordered regions such as filopodia. These findings show that C-laurdan can be successfully employed for studies of membrane lateral organization using a conventional confocal microscope and can open the possibility of studying a wide variety of membrane-related processes.

Intracellular localization of organized lipid domains of C16-ceramide/cholesterol

Lipid microdomains, also called lipid rafts, consisting of sphingolipids and cholesterol, play important roles in membrane trafficking and in signaling. Despite years of study of the composition, size, half-life and dynamic organization of these domains, many open questions remain about their precise characteristics. To address some of these issues, we have developed a new experimental approach involving the use of specific monoclonal antibodies as recognition tools. One such antibody was raised against a homogeneous, mixed, ordered monolayer phase comprised of 60:40 mol% cholesterol:C16-ceramide, and has been used previously to demonstrate the existence of C16-ceramide/cholesterol domains in the membranes of cultured cells. We now use a combination of quantitative fluorescence microscopy, immuno-transmission electron microscopy and immuno-scanning cryo-electron microscopy, optimized for the study of intracellular lipid antigens. In a variety of cultured cells, C16-ceramide/cholesterol structural domains were found at high levels in late endosomes and in the trans-Golgi network, but were not found at statistically significant levels in early endosomes, lysosomes or the endoplasmic reticulum. We discuss the relevance of these results to understanding the role of lipid lateral organization in biological membranes.

Sterol affinity for bilayer membranes is affected by their ceramide content and the ceramide chain length

It is known that ceramides can influence the lateral organization in biological membranes. In particular ceramides have been shown to alter the composition of cholesterol and sphingolipid enriched nanoscopic domains, by displacing cholesterol, and forming gel phase domains with sphingomyelin. Here we have investigated how the bilayer content of ceramides and their chain length influence sterol partitioning into the membranes. The effect of ceramides with saturated chains ranging from 4 to 24 carbons in length was investigated. In addition, unsaturated 18:1- and 24:1-ceramides were also examined. The sterol partitioning into bilayer membranes was studied by measuring the distribution of cholestatrienol, a fluorescent cholesterol analogue, between methyl-beta-cyclodextrin and large unilamellar vesicle with defined lipid composition. Up to 15 mol% ceramide was added to bilayers composed of DOPC:PSM:cholesterol (3:1:1), and the effect on sterol partitioning was measured. Both at 23 and 37 degrees C addition of ceramide affected the sterol partitioning in a chain length dependent manner, so that the ceramides with intermediate chain lengths were the most effective in reducing sterol partitioning into the membranes. At 23 degrees C the 18:1-ceramide was not as effective at inhibiting sterol partitioning into the vesicles as its saturated equivalent, but at 37 degrees C the additional double bond had no effect. The longer 24:1-ceramide behaved as 24:0-ceramide at both temperatures. In conclusion, this work shows how the distribution of sterols within sphingomyelin-containing membranes is affected by the acyl chain composition in ceramides. The overall membrane partitioning measured in this study reflects the differential partitioning of sterol into ordered domains where ceramides compete with the sterol for association with sphingomyelin.

Sterol Affinity for Bilayer Membranes is Affected by their Ceramide Content and the Ceramide Chain Length

It is known that ceramides can influence the lateral organization in biological membranes. In particular ceramides have been shown to alter the composition of cholesterol and sphingolipid enriched nanoscopic domains, by displacing cholesterol, and forming gel phase domains with sphingomyelin. Here we have investigated how the bilayer content of ceramides and their chain length influence sterol partitioning into the membranes. The effect of ceramides with saturated chains ranging from 4 and 24 carbons in length was investigated.

Asymmetry determines the effects of natural ceramides on model membranes

Ceramides can dramatically influence the lateral organization of biological membranes. In particular, ceramide-induced alterations of protein-lipid domains can be involved in several cellular processes, ranging from senescence to immune response. In this context, an important role is played by the length of the fatty acid bound to the sphingosine moiety. Asymmetric, heterogeneous ceramides, with more than 20 or less than 16 carbon atoms in the fatty acyl chain, in fact exert diverging effects in vivo if compared to their symmetric counterparts. In this work, we investigated the role of ceramide asymmetry and heterogeneity in model membranes showing raft-like phase separation, using a combination of fluorescence imaging, atomic force microscopy, fluorescence correlation spectroscopy and differential scanning calorimetry. We show that ceramide produced enzymatically from natural mixtures of sphingomyelin can dramatically alter the mixing behaviour of proteins and lipids in the membrane, inducing a homogenization of the bilayer. Furthermore, we characterized the physical properties of coexisting lipid phases at equilibrium in membranes with varying ceramide content, emphasizing the differences between symmetric-homogeneous and asymmetric-heterogeneous ceramides. While symmetric ceramides always produce enhanced order, asymmetric ceramides display a more complex behavior similar to that of cholesterol. Our results might help contribute to a more precise understanding of the rearrangements induced by different kinds of ceramide generation in cellular membranes.

Methods for Quantification of Lateral Organization in Biological Membranes

Methods for Quantification of Lateral Organization in Biological Membranes

Raft Domain Reorganization Driven by Short- and Long-Chain Ceramide: A Combined AFM and FCS Study

Naturally occurring long-chain ceramides (Cer) are known to alter the lateral organization of biological membranes. In particular, they produce alterations of microdomains that are involved in several cellular processes, ranging from apoptosis to immune response. In order to induce similar biological effects, short-chain Cer are extensively used in in vivo experiments to replace their long-chain analogues. In this work, we used the combined approach of atomic force microscopy (AFM) and fluorescence correlation spectroscopy (FCS) to investigate the effect of Cer chain length in lipid bilayers composed of sphingomyelin, dioleoyl-phosphatidylcholine, and cholesterol. Our results show that only long-chain Cer, like C18 and C16, are able to segregate from the liquid-ordered phase, forming separate Cer-enriched domains. Conversely, short-chain Cer do not form a separate phase but alter the physical properties of the liquid-ordered domains, decreasing their stability and viscosity and perturbing the lipid packing. These differences may contribute to the explanation of the different physiological effects that are often observed for the long- and short-chain Cer.

Effects of Ceramide on Liquid-Ordered Domains Investigated by Simultaneous AFM and FCS

The sphingolipid ceramides are known to influence lipid lateral organization in biological membranes. In particular, ceramide-induced alterations of microdomains can be involved in several cell functions, ranging from apoptosis to immune response. We used a combined approach of atomic force microscopy, fluorescence correlation spectroscopy, and confocal fluorescence imaging to investigate the effects of ceramides in model membranes of biological relevance. Our results show that physiological quantities of ceramide in sphingomyelin/dioleoylphosphatidylcholine/cholesterol supported bilayers lead to a significant rearrangement of lipid lateral organization. Our experimental setup allowed a simultaneous characterization of both structural and dynamic modification of membrane microdomains, induced by the presence of ceramide. Formation of similar ceramide-enriched domains and, more general, alterations of lipid-lipid interactions can be of crucial importance for the biological function of cell membranes.