Lipid Domain Formation Research Articles

Phospholipid Architecture of the Bovine Milk Fat Globule Membrane Using Giant Unilamellar Vesicles as a Model

Giant unilamellar vesicles (GUVs) were constructed using an electroformation technique to mimic the morphology of the native milk fat globule membrane (MFGM) for the purpose of structural investigation. Bovine milk derived phospholipids were selected to manufacture GUVs which were characterized by confocal laser scanning microscopy after fluorescent staining. Circular nonfluorescent dark regions were observed in a 3/7 (mol/mol) surface mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3 phosphoethanolamine. Linear shaped dark lipid domains were found in GUVs containing sphingomyelin (SM) in the absence of cholesterol. The dark regions were interpreted as a gel phase formed by a high gel-liquid phase transition temperature (Tm) of DPPC and SM. This study provides a strategy for investigating the lipid structural organization within the native MFGM using a model lipid bilayer system and reveals that a SM and cholesterol association network is not the only requirement for nonfluorescent lipid domain formation and that PE is preferably located in the inner leaflet of the phospholipid bilayer.

Sphingolipid symmetry governs membrane lipid raft structure

Lipid domain formation in membranes underlies the concept of rafts but their structure is controversial because the key role of cholesterol has been challenged. The configuration of glycosphingolipid receptors for agonists, bacterial toxins and enveloped viruses in plasma membrane rafts appears to be an important factor governing ligand binding and infectivity but the details are as yet unresolved. I have used X-ray diffraction methods to examine how cholesterol affects the distribution of glycosphingolipid in aqueous dispersions of an equimolar mixture of cholesterol and egg-sphingomyelin containing different proportions of glucosylceramide from human extracts. Three coexisting liquid-ordered bilayer structures are observed at 37°C in mixtures containing up to 20mol% glycosphingolipid. All the cholesterol was sequestered in one bilayer with the minimum amount of sphingomyelin (33mol%) to prevent formation of cholesterol crystals. The other two bilayers consisted of sphingomyelin and glucosylceramide. Asymmetric molecular species of glucosylceramide with N-acyl chains longer than 20 carbons form an equimolar complex with sphingomyelin in which the glycosidic residues are arranged in hexagonal array. Symmetric molecular species mix with sphingomyelin in proportions less than equimolar to form quasicrystalline bilayers. When the glycosphingolipid exceeds equimolar proportions with sphingomyelin cholesterol is incorporated into the structure and formation of a gel phase of glucosylceramide is prevented. The demonstration of particular structural features of ceramide molecular species combined with the diversity of sugar residues of glycosphingolipid classes paves the way for a rational approach to understanding the functional specificity of lipid rafts and how they are coupled across cell membranes.

Fluorescence correlation and lifetime correlation spectroscopy applied to the study of supported lipid bilayer models of the cell membrane

Supported Lipid Bilayers (SLBs) are versatile models capable of mimicking some of the key properties of the cell membrane, including for example lipid fluidity, domain formation and protein support, without the challenging complexity of the real biological system. This is important both from the perspective of understanding the behaviour and role of the lipid membrane in cell structure and signalling, as well as in development of applications of lipid membranes across domains as diverse as sensing and drug delivery. Lipid and protein diffusion within the membrane is vital to its function and there are several key experimental methods used to study membrane dynamics. Amongst the optical methods are Fluorescence Recovery After Photobleaching (FRAP), single particle tracking and Fluorescence Correlation (and Fluorescence Lifetime Correlation) Spectroscopy (FCS/FLCS). Each of these methods can provide different and often complementary perspectives on the dynamics of the fluid membrane. Although FCS is well established, FLCS is a relatively new technique and both methods have undergone a number of extensions in recent years which improve their precision and accuracy in studying supported lipid bilayers, most notably z-scan methods. This short review focusses on FCS and FLCS and their recent applications, specifically to artificial lipid bilayer studies addressing key issues of cell membrane behaviour.

Characterisation of Coexisting Liquid Phases in Mixtures of Dipalmitoylphosphatidylcholine and Cholesterol

Since their introduction, lipid rafts have been considered to be vital for various processes, such as membrane trafficking and signal transduction. While there is growing evidence for their existence and importance, the understanding of their emergence and physical properties is still limited.Perhaps surprisingly, the understanding of similar issues is limited also in much simpler model systems. One of the most thoroughly studied cases is the binary mixture of dipalmitoylphosphatidylcholine (DPPC) and cholesterol, which despite its simplicity shows a spectrum of phases and their coexistence regions. Even though its phase behaviour is well established, the structural and dynamic details of the nanoscale domains in the coexistence region remain largely unknown.To shed light on these features, we used atomistic molecular dynamics simulations to consider the phase behaviour of the DPPC-cholesterol system, with a focus on domain properties. Extensive simulations performed at varying temperatures and cholesterol concentrations highlighted that the experimental phase diagram was well reproduced by the atomistic simulation model. Importantly, under appropriate conditions the lipids were found to separate into two different liquid phases with clearly visible boundaries. The simulation data was used to analyse the driving forces for the formation of lipid domains, as well as their dynamics. In light of the results for this simple model system, we discuss how much can we expect to learn from related phenomena in more complex membrane systems.

Kinetic Mechanism of Phase Separation in Stratum Corneum Models by IR Spectroscopy

The primary barrier to permeability in mammalian skin resides within the ∼15 μm thick outermost layer, the stratum corneum (SC) which is formed from anucleated corneocytes embedded in a lamellar lipid matrix. Studies of lipid structural organization in human SC have begun to provide a basis for understanding barrier function with the potential for elucidating how skin organization is altered in disease states. In contrast, the kinetics and mechanisms of the development of lamellar structures, lipid packing motifs, and domain formation have not been widely probed. Yet, molecular level information concerning the mechanisms and kinetics of lipid domain formation would help define potential pathways for permeation of hydrophobic species as well as to possibly provide clues as to why a variety of ceramides and additional chemical species are present in the SC. In addition, understanding the formation and dissipation kinetics of domains formed from particular ceramide and fatty acid species would aid in defining biological processes such as epidermal desquamation. The current study demonstrates the feasibility of utilizing IR spectroscopy for studying lateral phase separation and the development of the early stages of lamellar structure in skin lipid models formed from two fairly standard ternary equimolar mixtures, namely ceramide[NS]/stearic acid/cholesterol and ceramide[AS]/stearic acid/cholesterol mixtures. The IR measurements show that the following sequence of events occurs in ceramide[NS]/stearic acid/cholesterol mixtures: (1) rearrangements of H-bonds in the ceramide component, (2) formation of small ceramide domains and orthorhombic phases, followed by (3) rearrangement of H-bonds in stearic acid, and (4) formation of relatively large and pure orthorhombic domains of stearic acid, probably indicative of the onset of lamellar phase formation. Notably different kinetic behavior is observed for the stearic acid component in the ceramide[AS] mixture.

Le sport m’a tué : évaluation transversale des comportements de photoprotection chez 254 sportifs de haut niveau

Glucosylceramide (GlcCer), a relevant intermediate in the pathways of glycosphingolipid metabolism, plays key roles in the regulation of cell physiology. The molecular mechanisms by which GlcCer regulates cellular processes are unknown, but might involve changes in membrane biophysical properties and formation of lipid domains. In the present study, fluorescence spectroscopy, confocal microscopy and surface pressure–area (π–A) measurements were used to characterize the effect of GlcCer on the biophysical properties of model membranes. We show that C16:0-GlcCer has a high tendency to segregate into highly ordered gel domains and to increase the order of the fluid phase. Monolayer studies support the aggregation propensity of C16:0-GlcCer. π–A isotherms of single C16:0-GlcCer indicate that bilayer domains, or crystal-like structures, coexist within monolayer domains at the air–water interface. Mixtures with POPC exhibit partial miscibility with expansion of the mean molecular areas relative to the additive behavior of the components. Moreover, C16:0-GlcCer promotes morphological alterations in lipid vesicles leading to formation of flexible tubule-like structures that protrude from the fluid region of the bilayer. These results support the hypothesis that alterations in membrane biophysical properties induced by GlcCer might be involved in its mechanism of action.

Lipid Segregation and Membrane Budding Induced by the Peripheral Membrane Binding Protein Annexin A2*

The formation of dynamic membrane microdomains is an important phenomenon in many signal transduction and membrane trafficking events. It is driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Here we analyzed the ability of one peripherally associated membrane protein, annexin A2 (AnxA2), to induce the formation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-rich domains in giant unilamellar vesicles (GUVs) of complex lipid composition. AnxA2 is a cytosolic protein that can bind PI(4,5)P2 and other acidic phospholipids in a Ca(2+)-dependent manner and that has been implicated in cellular membrane dynamics in endocytosis and exocytosis. We show that AnxA2 binding to GUVs induces lipid phase separation and the recruitment of PI(4,5)P2, cholesterol and glycosphingolipids into larger clusters. This property is observed for the full-length monomeric protein, a mutant derivative comprising the C-terminal protein core domain and for AnxA2 residing in a heterotetrameric complex with its intracellular binding partner S100A10. All AnxA2 derivatives inducing PI(4,5)P2 clustering are also capable of forming interconnections between PI(4,5)P2-rich microdomains of adjacent GUVs. Furthermore, they can induce membrane indentations rich in PI(4,5)P2 and inward budding of these membrane domains into the lumen of GUVs. This inward vesiculation is specific for AnxA2 and not shared with other PI(4,5)P2-binding proteins such as the pleckstrin homology (PH) domain of phospholipase Cδ1. Together our results indicate that annexins such as AnxA2 can efficiently induce membrane deformations after lipid segregation, a mechanism possibly underlying annexin functions in membrane trafficking.

Spatial partitioning improves the reliability of biochemical signaling

Spatial heterogeneity is a hallmark of living systems, even at the molecular scale in individual cells. A key example is the partitioning of membrane-bound proteins via lipid domain formation or cytoskeleton-induced corralling. However, the impact of this spatial heterogeneity on biochemical signaling processes is poorly understood. Here, we demonstrate that partitioning improves the reliability of biochemical signaling. We exactly solve a stochastic model describing a ubiquitous motif in membrane signaling. The solution reveals that partitioning improves signaling reliability via two effects: it moderates the nonlinearity of the switching response, and it reduces noise in the response by suppressing correlations between molecules. An optimal partition size arises from a trade-off between minimizing the number of proteins per partition to improve signaling reliability and ensuring sufficient proteins per partition to maintain signal propagation. The predicted optimal partition size agrees quantitatively with experimentally observed systems. These results persist in spatial simulations with explicit diffusion barriers. Our findings suggest that molecular partitioning is not merely a consequence of the complexity of cellular substructures, but also plays an important functional role in cell signaling.

Influence of Calcium on Lipid Domain Formation in Agarose Supported Lipid Bilayers

Cell membranes are complex entities whose local structure modulates important biological functions. Because of their complexity, it is difficult to observe a simple cause-and-effect relationship regarding just one aspect of cell membrane. using artificial systems, only an intended aspect of cell membrane, such as the formation of cholesterol-stabilized nanodomains can be captured in a significantly simpler system. Recently, it was shown that agarose supported lipid bilayers are decoupled sufficiently from the glass support to allow the formation of cholesterol stabilized nanodomains. In cells, these domains are too small to be directly observed. Hence, we study them using bimFCS, a camera based FCS technique, which measures diffusion of membrane markers over many length scales simultaneously. By tracing fluorescently labeled lipids such as PIP2 and Cholesterol, which interact with domains, bimFCS measures changes in the interaction of the tracers and the domains. This provides information on the stability and the size of the domains. We use the agarose supported model system in conjunction with bimFCS to study the effect of calcium ions on PIP2 containing cholesterol-stabilized nanodomains. It has been proposed that the divalent calcium ions may cross-link the negatively charged head groups and stabilize the domains. We measure the interaction of calcium with PIP2 in simple SOPC bilayers, and study more complex systems of DOPC/SM/Chol/PIP2 to model the effects of PIP2 onto cholesterol-stabilized nanodomains. We perform bimFCS measurements before and after the addition of physiologically relevant concentrations of calcium from 0.1μM to 1.8mM to study the dynamic changes of the domains.

Developing CHARMM-Compatible Lipid Parameters for Ceramides and United Atom Chains

Biological membranes form a barrier to protect the cell from its environment and consist of a wide variety of lipids. In molecular simulations, accurate force field (FF) parameters are crucial for representing biological membranes. Although all-atom FFs, such as CHARMM36 (C36), can now accurately represent a variety of lipid chains (saturated, unsaturated, and branched) and head groups (PC, PE, PG, and PS), parameters for ceramide lipids are lacking. These lipids are common in biology (neuronal and ocular lens membranes) and are involved in lipid domain formation with cholesterol. Quantum mechanical calculations are used to develop torsional profiles and partial charges of the head group. Molecular dynamics (MD) simulations of palmitoylsphingomyelin (PSM) are used to test new parameters in comparison with available deuterium order parameters. The goal of this new lipid parameter set is to obtain the proper phase of PSM as a function of temperature, where previous models have failed. The development of a CHARMM-compatible united atom (UA) chain model will also be presented, referred to here as C36-UA. This is an extension of previous work (Hénin et al. JPC, 112: 7008 (2008)) to the C36 FF. MD simulations of pure and mixed bilayers with cholesterol demonstrate a UA-chain model that is accurate and compatible with the CHARMM family of force fields. MD simulations of micelle formation of short-chain lipids and single-chained detergents are used to demonstrate the ability of C36-UA to self-assemble lipids. MD simulations with C36-UA allow for timescales not approachable with all-atom models without a loss of accuracy, which may have applications to lipid organization and studies with membrane-associated proteins.

Biodegradable polymer-lipid monolayers as templates for calcium phosphate mineralization.

A combination of poly([R]-3-hydroxy-10-undecenoate) (PHUE), a biodegradable polymer from the group of polyhydroxyalkanoates (PHAs), and lipids of different head groups was used to support the growth of calcium phosphate, the main component of mammalian bones. Crystallization took place under two-dimensional films (Langmuir monolayers). The addition of a negatively charged lipid, 1,2-dioleoyl-sn-glycero-3-phospho-l-serine, to a PHUE film led to the formation of lipid domains (rich in negative charge), and resulted in excellent mineralization control: crystals with uniform size and morphology were formed. The results show that carefully optimized combinations of materials can lead to better control of calcium phosphate crystallization compared to one-component organic scaffolds.

Is the fluid mosaic (and the accompanying raft hypothesis) a suitable model to describe fundamental features of biological membranes? What may be missing?

The structure, dynamics, and stability of lipid bilayers are controlled by thermodynamic forces, leading to overall tensionless membranes with a distinct lateral organization and a conspicuous lateral pressure profile. Bilayers are also subject to built-in curvature-stress instabilities that may be released locally or globally in terms of morphological changes leading to the formation of non-lamellar and curved structures. A key controller of the bilayer’s propensity to form curved structures is the average molecular shape of the different lipid molecules. Via the curvature stress, molecular shape mediates a coupling to membrane-protein function and provides a set of physical mechanisms for formation of lipid domains and laterally differentiated regions in the plane of the membrane. Unfortunately, these relevant physical features of membranes are often ignored in the most popular models for biological membranes. Results from a number of experimental and theoretical studies emphasize the significance of these fundamental physical properties and call for a refinement of the fluid mosaic model (and the accompanying raft hypothesis).

Controlling Bilayer Curvature and Membrane Protein Density using Droplet Interface Bilayers

A Droplet Interface Bilayer (DIB) forms when nanolitre aqueous droplets are contacted together in an oil solution in the presence of phospholipids: A lipid monolayer forms at each oil-water interface, and by bringing together two monolayers a bilayer is created.We have recently extended this methodology to modulate both two-dimensional protein concentration[1] and membrane curvature.Manipulation of the axial position of the droplet relative to a hydrogel substrate controls the size of the bilayer formed at the interface; this enables the surface density of integral membrane proteins to be controlled. We are able to modulate the surface density of integral membrane proteins over a range of 4 orders of magnitude within a timeframe of a few seconds. By imaging DIBs on curved substrates we have observed correlation of lipid domain formation with sites of intrinsic curvature. This technique provides a new method for dictating the curvature of artificial bilayers, enabling single molecule measurements on the role of curvature in membrane organization and function.[1] Gross LCM, Castell OK, Wallace MI. Nano Letters (2011) http://dx.doi.org/10.1021/nl201689v

Curvature Driven Membrane Organisation Revealed by Single Molecule Imaging in Droplet Interface Bilayers

Membrane curvature plays a crucial role in lipid and protein sorting within the cell membrane [1] giving rise to spontaneous self-organisation and localisation of complex biological macromolecules. Such spatial organisation in response to, and in generating, membrane curvature contributes to many important biological processes such as neurotransmission, trafficking and cell division [2]. Despite this importance, the majority of in-vitro studies are performed in planar or pseudo-planar (GUV) bilayer systems. As such, there is a need for novel strategies to more closely mimic the curvature of cellular membranes, whilst allowing for correlated measurements of protein function.Here, we create Droplet Interface Bilayers (DIBs) [3] on an underlying curved substrate, resulting in the morphology of the underlying substrate being reflected in the curvature of the bilayer. Total Internal Reflection Fluorescence (TIRF) imaging reveals the correlation of lipid domain formation at sites of intrinsic curvature and the preferential localisation and pore formation by antimicrobial peptides at regions of positive curvature. This technique provides a new method for dictating the curvature of artificial bilayers, enabling single molecule studies on the role of curvature in membrane organisation and function.1. Ramamurthi, K. S. et al. Science 2009 323, 1354.2. McMahon, H. T. et al. Nature 2005 438, 590.3. Thompson, J. R. et al. Nano Letters 2007 7, 3875.

Förster Resonance Energy Transfer (FRET) between Heterogeneously Distributed Probes: Application to Lipid Nanodomains and Pores

The formation of membrane heterogeneities, e.g., lipid domains and pores, leads to a redistribution of donor (D) and acceptor (A) molecules according to their affinity to the structures formed and the remaining bilayer. If such changes sufficiently influence the Förster resonance energy transfer (FRET) efficiency, these changes can be further analyzed in terms of nanodomain/pore size. This paper is a continuation of previous work on this theme. In particular, it is demonstrated how FRET experiments should be planned and how data should be analyzed in order to achieve the best possible resolution. The limiting resolution of domains and pores are discussed simultaneously, in order to enable direct comparison. It appears that choice of suitable donor/acceptor pairs is the most crucial step in the design of experiments. For instance, it is recommended to use DA pairs, which exhibit an increased affinity to pores (i.e., partition coefficients KD,A > 10) for the determination of pore sizes with radii comparable to the Förster radius R0. On the other hand, donors and acceptors exhibiting a high affinity to different phases are better suited for the determination of domain sizes. The experimental setup where donors and acceptors are excluded from the domains/pores should be avoided.

Effect of glucosylceramide on the biophysical properties of fluid membranes

Glucosylceramide (GlcCer), a relevant intermediate in the pathways of glycosphingolipid metabolism, plays key roles in the regulation of cell physiology. The molecular mechanisms by which GlcCer regulates cellular processes are unknown, but might involve changes in membrane biophysical properties and formation of lipid domains. In the present study, fluorescence spectroscopy, confocal microscopy and surface pressure–area (π–A) measurements were used to characterize the effect of GlcCer on the biophysical properties of model membranes. We show that C16:0-GlcCer has a high tendency to segregate into highly ordered gel domains and to increase the order of the fluid phase. Monolayer studies support the aggregation propensity of C16:0-GlcCer. π–A isotherms of single C16:0-GlcCer indicate that bilayer domains, or crystal-like structures, coexist within monolayer domains at the air–water interface. Mixtures with POPC exhibit partial miscibility with expansion of the mean molecular areas relative to the additive behavior of the components. Moreover, C16:0-GlcCer promotes morphological alterations in lipid vesicles leading to formation of flexible tubule-like structures that protrude from the fluid region of the bilayer. These results support the hypothesis that alterations in membrane biophysical properties induced by GlcCer might be involved in its mechanism of action.

Phosphatidylinositol 4,5-Bisphosphate (PtdIns(4,5)P2) Specifically Induces Membrane Penetration and Deformation by Bin/Amphiphysin/Rvs (BAR) Domains

Cellular proteins containing Bin/amphiphysin/Rvs (BAR) domains play a key role in clathrin-mediated endocytosis. Despite extensive structural and functional studies of BAR domains, it is still unknown how exactly these domains interact with the plasma membrane containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) and whether they function by a universal mechanism or by different mechanisms. Here we report that PtdIns(4,5)P(2) specifically induces partial membrane penetration of the N-terminal amphiphilic α-helix (H(0)) of two representative N-BAR domains from Drosophila amphiphysin (dAmp-BAR) and rat endophilin A1 (EndoA1-BAR). Our quantitative fluorescence imaging analysis shows that PtdIns(4,5)P(2)-dependent membrane penetration of H(0) is important for self-association of membrane-bound dAmp-BAR and EndoA1-BAR and their membrane deformation activity. EndoA1-BAR behaves differently from dAmp-BAR because the former has an additional amphiphilic α-helix that penetrates the membrane in a PtdIns(4,5)P(2)-independent manner. Depletion of PtdIns(4,5)P(2) from the plasma membrane of HEK293 cells abrogated the membrane deforming activity of EndoA1-BAR and dAmp-BAR. Collectively, these studies suggest that the local PtdIns(4,5)P(2) concentration in the plasma membrane may regulate the membrane interaction and deformation by N-BAR domain-containing proteins during clathrin-mediated endocytosis.

Scattering techniques in biology—Marking the contributions to the field from Peter Laggner on the occasion of his 68th birthday

This special issue of the European Biophysics Journal marks the contributions of Peter Laggner to molecular biophysics and X-ray and neutron scattering techniques on the occasion of his 68th birthday. Actually, Peter Laggner is not a person who likes to dwell in the past, or talk of the good old days. ‘‘Why look back, if one can look forward?’’, is one of his most popular quotes. Well, we hope that he will forgive us on this one occasion. So, let us look back and recall some of the past from a rich life with many things to remember, but, to make it bearable, we will use an element of humor. Born in Piberbach, Upper Austria, Peter went to Graz to study chemistry and physics. During his PhD, he started with his so successful combination of small-angle X-ray scattering (SAXS), which he learned from the Austrian pioneer in SAXS, Otto Krakty, and biophysics, taught to him by Anton Holasek, then head of the Medical Biochemistry Institute, University of Graz. For his thesis on ‘‘Structure of Antigen Antibody Complexes in Solution by SAXS’’ Peter had an urgent need for blood. Therefore, this part of his career truly was bloody. Using his own car he would go to the slaughterhouse to fetch swine blood in buckets and then drive back to his laboratory. Occasionally blood spilled over. Who cares? In the laboratory the tedious job of precipitation and centrifugation awaited him, accompanied by anxious moments when the rotor lifted and the centrifuge started to move around. By dawn his eyes were blood-shot, but the proteins were prepared and ‘‘real science’’ could start. Soon afterwards lipoproteins—high-density (HDL) and low-density lipoproteins (LDL)—caught Peter’s scientific interest. Again, blood had to be isolated, and trouble started from the beginning. This time the blood had to be of human origin. Thus, healthy normolipidemic volunteers were required. By chance, PhD students were ‘‘lucky’’ to act as donors for Peter’s blood pool, but the most fascinating thing was, however, to use one’s own blood, as he always told us. Later, lipoprotein subspecies came in from John Chapman in Paris, making life much easier. However, whenever whole blood was harvested in our laboratory, Peter was among the first to put his name on the list of donors, eager to support our common efforts to solve the structure of LDL. One day, early in the morning, Peter arrived at the laboratory with a bottle of warm blood in his hands and laughed, ‘‘Hey girls, I have been at the doctor and thought I could bring you some fresh blood!’’, remembers Ruth Prassl. Peter’s pioneering work on lipoproteins has significantly shaped our current understanding of lipoprotein structure and dynamics. In addition to his research focus on LDL, Peter devoted a large part of his endeavor towards understanding complex biological membranes. Early on, he considered the membrane as a dynamic entity with spatial and temporal fluctuations in its local composition. So, he initiated structural studies on lipid polymorphism and domain formation, long before the term ‘‘membrane rafts’’ became popular. A particularly important study concerns the demonstration of chain interdigitated bilayers in ether-chain lipid membranes. He also pioneered lipid interactions with membrane-active peptides, showing that melittin from bee venom acts on membrane collective properties at concentrations as low as 1/1,000 (peptide/lipid molar ratio). G. Pabst (&) R. Prassl H. Amenitsch M. Rappolt K. Lohner Institute of Biophysics and Nanosystems Research, 8042 Graz, Austria e-mail: georg.pabst@oeaw.ac.at

Dynamic clustering of lipids in hydrated two-component membranes: results of computer modeling and putative biological impact

Delineation and analysis of lateral clustering of lipids in model bilayers is an important step toward understanding of the physical processes underlying formation of lipid domains and rafts in cell membranes. Computer modeling methods represent a powerful tool to address the problem since they can detect clusters of only few lipid molecules – this issue still resists easy characterization with modern experimental techniques. In this work, we propose a computational method to detect and analyze parts of membrane with different packing densities and hydrogen bonding patterns. A series of one- and two-component fluid systems containing lipids with the same polar heads and different acyl chains, dioleoylphosphatidylcholine (18:1) and dipalmitoylphosphatidylcholine (16:0), or with same acyl chains and different polar heads, dioleoylphosphatidylserine (18:1) and dioleoylphosphatidylcholine (18:1), were studied via molecular dynamics simulations. Four criteria of clustering were considered. It was shown that the water–lipid interface of biomembranes represents a highly dynamic and “mosaic” picture, whose parameters depend on the bilayer composition. Some systems (e.g. with 20–30% of the anionic lipid) demonstrate unusual clustering properties and demand further investigation at molecular level. Lateral microheterogeneities in fluid lipid bilayers seem to be among the most important factors determining the nature of the membrane–water interface in a cell.

A comparison of the behavior of cholesterol, 7-dehydrocholesterol and ergosterol in phospholipid membranes

A molecular description of the effect of incorporation of cholesterol (CHOL), 7-dehydrocholesterol (7DHC) and ergosterol (ERGO) on the structure of DPPC or EggPC liposomes is provided. Data obtained from ATR-IR spectroscopy, detergent solubility and zeta potential measurements show that the insertion of the various sterols alters the packing arrangement of the tails and headgroup of the PC lipids and may lead to lipid domain formation. On a molecular basis, the differences in lipid packing architecture are traced to differences between the ring and tail structure of the three sterols and these differences in structure produce different effects in DPPC liposomes in the gel phase and EggPC liposomes in the fluid phase. Specifically, CHOL has a relatively flat and linear structure and among the three sterols, shows the strongest molecular interactions with DPPC and EggPC lipids. An extra double bond in the fused ring of 7DHC hinders a tightly packing arrangement with DPPC lipids and leads to less domain formation than CHOL whereas 7DHC clearly produces more lipid domain formation in EggPC. ERGO produces similar structural changes to 7DHC in the tail and headgroup region of DPPC. Nevertheless, ERGO incorporation into DPPC liposomes produces more domain formation than 7DHC.