© The Authors Journal compilation © 2015 Biochemical Society Essays Biochem. (2015) 57, 1–19: doi: 10.1042/BSE0570001

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Lipid domains in model membranes: a brief historical perspective Ole G. Mouritsen*1 and Luis A. Bagatolli† *MEMPHYS-Center for Biomembrane Physics, Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark, DK-5230 Odense M, Denmark †Membrane Biophysics and Biophotonics Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark

Abstract All biological membranes consist of a complex composite of macromolecules and macromolecular assemblies, of which the fluid lipid-bilayer component is a core element with regard to cell encapsulation and barrier properties. The fluid lipid bilayer also supports the functional machinery of receptors, channels and pumps that are associated with the membrane. This bilayer is stabilized by weak physical and colloidal forces, and its nature is that of a self-assembled system of amphiphiles in water. Being only approximately 5 nm in thickness and still encapsulating a cell that is three orders of magnitude larger in diameter, the lipid bilayer as a material has very unusual physical properties, both in terms of structure and dynamics. Although the lipid bilayer is a fluid, it has a distinct and structured trans-bilayer profile, and in the plane of the bilayer the various molecular components, viz different lipid species and membrane proteins, have the capacity to organize laterally in terms of differentiated domains on different length and time scales. These elements of small-scale structure and order are crucial for the functioning of the membrane. It has turned out to be difficult to quantitatively study the small-scale structure of biological membranes. A major part of the insight into membrane micro- and nano-domains and the concepts used to describe them have hence come from studies of simple lipid bilayers as models of membranes, by use of a wide range of theoretical, experimental and simulational approaches. Many questions remain to be answered as to which extent the result from model studies can carry over to real biological membranes.

To whom correspondence should be addressed (email [email protected]).

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Keywords: cholesterol, correlation length, fluorescence microscopy, lateral structure, lipid bilayer, lipid– protein interaction, membrane, non-equilibrium, raft.

Introduction: models of membranes The power of models as frameworks for progress, experimentally, theoretically and computationally, cannot be underestimated in science in general and in molecular life sciences in particular [1]. Models help to shape our intuition, they make quantitative predictions possible, they guide experiments and the subsequent analysis and interpretation of the data, and they make it possible to communicate our understanding about complex phenomena. Models have their virtues, but also their drawbacks. If one becomes too preoccupied with a particular model or use a model beyond its sphere of application, a model can severely hamper progress in the field. The history of membrane modelling bears witnesses to these facts [2,3]. The modern history of membrane models started in 1925 when Gorter and Grendel [4] discovered, when extracting the lipids from red blood cells and spreading them on an air/water interface, that the lipids formed a monomolecular film with an area that was approximately two times the surface area of the blood cells. Although there was some fortuitous cancellation of errors in this work, this first indication of the biological membrane being a bimolecular structure has become the cornerstone of all subsequent membrane modelling [5]. Later, other workers introduced proteins in the bilayer model, for example, as a spread on the lipid polar head groups on the two sides of the bilayer [6] or as a stratified layer sandwiching the lipid bilayer [7]. The first tangible and direct proof of the bimolecular nature of lipid membranes came in 1964, when Bangham and Horne [8], using electron microscopy, found that phospholipids organize in closed bilayer structures, that is, bags, first called bangasomes and later liposomes. Liposomes have since become one of the most popular model systems of membranes [9]. In 1972, Singer and Nicolson [10] proposed the fluid-mosaic model that still stands as the membrane model (Figure 1a). In this model, proteins are associated with the membrane as either integral, traversing entities or being peripherally attached to the fluid bilayer, exposing the bilayer as a kind of mosaic structure. The notion of fluidity and the absence of long-range order are the most important concepts in the Singer–Nicolson model. Although it was explicitly stated in the 1972 paper that “the absence of long-range order should not be taken to imply an absence of short-range order in the membrane,” many workers have interpreted the fluidity condition as being equivalent with the lack of lateral order in the bilayer matrix, assuming the molecular components of the membrane to be either randomly organized or even homogeneously distributed. This and other relevant details have been discussed in a recent review by Garth Nicolson where the fluid-mosaic model is revisited [11]. As we shall come back to shortly, despite the fluidity condition, fluid bilayers do have elements of local order and lateral structure. A number of refinements of the fluid-mosaic models have been put forward, for example, the model by Israelachvili [12] (Figure 1b), which introduced elements of curvature and aspects of lipid–protein interactions. Simultaneously Gebhardt et al. [13] emphasized the relation between domain structure and local curvature in lipid bilayers and biological membranes. The impact of lipid–protein interactions on membrane structure were considered in a © The Authors Journal compilation © 2015 Biochemical Society

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Figure 1. Schematic illustrations of some membrane models (a) The Singer–Nicolson fluid-mosaic model [10]. (b) The Israelachvili model [12]. (c) The mattress model [17]. (d) The raft model [20]. Courtesy of Dr Kai Simons. (e) The picket-fence model [28]. Courtesy of Dr Aki Kusumi.

landmark paper by Marčelja [14] and later reviewed in a classic paper by Sackman [15]. More complex membrane models were devised to highlight the importance of the cytoskeleton and the glycocalyx [16]. The importance of hydrophobic matching between integral membrane proteins and the thickness of the lipid bilayer was considered in the so-called mattress model [17] (Figure 1c). This model proposed matching to provide a driving force for lateral organization of lipids and proteins [18]. The current models of biological membranes are to a large extent biased by the proposal by Simons and van Meer [19], and later developed by Simons and Ikonen [20], of the existence of certain small-scale domains, called ‘rafts’, in live cell membranes (Figure 1d). These authors were largely influenced by previous work proposing that lipid compositional heterogeneity may play a role in the modulation of relevant physical properties of natural membranes. Lipid lateral segregation, which might arise under particular conditions plausibly found in physiological states, would be one of these [13,21]. Furthermore, membrane regions induced by lipid–protein interactions were also proposed as a physical basis for membrane-mediated processes [14,16,17]. These and other questions as well as different theoretical possibilities were addressed by various researchers on several occasions [22–25]. Although these rafts originally were defined operationally by ice-cold detergent-extractionresistant fractionation, a method that later was pointed out to lead to artifacts [26], the proposal of rafts literally started a ‘raft-rush.’ The rafts were surmised to be some kind of platform © 2015 Biochemical Society

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supporting a range of biological functions by being differentiated from the bulk of the membrane. Cholesterol and sphingolipids were claimed to be enriched in these rafts, leading to a local membrane structure that helps to segregate proteins such as glycosyl-phosphatidylinositolanchored proteins. The rafts are now believed, for example, to play a role in membrane-protein sorting and the formation of signalling complexes [27]. Among several other models accounting for compartmentalization of membranes, special mention should be made of the picket-fence model by Kusumi and colleagues (Figure 1E), which proposes a partitioning of the membrane driven by the membrane-associated actin-skeleton and transmembrane proteins anchored to this skeleton [28]. The picket-fence model has been called into question by some workers, for example with reference to problems related to imaging of three-dimensional motion on two-dimensional planes [29]. Ever since the raft proposal it has been an issue first to prove the existence of rafts in cell membranes, and then to characterize them in terms of size distribution and lifetime. Currently there is no clear consensus, and it has turned out that as new experimental techniques emerge with better resolution in space and time, researchers find smaller and smaller structures that live for shorter and shorter times [30]. It has been like opening Pandora’s box. As highlighted in Figure 1(D), a raft can be anything from a small transient cluster of molecules, an aggregate of more long-lived nature, to a large domain that resembles a thermodynamic phase. The proposal of rafts in membranes has on the one hand led to a lot of activity, but on the other hand the many different uses of the term without a firm scientific basis has somewhat discredited the field. In the present authors’ opinion, this situation is to some extent caused by a misinterpretation of the fluidity concept in the Singer–Nicolson fluid-mosaic model. Many workers have read and interpreted the picture in Figure 1(a) as though that there is no local and small-scale structure in the lipid-bilayer component of the membrane despite the warning in Singer and Nicolson’s original paper [10]. However, as we shall describe below, one would in fact expect, based on fundamental reasoning, that such small-scale structures, often of dynamic nature, do exist in all interacting many-body systems, and it should come as no surprise that membranes also possess such structures. The challenge is of course to find ways of measuring them in space and time and link these structures to biological function and regulation.

Lipids move centre stage The lesson from fundamental studies of liquids and liquid crystals is that the phases of such systems, although inherently dominated by long-range disorder, has distinct elements of order on small length scales. The molecular interactions lead by default to correlations in space and time that are best described by a general set of spatial correlation functions gAB(rij,t) =  −   for pairs of molecules, A and B, displaced by a distance rij. In the general case, the correlation function decays exponentially, gAB(rij) ~ exp[−rij/ξ], by a characteristic correlation (or coherence) length, ξ, that is a function of temperature and composition. ξ is the proper measure of the size of a domain [31]. In principle, the correlation function and ξ can be extracted from in-plane scattering experiments (e.g. neutron or X-ray scattering), but performing such experiments are not easy. We shall return to this below in our description of cholesterol-induced ordering of fluid lipid bilayers. The correlation function describes how order arises locally out of global disorder in a fluid system. The correlation function also describes correlations in fluctuations, for example, © The Authors Journal compilation © 2015 Biochemical Society

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fluctuations in local density (e.g. membrane area per molecule) and fluctuations in local composition that may be very different from the global composition. Close to critical points, the correlation length ξ can become very large, indicating that the system is correlated over large length scales. This fact points to a particular, often overlooked, aspect of lipid behaviour in membranes [1]. Owing to lipid interactions, direct as well as indirect, the lipid bilayer membrane displays co-operative behaviour leading to macroscopic thermodynamic phases, phase transitions and co-operative fluctuations that manifest themselves in small-scale domain formation [32] The co-operative behaviour of the lipid bilayer implies that even in the fluid state it will neither display homogeneous nor random behaviour. It will have to possess some kind of local structure and ordering. The physical chemistry of lipids is well researched and most of the fundamental work was performed during the 1960s to 1980s. Unfortunately, a lot of this work seems to be forgotten or remains unknown to many life scientists. The renewed interest in lipids and the increased understanding of the importance of lipids in biology [33] has brought lipids back to centre stage [34,35]. A monumental testimony to decades of careful and quantitative studies of lipids and lipid bilayers can be found in the new edition of Handbook of Lipid Bilayers [36].

Models of lipid membranes A range of simple experimental and theoretical models is being used to study general behaviour of lipid bilayers, including domain formation and small-scale structure. In the present chapter we focus on experimental models, some of which are illustrated in Figure 2. Multilamellar and unilamellar vesicles (Figure 2a) are well-characterized membrane models that can be composed of one- or many-component-lipid mixtures, possibly reconstituted with membrane proteins. GUVs (giant unilamellar vesicles) are of particular advantage for studying lipid domains on length scales above 300 nm using light microscopy, for example, fluorescence microscopy involving particular lipid-analogue probes [37]. Whereas GUVs represent free-standing bilayers, lipid bilayers on solid supports can be influenced by the proximity of the support, which may affect domain formation. Supported bilayers, however, enable the application of techniques that allow smaller-scale structures to be imaged, for example, by using AFM [38,39], which provides lateral resolution below the diffraction limit of light. By employing techniques involving double-supported bilayers, free-standing membranes can be modelled by the top bilayer (Figure 2b). Recently, protocols for fixating the unperturbed structure of GUVs by exploding them on solid supports have been devised [40], which may possibly open up a new avenue in studying small-scale structures in lipid bilayers by microscopy. Solid-supported bilayers also lend themselves to structural analysis by scattering techniques [41].

Macro-, meso- and nano-domains in lipid bilayers In Figure 3 are shown schematically some possibilities for large- and small-scale structuring and domain formation in lipid bilayers. Being a liquid crystal, a lipid bilayer has at least two fundamental degrees of freedom that need to be taken into account when describing its lateral © 2015 Biochemical Society

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Figure 2. ​Schematic illustrations and microscopy images of lipid-bilayer systems that are simple models of membranes (a) Upper panels: MLVs (multilamellar vesicles) and unilamellar liposomes and vesicles of different sizes [SUV (small unilamellar vesicle), ~30 nm diameter; LUV (large unilamellar vesicle), ~100 nm diameter; and GUV (giant unilamellar vesicle) of diameters between 5 and 100 µm]. Lower panels: The larger of these structures can be observed using fluorescence imaging (MLVs and GUVs). Note that, of the unilamellar vesicles, only GUVs can be observed using light microscopy, because SUVs and LUVs have dimensions below the resolution limit of light, which is approximately 300 nm radial. (b) Upper panel: sketch of single- and double-supported planar membrane bilayers on mica (an atomically flat substrate) with the corresponding fluorescent image shown above for a bilayer made of DOPC imaged by the fluorescent dye DiIC18. The lower panel shows a typical example of a membrane domain observed by AFM in a planar membrane composed of a 1:1 DOPC/DPPC mixture [101]. Below the AFM image is shown a height contour corresponding to the scanned line shown in the AFM image. The scan shows that the domains of the solid phase (light) are thicker that the surrounding membrane in the liquid-disordered phase [39]. Courtesy of Dr Adam C. Simonsen.

Figure 3. ​Schematic illustration of the different possibilities for lateral organization and lateral heterogeneity in different lipid-bilayer phases (a) Single equilibrium macroscopic phase with small-scale fluctuating domains of another structure/composition (phase). (b) Same as (a) but inverted. (c) Equilibrium macroscopic phase separation between two phases, each of which harbours small-scale fluctuating domains of the other phase. © The Authors Journal compilation © 2015 Biochemical Society

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structure in terms of phase transitions and phase equilibria: a translational (or positional) degree of freedom and an orientational (or internal related to the acyl chains) degree of freedom. The co-operativity in the coupling of these degrees of freedom leads to phase transitions, of which the so-called main phase transition [42] is of particular importance for lipid-domain formation. For a one-component lipid bilayer, the main transition separates a phase that is ordered both in terms of translational degrees of freedom and internal degrees of freedom (the solid-ordered phase, or gel phase) from a phase that is disordered in terms of both degrees of freedom (the liquid-disordered phase, or fluid phase). For lipid bilayers with two or more different lipid components, more complex phase behaviour develops with coexisting phases described by thermodynamic phase diagrams. In the particular case of bilayers with cholesterol, which we shall address below, a new intermediate phase, called liquid-ordered phase, is induced [43,44]. A thermodynamic phase in equilibrium refers to a macroscopic state of matter that, however, need not be random or homogeneous. Owing to the underlying phase transitions and phase equilibria of lipid bilayers [1], the phase can harbour small-scale structures, that is, domains of different density (for one-component systems) or different density/composition (for bilayers with two or more components). These phenomena, which have also been referred to as dynamic lateral heterogeneity [45], are illustrated schematically in Figure 3. The smallscale structures can in principle have sizes stretching from the nanoscale to the micro- and meso-scale. In equilibrium, the correlation length controls the extent of these structures. If the system is not in equilibrium, for example, subsequent to a change in thermodynamic conditions such as temperature [46], or under the influence of external constraints (e.g. curvature and finite-size effects) [47,48], complete phase separation may not develop, and the concept of thermodynamic phases and phase diagrams becomes murky.

Domains in one-, two- and several-component lipid bilayers Since the 1970s, major research efforts have been presented to elucidate lipid domains in model membranes. In particular, experimental studies of phase transitions and phase coexistence in artificial lipid mixtures are numerous and have involved a wide range of techniques, such as fluorescence spectroscopy [49–54], infrared spectroscopy [55], electron-spin-resonance spectroscopy [56], nuclear magnetic resonance spectroscopy [57,58], differential-scanning calorimetry [54,59–62], X-ray techniques [63–65] and neutron scattering [64] to mention a few. All this information lack direct spatially resolved information, something that can be circumvented with strategies involving imaging techniques. Some of the first fluorescence microscopy studies of lateral structure in free-standing membranes are those by Haverstick and Glaser [66] who reported the first visualization of lipid domains in GUVs. These authors directly visualized Ca2+-induced lipid domains, at constant temperature, in GUVs composed of mixtures of unsaturated diacyl PCs (phosphatidylcholines) and diacyl PEs (phosphatidylethanolamines) mixed with acidic phospholipids [diacyl PA (phosphatidic acid)], as well as GUVs composed of natural lipids from erythrocyte membranes and erythrocyte ghosts [67]. Nevertheless, it was not until 1999–2001 that seminal papers appeared in the literature using fluorescence microscopy techniques, in particular © 2015 Biochemical Society

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two-photon excitation fluorescence microscopy or laser-scanning confocal fluorescence microscopy combined with FCS (fluorescence correlation spectroscopy) [68] to show spatially resolved information of lipid-induced lateral heterogeneity in GUVs composed of different one-component systems, binary mixtures, and ternary lipid mixtures containing phospholipids, sphingolipids, and cholesterol at different temperatures and composition [69,70]. These papers showed for the first time images of different micrometre-sized lipid domains in bilayers, including dynamical information about the coexisting membrane regions. The presence of distinct membrane domains was connected with coexistence of equilibrium thermodynamic phases, that is, solid-ordered/liquid-disordered or liquid-ordered/ liquid-disordered phases. Particular features of these domains were reported to be their shape (e.g. elongated, flower shaped, snowflake-like and circular), and their size was found to depend on lipid composition and temperature. In all cases the domains were found to span the lipid bilayer [37]. This observation cannot be generalized to all types of lipid-bilayer systems, since it has been shown that lipid domains in supported planar membranes can be decoupled, that is, domains can form independently in the two leaflets of the bilayer due to the interaction with the substrate [71,72]. Several studies exploring and characterizing domain coexistence in different lipid mixtures were reported later using similar approaches, particularly using wide-field fluorescence microscopy as an alternative technique [73]. Most of these studies explored ‘canonical’ raft mixtures, generally composed of unsaturated phospholipids, single or natural mixtures of sphingomyelin, and cholesterol [although in some cases sphingomyelin was replaced by DPPC (dipalmitoylphosphatidylcholine)] [74]. In addition to revealing lateral membrane domains, the experimental data from fluorescence microscopy was shown to allow the construction of phase diagrams from the visual appearance of the domains [73]. Moreover, the partitioning of particular membrane proteins (e.g. those relevant to the raft hypothesis) into membranes displaying liquid-ordered/liquid-disordered phase coexistence has been also explored using GUVs [75]. An illustration of phase separation observed by multiphoton excitation fluorescence microscopy applied to a GUV composed of two different lipids is provided in Figure 4. The figure shows the thermodynamic phase diagram and the different lateral structures that develop at representative points across the binary phase diagram. It is seen that the solid-ordered phase establishes itself with progressively larger domains as the temperature is lowered below the liquidus line. Owing to curvature constraints, the solid phase below the solidus line does not fill the entire surface of the GUV. Figure 5 shows a gallery of GUVs of different compositions illustrating some of the variation in lateral structure of multi-component membranes. A comparison is made below between an artificial lipid mixture containing cholesterol and a natural extract of lung surfactants [76]. Pulmonary surfactant is mainly composed of phospholipids and small amounts of specifically associated proteins (SP-B and SP-C). Among the phospholipids, significant amounts of DPPC and phosphatidylglycerol are present, both of which are unusual species in most animal membranes. Mono-unsaturated PCs, phosphatidylinositol and neutral lipids including cholesterol are also present in varying proportions. The comparison in Figure 5 indicates that the lateral structure in lung-surfactant membranes is likely to be controlled by the lipids and that cholesterol is a very important regulator. Upon extraction of cholesterol, the domain pattern is changed completely as shown in Figures 5(c) and 5(d). © The Authors Journal compilation © 2015 Biochemical Society

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Figure 4. ​Experimental phase diagram for a two-component lipid-bilayer membrane composed of mixtures of DPPC and DPPE Along the vertical line are indicated different temperature points across three different phase regimes (liquid-disordered, liquid-disordered/solid-ordered coexistence, and solid-ordered). The fluorescence images corresponding to the five different points are shown to the left [69]. The fluorescent probe is rhodamine-DPPE (dipalmitoylphosphatidylethanolamine) and the images were acquired using multiphoton excitation fluorescence microscopy.

Light microscopy does not provide direct information about the size of lipid domains below the diffraction limit. However, indirect information about physical features of smaller domains in the nanometre range can be obtained by, for example, fluorescence spectroscopy [77,78], FCS [79] or stimulated emission depletion FCS [80].

The peculiar case of cholesterol-induced order Cholesterol plays a particular role in the lateral ordering of biological membranes. All plasma membranes of eukaryotes contain large amounts of higher sterols, approximately 20–30 mol%; cholesterol in animals, ergosterol in fungi, and various phytosterols in plants and algae. Hence, in the case of animals, cholesterol is the single most abundant molecule in plasma membranes. The phase behaviour of cholesterol in membranes has turned out to be a particularly elusive problem [44] and only in a few cases are the phase diagrams known with some certainty. It was suggested in 1987 that cholesterol modulates the phase behaviour of lipid bilayers by inducing an intermediate phase, the so-called liquid-ordered phase that is translationally disordered, but has a high degree of acyl-chain order [43]. The nature of the liquid-ordered phase and in particular of small domains with aspects of liquid-ordered nature is difficult to assess and would in most cases require use of, for example, NMR techniques [81]. Whereas several ternary [82] and even quaternary [83] phase diagrams involving cholesterol have been mapped out, the case of binary phospholipid-cholesterol mixtures © 2015 Biochemical Society

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Figure 5. F ​ luorescence microscopy images of selected GUVs made of artificial ternary mixtures compared with natural pulmonary surfactant extracts from pig lung (a) 2:2:1 DOPC/DPPC/cholesterol mixture displaying liquid-ordered/liquid-disordered coexistence and a domain structure that is similar to the one in (b) for a mixture of native lipids and associated proteins (SP-A and SP-B). (c) and (d) Images corresponding to the systems in (a) and (b), respectively, but after cholesterol extraction by methyl-β-cyclodextrin. The membranes are labelled with DiIC18 (red) and BODIPY-PC (green). The yellow colour in the liquid-disordered-like domains is due the presence of DiIC18, which is preferentially labelled by the green probe BODIPY-PC [75]. BODIPY, boron-dipyrromethene.

is still in dispute [44], in particular with respect to the existence of a liquid-disordered/­ liquid-ordered phase coexistence region. Figure 6(a) shows a generic version of the phase diagram for lipid-cholesterol mixtures, indicating the existence of a putative region of liquid-disordered/liquid-ordered phase coexistence. The liquid-ordered phase and the proximity of this phase to the coexistence region is now believed to be of importance to the functioning of cell membranes, and the whole raft hypothesis is built on the existence of small-scale structures in which cholesterol is enriched, leading to a local environment that resembles the liquid-ordered phase [84]. Recent progress in applying neutron diffraction to solid-supported lipid-cholesterol bilayers have now provided hard evidence for the existence of small, dynamic domains of ordered domains enriched in cholesterol (up to 66 mol%) on length scales of approximately 10 nm and of lifetimes in the range of up to approximately 100 ns (Figure 6b) [85]. These findings are in accordance with early minimal-model Monte Carlo simulations (Figure 6c) [86] and recent Molecular Dynamics calculations (Figure 6d) [87]. © The Authors Journal compilation © 2015 Biochemical Society

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Figure 6. ​Cholesterol in lipid bilayers (a) Generic phase diagram for a binary mixture of cholesterol and phospholipids. (b) Smallscale structure in the liquid-ordered phase as obtained from in-plane diffraction [85]. Courtesy of Dr Maikel C. Rhainstädter. (c) Local structure of cholesterol (solid circles) in the liquidordered phase (disordered lipid chain shown as an open circle, ordered lipid chains shown as  +) as obtained from minimal-model Monte Carlo simulations [86]. (d) Liquid-ordered domains enriched in cholesterol (shown in green) as obtained from coarse-grained Molecular Dynamics simulations [87]. Courtesy of Dr Friederike Schmid.

Domains induced by lipid–protein interactions The schematic illustration of the raft phenomenology in Figure 1(d) anticipates that domains or rafts enriched in cholesterol will display larger membrane thickness than that of the surrounding membrane because of cholesterol’s unique ability to order lipid-acyl chains. The local thickening of the membrane puts mechanical constraints on the integral membrane proteins via the hydrophobic matching condition [17], leading to ways of regulating function, for example, by changing internal protein conformational states, by altering mode of protein acylation, or by modifying the local lipid environment around the protein (Figure 7a). It was already anticipated in a classical paper by Sackmann [16] that such phenomena could support triggering mechanisms, some of which have been extensively studied by Andersen and colleagues using gramicidin A as a mechanical transducer to measure quantitatively the effects of the lipid–protein interactions in model as well as biological membranes [88]. With due consideration of hydrophobic matching, small-scale lipid domains and the associated correlation length present themselves as unique features of membrane organization and become a clear manifestation of lipid–protein interactions. On the one side, specialized lipid domains may aid to segregate proteins and provide for indirect lipid-mediated protein– protein interactions. On the other side, proteins may ‘harvest’ virtual lipid domains, characterized by the correlation function, and nucleate and stabilize these, for example, in the form of a capillary condensate or wetting layer (Figure 7b) [89]. The longer the coherence length of the lipid fluctuations, the larger the region over which proteins can influence each other via the lipid-mediated, indirect protein–protein interactions. © 2015 Biochemical Society

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Figure 7. ​Schematic illustrations of lateral organization of lipid bilayers with embedded transmembrane proteins (a) Hydrophobic matching between hydrophobic bilayer thickness and hydrophobic length of transmembrane protein. (b) Capillary condensation of a lipid domain between two transmembrane proteins. (c) Computer simulation images of a phase-separated binary membrane (red and green phase) with transmembrane proteins (yellow and black dots) in the absence of a drive (left panel) and in the presence of drives of increasing intensity (middle and right panels) leading to a steady-state non-equilibrium restructuring of the membrane into dynamic domains [90].

Non-equilibrium membranes Almost all studies of model membranes have been carried out under equilibrium or near-equilibrium conditions, and membrane phase behaviour is conventionally interpreted in terms of equilibrium thermodynamics. Often, however, the model membranes are not in true equilibrium, but rather in a kind of kinetically constrained metastable state. An example of this is the observation of finite-size domains in membranes that in principle should develop into macroscopic phase separation (Figure 4). Still, membranes in live cells are active and either in a steady-state or far-from-equilibrium situation. The study of small-scale structures and lipid domains in model membranes, for example, those that are being quenched in temperature and undergoing a process of restructuring [46] or those that are driven out of equilibrium due to the functioning of membrane-bound proteins and other active membrane processes [89–93], is still in its infancy. Measurements of the mechanical properties of GUVs with incorporated active transmembrane proteins, for example, Na+,K+-ATPase [94], indicate strong perturbation of the bilayer due to the active proteins, leading to softening and long-time correlations in the fluctuation spectrum of the thermal GUV undulations. These findings may indirectly suggest changes of bilayer structure and the presence of domain formation, but that remains to be proven. Computer-simulation calculations on simple, phase-separated binary lipid bilayers with transmembrane proteins that perform a function implying a time-dependent variation in the hydrophobic matching condition [90] have shown that lipid domains become modulated when the protein activity is turned on, and that the characteristic domain size is directly controlled by the strength of the drive as illustrated in Figure 7(c). Further studies along these lines may shed more light on the extent to which the putative rafts in cell membranes owe their existence to active proteins that create local order out of a disordered membrane matrix. © The Authors Journal compilation © 2015 Biochemical Society

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Another example of non-equilibrium modulation and remodelling of lipid bilayers in the presence of active proteins is that of phospholipase A2 action that is controlled by the lateral structure of the bilayer substrate [95]. This system is out of equilibrium due to the changing composition of the bilayer substrate incurred by the turnover of diacyl phospholipids into free fatty acids and lysolipids. This condition implies that the system traverses what corresponds to a non-equilibrium path in a ternary phase diagram. On the one side, the activity of the enzyme is controlled by the small-scale structure, and on the other side the enzymatic turnover of lipid species leads to a restructuring of the lateral organization of the bilayer [96]. A similar phenomenon has been described for a rare enzyme from Loxosceles spider venom termed sphingomyelinase D, which transforms sphingomyelin to ceramide 1-phosphate [97].

Biological membranes One of the major challenges is to correlate membrane heterogeneity and lipid-domain formation observed in compositionally simple model membranes (membranes composed of few lipid species) to compositionally complex mixtures (natural lipid extracts or native membranes) [35,98]. Because of the complex composition of natural membranes this correlation is not straightforward if based on data from bulk experimental techniques applied to solutions containing many liposomes (or cells). The spatially resolved information provided by fluorescence microscopy or AFM on membrane models for individual systems (e.g. individual GUVs) is therefore key to examine this type of correlation. Since GUVs can be prepared using both artificial lipid mixtures and innate membranes, correlations among compositionally simple and complex model membranes are feasible. For example, when innate pulmonary surfactant membranes (full composition in this case with respect to lipids and proteins) are compared with a relevant artificial lipid mixture [DOPC (dioleoyl phosphatidylcholine)/DPPC/cholesterol mixture], a similar lateral organization is observed in the two systems, suggesting the presence of liquid-ordered/liquid-disordered-like phase coexistence in the innate material (Figure 5). Additionally, extraction of cholesterol (but not extraction of the membrane proteins) [54] affects the observed membrane lateral pattern in a similar manner to that in the simple model mixture. Studies of the spreading capability of lung surfactant films with and without cholesterol indicates that the mechanical properties of the lung film requires the presence of cholesterol, suggesting that lateral structure and lipid-domain formation may be important for proper lung function [76]. Pulmonary surfactant membranes could well be one of the first membranous systems reported where the coexistence of specialized membrane domains may exist as a structural basis for its function. Similarities between artificial systems and native membranes have been observed in other cases, such as membranes composed of brush border lipid extracts from renal cortical tissue of adult Sprague Dawley rats [70] or in GUVs directly generated from natural plasma membranes, the so-called giant plasma membrane vesicles [98] that display similarities with lateral structures in ternary lipid mixtures containing glycerophospholipid, sphingomyelin and cholesterol. It appears that the presence of key lipid species at particular concentrations in specific biological membranes can trigger phenomena that can be linked to the presence of lateral phase coexistence and lipid-domain formation. However, caution should be exercised in © 2015 Biochemical Society

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generalizing information about the lateral structure of membranes. Biological membranes are adapted to different functions, they have vastly different compositions and they are generally functional under non-equilibrium conditions.

Conclusion Biological membranes are self-assembled many-body systems, and the various molecular interactions among lipids and proteins imply a rich co-operative behaviour on different length and time scales. Lipid-domain formation is therefore the rule rather than the exception. The nature of the domains can be studied by various microscopy techniques, for example, fluorescence microscopy and atomic force microscopy, leading to information from the nanoscale, via the mesoscale, to macroscopic length scales. The lateral structure can conveniently be investigated quantitatively by means of a range of model systems, involving bilayer membranes of different morphologies and compositions. The connection between model membranes and real biological membranes, not least in the context of domain and raft formation, is complex and only incompletely understood [35,99,100]

Summary • • • • •

• •

Lipid bilayer membranes are simple models of real biological membranes. The many-body character of lipid bilayers is a source of lateral organization and domain formation on different length and time scales. Giant unilamellar vesicles and solid-supported bilayers constitute convenient systems to study fundamental aspects of lateral membrane organization. Cholesterol plays a unique role in the formation of lipid domains that are fluid (liquid) but still possess a high degree of order (liquid-ordered structures). Lipid–protein interactions provide a mechanism for membrane domain formation and the formation of differentiated regions in the plane of the membrane. Active proteins and enzymes in membranes lead to restructuring and remodelling of the lateral structure. The connection between the lateral organization of model membranes and that in functional real membranes is still not clear.

The work of the authors is supported in part by the Danish Research Councils [grant number 11-107269].

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Lipid domains in model membranes: a brief historical perspective.

All biological membranes consist of a complex composite of macromolecules and macromolecular assemblies, of which the fluid lipid-bilayer component is...
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