Metabolic Precursor of Cholesterol Causes Formation of Chained

Jan 19, 2016 - ... Bonchev Str., Sofia 1113,. Bulgaria. §. National University of Science and Technology “MISiS”, 4 Leninskiy prospect, Moscow 11...
0 downloads 0 Views 9MB Size
Download PDF
Article pubs.acs.org/Langmuir

Metabolic Precursor of Cholesterol Causes Formation of Chained Aggregates of Liquid-Ordered Domains Galya Staneva,*,‡ Denis S. Osipenko,† Timur R. Galimzyanov,†,§ Konstantin V. Pavlov,† and Sergey A. Akimov*,†,§ †

A.N. Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, 31/4 Leninskiy prospekt, Moscow 119071, Russia ‡ Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, 21 Academic G. Bonchev Str., Sofia 1113, Bulgaria § National University of Science and Technology “MISiS”, 4 Leninskiy prospect, Moscow 119049, Russia ABSTRACT: 7-Dehydrocholesterol, an immediate metabolic predecessor of cholesterol, can accumulate in tissues due to some metabolic abnormalities, causing an array of symptoms known as Smith−Lemli−Opitz syndrome. Enrichment of cellular membranes with 7-dehydrocholesterol interferes with normal cellsignaling processes, which involve interaction between rafts and formation of the so-called signaling platforms. In model membranes, cholesterol-based ordered domains usually merge upon contact. According to our experimental data, ordered domains in the model systems where cholesterol is substituted for 7-dehydrocholesterol never merge on the time scale of the experiment, but clusterize into necklace-like aggregates. We attribute such different dynamical behavior to altered properties of the domain boundary. In the framework of thickness mismatch model, we analyzed changes of interaction energy profiles of two approaching domains caused by substitution of cholesterol by 7-dehydrocholesterol. The energy barrier for domain merger is shown to increase notably, with simultaneous appearance of another distinct local energy minimum. Such energy profile is in perfect qualitative agreement with the experimental observations. The observed change of domain dynamics can impair proper interaction between cellular rafts underlying pathologies associated with deviations in cholesterol metabolism.

■

INTRODUCTION Lateral distribution of lipids in the bilayer membranes containing sterols has been an attractive research subject for decades. Though phase separation with formation of immiscible 2D liquids in the bilayer membrane has been reported for cholesterol-containing binary mixtures,1,2 ternary mixtures (a sterol and two lipids, typically with large differences in carbon chain melting temperatures) have attracted more attention,3−5 especially after the coexistence of phases was directly visualized for the first time in 2001.6,7 Interest in phase separation in lipid membranes was largely stimulated by the concept of “lipid rafts”, according to which colocalization of certain proteins preferring liquid-ordered (Lo) phase is important for their biological function.8,9 In model membranes, raftlike Lo domains could be formed in ternary lipid mixture composed of sphingomyelin (and/or saturated phosphatidylcholine (PC))/unsaturated PC/ cholesterol. Rafts have always been viewed as specific bilayer domains of lipid membranes, an unstated assumption being that the raft boundary on one side of the membrane is a mirror image of that on the other side. However, our recent theoretical analysis,10,11 and analysis of the experimental results reported in the present paper demonstrate that it is not necessarily true. Besides the apparent purely academic interest, this concept © 2016 American Chemical Society

might have far-reaching consequences for our understanding of a broad range of phenomena associated with phase separation in cellular membranes, protein−lipid interactions, and functioning of transmembrane proteins in biological cells. There is increasing bulk of evidence that in vivo such phase separation is crucial for proper function of many membrane proteins, partitioning preferentially into rafts, into nonraft phase, or even preferring to stay near the phase separation boundary.12−15 For example, many receptors have been shown to assemble into so-called signaling platforms, proteo-lipidic formations differing notably from the rest of the membrane in terms of their lipid composition and protein content.12 Cholesterol is a major structural component of detergent-resistant membrane domains, which are known to consist of Lo phase7 and are commonly identified with rafts. The term “raft” refers to a broad spectrum of objects with different chemical and physical properties. What they do have in common is “higher than membrane-average” degree of ordering and thickness,7,16,17 and resistance to detergents.10,17 What can be only hypothesized is that these physicochemical Received: October 29, 2015 Revised: January 15, 2016 Published: January 19, 2016 1591

DOI: 10.1021/acs.langmuir.5b03990 Langmuir 2016, 32, 1591−1600

Article

Langmuir

Figure 1. Chemical structures of cholesterol (A) and 7-dehydrocholesterol (B). The structures differ only in the type of C7−C8 bond.

micrometer-sized, and can therefore be visualized with optical microscope. Their near-circular shape is indicative of significant line tension of the boundary, which drives coalescence of small domains, thus minimizing total length of phase separation boundary.5,6,31 Herein, we report the results concerning behavior and dynamics of Lo domains formed in the presence of either of the two sterols in the model membranes of giant unilamellar vesicles (GUV). Lo phase containing 7DHC appears more rigid than the cholesterol-based phase: the microscopic Lo domains formed in the 7DHC-containing lipid membranes fuse extremely rarely, but tend to cluster, forming “necklaces” of domains staying in contact with each other (Figure 6). The necklace-like aggregates move concertedly along the membrane surface. This implies that 7DHC induces changes in overall physicochemical properties of the membrane, including raft structure, organization and dynamics. The observed formation of necklace-like domain aggregates in 7DHC-containing membranes as opposed to fusion of approaching domains in cholesterol-containing membranes most likely conditioned by different structures of domains boundary in these two systems, resulting in different interaction energy profiles of approaching domains. Lipid bilayer of Lo domains is experimentally shown to be about 0.5−1 nm thicker than the bilayer of the surrounding liquid-disordered (Ld) membrane.16−18 Due to high water−lipid tails surface tension, the thickness mismatch should most likely be compensated by elastic deformations.32−34 The energy of deformations represents elastic contribution to the line tension of the boundary between the raft domain and the surrounding membrane. Recently, having applied the thickness mismatch concept and continuum elasticity theory35 to calculate energy of elastic deformations, we have shown that a certain lateral misalignment of Lo domains in two leaflets of the membrane yields minimal lipid deformation energy.10,11 More precisely, minimum line tension between the thicker (ordered) and the thinner (disordered) domains is achieved when the boundaries of Lo domains in the opposing leaflets are laterally shifted by a few nanometers relative to each other. Such an equilibrium shift of Lo monolayer domain boundaries in bilayer raft was also observed in recent molecular dynamics studies.36−39 Here we consider interaction of bilayer domains composed of two incompletely aligned monolayer domains in membranes of different lipid composition. In our “elastic” approach different lipid compositions are modeled by different elastic properties of Lo and Ld phases: spontaneous curvatures and splay moduli. We apply this theoretical analysis to explain the observed intriguing behavior of Lo phase domains in the membranes containing 7DHC based on fundamental physical principles and hypothesize that it is the change of these physical properties of cellular membranes that underlies the symptoms of medical conditions associated with increased content of 7DHC in tissues.

properties define important biological functions: solubility and folding of membrane proteins, coalescence into signaling platforms, local mechanical and electrical properties, for example, bending rigidity, dielectric permittivity, water penetrability, and so forth. The importance of these islets of liquid-ordered phase for proper functioning of many membrane proteins, and specifically for proper assembly of signaling platforms, has been reported by many authors based on experimental (in vitro and in vivo) findings18−20 and theoretical considerations.21 Defects in cholesterol metabolism are implicated in many diseases of central nervous system, such as Smith−Lemli−Opitz syndrome (SLOS), Neumann−Pick type C disease, and Alzheimer’s disease.22 SLOS is a multiple malformation syndrome due to an inborn error of cholesterol synthesis caused by mutations of 7-dehydrocholesterol reductase (DHCR7) impairing the reduction of 7-dehydrocholesterol (7DHC) to cholesterol in the final step of cholesterol biosynthesis. Phenotypically, SLOS ranges from a mild disorder with behavioral and learning problems or autistic characteristics to a lethal malformation syndrome. Mutations of DHCR7 result in decreased cholesterol and increased dehydrocholesterol levels.23 Both insufficient availability of cholesterol and toxicity of 7DHC can underlie the physiological conditions characterizing the ailment, and there are lines of circumstantial evidence in favor of either of the assumptions. 7DHC is normally present in healthy organisms, serving as a cholesterol precursor in serum and many tissues, and functioning as provitamin-D3 in the skin. It is also found in the milk of several mammalian species.24 Normal cholesterol level does not exclude SLOS, whereas elevated 7DHC in blood or tissues is an ultimate confirmation of the diagnosis. However, SLOS treatment usually involves dietary cholesterol supplementation.25−27 In summary, both low cholesterol and elevated 7DHC appear to factor into the condition referred to as SLOS in a complex and cooperative manner. Mechanistically, it is natural to expect that shortage of a major component of plasma membranes and myelin sheath would affect morphogenesis on the cellular level; on the other hand, high content of 7DHC in cellular membranes was shown to interfere with certain cell signaling pathways.28,29 Thus, behavior of lipid membranes containing 7DHC in high concentrations is of great biological importance. Chemically, the only difference of 7DHC from cholesterol is one extra double bond C7−C8 obstructing rotation of the rings A and B around the C8−C9 bond (Figure 1). From comparison of domain melting temperatures, Megha and London concluded that replacement of cholesterol by 7DHC increases stability of rafts.30 Liquid-ordered domains occurring in artificial lipid membranes of simple composition (typically 3 or 4 lipid components including cholesterol) as a result of phase separation induced by temperature decrease are widely utilized as a convenient model of the Lo domains of multicomponent proteolipid plasma membranes of biological cells.6−8 These model domains are 1592

DOI: 10.1021/acs.langmuir.5b03990 Langmuir 2016, 32, 1591−1600

Article

Langmuir

■

described by director divergence at the neutral surface, div(n). We assume the deformations small and calculate elastic energy in the quadratic approximation on the deformations. Elastic energy of a unit area of the neutral surface can be expressed as35

EXPERIMENTAL SECTION

Materials. Lipids were used without further purification: Egg yolk Lα-phosphatidylcholine (EPC), egg yolk sphingomyelin (ESM), cholesterol, and 7-dehydrocholesterol were obtained from Sigma (Sigma-Aldrich Chimie SARL, St. Quentin, France). Fluorescent phospholipid analogue Texas Red [1,2-dihexadecanoyl-sn-glycero-3phosphoethanolamine, triethylammonium salt (TR-DHPE)] was purchased from Invitrogen (Eugene, OR). Three different molar ratios of lipids in the ternary mixtures of EPC/ESM/sterol (45/45/10, 40/40/ 20, 34/33/33) known to yield phase separation with optically detectable microscopic rafts in vitro40,41 were used. Aqueous solutions were made with 0.5 mM Hepes buffer, (pH = 7.4, conductance σ = 59 μS/cm) procured from Sigma-Aldrich. In order to minimize lipid oxidation, during the preparatory stages, lipids were maintained in the inert atmosphere of nitrogen and protected against exposure to light as much as was reasonably achievable. Dissolved oxygen was purged from hydration buffer by nitrogen. Electroformation and Fluorescence Microscopy of Giant Unilamellar Vesicles. Giant unilamellar vesicles (GUV) were prepared using the electroformation method developed by Angelova and Dimitrov.42 The lipids were dissolved in diethyl ether/chloroform/ methanol (70/20/10 v/v) to achieve 0.5 mg/mL concentration of total lipid. The diameters of platinum electrodes were 0.8 mm and they were positioned parallel to each other at a distance of 3 mm. The solvent was evaporated, and then the electrodes were placed in a temperaturecontrolled quartz chamber, where the lipid layers were hydrated and GUV formation was induced by 10 Hz alternating voltage (amplitude increasing from 100 to 400 mV within 30 min) applied to the electrodes. GUVs were observed using a Zeiss Axiovert 135 microscope equipped with 63× long-working distance objective lens (LD Achroplan Ph2). Images were recorded using Zeiss AxioCam HSm CCD camera connected to an image recording and processing system (Axiovision, Zeiss). The lipid phase separation in GUVs was visualized by means of fluorescent microscopy with the use of Zeiss filter set 15 (Ex/Em = 546/ 590 nm). Protection against Oxidation of Lipid Species. In order to minimize formation of lipid peroxides through electrochemical reactions, Pt electrodes were used and the applied voltages were kept under 400 mV (peak to peak). To minimize photoinduced oxidation during the fluorescence imaging experiments, we followed recommendations of the classical papers, refs 43−45, for example, illumination by low intensity light, low dye concentrations, use of neutral density filters of up to OD = 1.0, and minimized exposure to light during all preparatory manipulations and imaging. Fluorescence imaging microscopy experiments were made with the image acquisition times under illumination ranging from 0.3 to 0.6 s and repetition times in the dark of 2 s. Image acquisition was started immediately from the beginning of illumination to avoid photobleaching and photosensitization.

■

w=

B B K (div(n) + J0 )2 − J02 + t2 2 2 2

(1)

where B is splay modulus, K is tilt modulus, and J0 is monolayer spontaneous curvature. We assume monolayers to be locally volumetrically incompressible. This assumption is justified by the large value of the bulk compression modulus. Condition of volumetric incompressibility can be written as35 h = h0 −

h02 div(n) 2

(2)

where h is the current monolayer thickness and h0 is the unstrained monolayer thickness. Phase separation can occur in each monolayer individually, and a “raft” can be viewed as consisting of two juxtaposed monolayer domains of Lo phase. We have recently demonstrated that the energy minimum is achieved when the domains forming a raft are not in complete lateral alignment, the magnitude of the shift depending on the difference of the mechanical properties of the Lo and Ld phases. Typical values of the shift are about 4 nm, which is comparable to the membrane thickness.10,11 If two rafts with domain boundaries arranged in this way approach each other, two configurations are possible: an antisymmetric configuration (Figure 2A), where domain boundaries in the lower monolayer are

Figure 2. Schematic representation of antisymmetric (A) and symmetric (B) configurations of interacting rafts. Lo phase is gray, and Ld phase is blue. The monolayer of Lo phase has larger thickness (hr) than the monolayer of Ld phase (hs). There are five zones (numbered 1 through 5 in (B)) characterized by different values of the upper and lower monolayer thicknesses.

THEORETICAL SECTION

Elastic Energy Functional. Membrane is considered as consisting of two monolayers of laterally continuous elastic liquid crystal. While calculating elastic contribution into domain boundary energy, we take into account tilt and splay deformations only, neglecting lateral compression/stretch because of the large value of the corresponding elastic modulus.46 Deformations are defined at the so-called neutral surface where splay and lateral compression/stretch deformations are energetically uncoupled; in other words, where the quadratic form expressing energy as a function of splay and lateral compression/stretch deformations is diagonal.47 Two such neutral surfaces exist, one per each monolayer of a lipid membrane, in the regions where lipid polar heads connect with hydrophobic tails. The surfaces are generally parallel to the monolayer−water interface. The state of each monolayer is described by a vector field of unit vectors n known as “directors”, characterizing average orientation of lipid molecules. The neutral surface shape is described by the vector field of unit normals N. Tilt deformation is characterized by the tilt vector t = n/(nN) − N ≈ n − N, defined as a local deviation of the director from the normal vector. Splay deformation creates inhomogeneity in the field of directors and is quantitatively

shifted in the same direction with respect to those of the upper monolayer, and a symmetric one (Figure 2B), where domain boundaries are shifted in opposite directions. In the present work, we calculate raft interaction energy for both configurations. However, according to our analysis, the energy profiles for symmetric and antisymmetric configurations coincide (Figure 3) up to the point when domains contact (i.e., when the third zone in Figure 2 vanishes). Since it is the range of distances between the rafts from infinity to the formation of contact between the domains of primary interest, we hereafter restrict our analysis to symmetric configurations (Figure 2B). The system under consideration is characterized by three parameters: (1) distance between the lower domains, L; (2) boundary shift a1 of the upper monolayer domain relatively to the boundary of the lower monolayer domain in the left raft; (3) boundary shift a2 of the upper monolayer domain relatively to the boundary of the lower monolayer domain in the right raft. The membrane can be subdivided into five zones, transition between which is accompanied by a change of thickness of either the upper or the lower monolayer in the unperturbed state. Variables related to the upper and the lower monolayers are denoted by 1593

DOI: 10.1021/acs.langmuir.5b03990 Langmuir 2016, 32, 1591−1600

Article

Langmuir

In the unidimensional case, vector values can be replaced with their projections on Ox-axis. For small deformations div(n) ≈ dnx/dx, where nx is the projection of the director n on Ox-axis. For the sake of notation simplicity, we will omit the bottom index “x” for vector projections on Ox-axis. The shape of a membrane can be described by three functions: the shape of the neutral surface of the upper monolayer Hu(x), neutral surface shape of the lower monolayer Hl(x), and monolayer interface shape M(x). In this notation, the condition of local volumetric incompressibility (2) yields Hu(x) − M(x) = hu0 −

hu0 2 n u′ , 2

M(x) − Hl(x) = hl0 −

hl0 2 nl′ 2

(3)

for the upper and lower monolayers, respectively. Here and below, the prime superscript denotes the first derivative with respect to x. Projections of the unit normal to neutral surfaces of the upper and the lower monolayers for small deformations are equal to

Figure 3. Dependence of raft interaction energy on the distance between their boundaries. Curve 1 (dashed) corresponds to the symmetric configuration, and curve 2 (solid) to the antisymmetric configuration (see Figure 2 for definition of these two configurations).

h

2

2

h

Nu ≈ Hu′ = M′ − u02 n u″, Nl ≈ − Hl′ = − M′ − l02 nl″. Taking into account these expressions and eq 3, substituting tilt vector projections into eq 1, integrating over the neutral surfaces of monolayers, and dividing the result by the length of the boundary along the Oy-axis, we obtain the following expression for the free energy functional of an arbitrarily deformed bilayer, referred to a unit of length along Oy-axis:

the indices “u” and “l” respectively; to denote values, corresponding to a certain zone, its number is used as an index. In the general case, raft line tension depends on the raft size,33 but for significantly large domains it can be assumed constant. Here we assume for the sake of simplicity that rafts are large compared to the characteristic length of deformations. In this case, raft boundary can be considered as a straight line and the problem becomes effectively one-dimensional. We introduce a Cartesian coordinate system, the center O of which is located at the monolayer interface halfway between the domains. Ox-axis is perpendicular to domain boundaries; Oy-axis is parallel to domain boundaries. Thus, the upper left domain boundary has the coordinate x = −L/2 − a1, the lower left x = −L/2, the lower right x = L/2, and the upper right x = L/2 + a2. When the lower domains merge, the distance between the upper domains D equals D0 = a1 + a2, whereafter we redefine L as L = D − D0 to smoothly continue L into negative values. Due to the translational symmetry along the Oy-axis, we refer all calculations to the unit of boundary length along this direction. In principle, loss of translational symmetry along Oy-axis can take place, resulting in periodic tilt along this axis. Tilt periodicity immediately follows from consideration of a circular domain under the condition of continuity of all projections of the director. Most probably, appearance of periodic tilt along Oy-axis would result in some undulations being superimposed on the shape of the domain boundary. This would lead to increase of the boundary length, and, consequently, of the boundary energy (in the second order on deformations). However, the energy increase can be compensated by relaxation of deformations due to additional degree of freedom: tilt projections along Oy-axis. Generally, the possibility of loss of translational symmetry and its energetic consequences is an independent theoretical problem, resolving which is beyond the scope of the present work. Nevertheless, if such “periodic” configuration of the domain boundary can be physically realized, it should be observed in molecular dynamics studies of membranes with Lo/Ld phase coexistence. Although molecular dynamics allows detecting even such subtle details of the domains boundary as several nanometers equilibrium shift of boundaries of monolayer domains composing the bilayer domains, considered in the present work (Figure 2), we are not aware of any work, in which stationary periodic variation (e.g., of the order parameter along the domain boundary) has been ever observed. This allows concluding that even if the equilibrium periodic tilt along the domain boundary takes place, its characteristic wavelength should be greater than the typical size of simulation box, that is, about 25 nm in the work ref 36. However, the characteristic length of deformations change along Ox-axis is substantially smaller; it is about ∼ h02/l = 4 nm (see eq 6). This means that deformations along Oy-axis are relatively slow (in spatial sense) and the system still can be considered as quasi-unidimensional.

W=

∫

⎧ ⎞2 ⎪B B h 2 K⎛ ⎨ u (n u′ + Ju )2 − u Ju 2 + ⎜n u + u0 n u″ − M′⎟ ⎪ 2 2 2⎝ 2 ⎠ ⎩

+

⎞2 ⎫ ⎪ h 2 Bl B K⎛ (nl′ + Jl )2 − l Jl 2 + ⎜nl + l0 nl″ + M′⎟ ⎬ dx ⎪ 2 2 2⎝ 2 ⎠⎭ (4)

Calculations were performed taking into account different values of splay moduli for monolayers of domain and surrounding membrane, and different values of spontaneous curvature of respective monolayers. Spatial Distribution of Deformations. To obtain the deformations of the membrane, we minimize the functional 4 with respect to the functions nu(x), nl(x), and M(x) in each of the five zones of the membrane (Figure 2). As a result, in each zone, we obtain three Euler− Lagrange differential equations: ⎧h 4 h 2 ⎪ u0 n u(4) + (hu0 2 − lu 2)n u″ + n u − u0 M‴ − M = 0, 2 ⎪ 4 ⎪ 2 ⎪ h4 h ⎨ l0 nl(4) + (hl0 2 − l l 2)nl″ + nl + l0 M‴ + M = 0, ⎪ 4 2 ⎪ 2 2 ⎪ hu0 ‴ hl0 ‴ n − nl + n u − nl − 2M″ = 0 ⎪ ⎩ 2 u 2

(5)

where lu,l = Bu,l /K . These equations can be solved analytically in the general case. In the first, the third, and the fifth zones (x < −L/2 − a1, −L/2 < x < L/2, and x > L/2 + a2) (Figure 2), the solutions are

n u(x) = c1 e(l +

l 2 − h0 2 )x / h0 2 2

2

+ c 2 e(l − 2

l 2 − h0 2 )x / h0 2 2

+ c3 e(−l − l − h0 )x / h0 + c4 e(−l + l − h0 c + 5 (x 2 − h0 2 + 2l 2) + c6x + c 7 , 2 nl(x) = c1 e(l +

l 2 − h0 2 )x / h0 2 2

2

+ c 2 e(l − 2

)x / h0 2

l 2 − h0 2 )x / h0 2 2

+ c3 e(−l − l − h0 )x / h0 + c4 e(−l + l − h0 c − 5 (x 2 − h0 2 + 2l 2) − c6x − c 7 , 2 c5 3 c6 2 M(x) = x + x + c 7x + c8 6 2 1594

2

2

)x / h0 2

(6)

DOI: 10.1021/acs.langmuir.5b03990 Langmuir 2016, 32, 1591−1600

Article

Langmuir

Figure 4. Fusion of the Lo domains (dark gray); the duration of the recording from (A) to (D) is about 3 s. The length of the bar shown in panel (A) is 20 μm. where c1,..., c8 are integration constants that can be determined from the boundary conditions. Membrane is assumed flat and parallel to an Oxyplane far away from the raft boundary. We choose the coordinate system so that the monolayer interface plane matches the Oxy-plane when x → −∞. In this case, the following boundary conditions hold:

⎧ n u,l(±∞) = 0, ⎪ ⎪ ⎨ M(−∞) = 0, ⎪ ⎪ M(+∞) = const ⎩

angles inside each ring, the paper does not report the amplitude of thermal fluctuations of torsion angles involving adjacent rings. Marrink and co-workers53 obtained that in coarse-grained MD the consideration of cholesterol steroid part as an absolutely rigid body results in spurious membrane instability. This instability vanishes if the steroid part is assumed to consist of two beads, sharing a common edge with allowed freedom to hinge on it with some effective restraining potential, V = Kθ(φ − φ0)2/2 (Kθ is force constant, φ and φ0 are the current and equilibrium dihedral angles, respectively). The force constant of the potential was obtained from comparison of the results of coarse-grained and all-atom MD simulations; the obtained value is Kθ = 50 kJ/(mol· rad2) ≈ 20kBT/rad2. Thus, assuming the characteristic thermal energy equal to 1kBT, we obtain that the angle between these two beads can thermally fluctuate with the average amplitude ∼18°, that is, the overall variation of the angle is about 36°. Further calculations were made for two values of spontaneous curvature of 7DHC: (1) JDChol1 = −0.2 nm−1; (2) JDChol2 = −0.1 nm−1; spontaneous curvature of cholesterol was considered equal to JChol = −0.494 nm−1.51 Additionally, we assumed that replacing cholesterol with 7DHC does not change distribution of other lipids between phases and used phase compositions determined in the ref 16. This assumption is compulsory. Even ternary phase diagrams for cholesterol-containing membranes are still debatable. The diagrams reported in different works vary significantly; they strongly depend on the method chosen to detect phase separation. To our knowledge, there is no consistent phase diagram of ternary lipid mixture containing 7DHC. Thus, the assumption of equal partitioning of cholesterol and 7DHC between domains and the surrounding membrane could be considered as the first approximation. This assumption is based on similarity of cholesterol and 7DHC chemical structures, though generally the present work is focused on investigation of difference of their physical properties. Overall, calculations of the domain interaction energy were carried out for the following spontaneous curvatures of monolayers of raft (Jr) and surrounding membrane (Js): (1) Js = Jr = 0; (2) membrane with cholesterol, Js = −0.122 nm−1, Jr = −0.117 nm−1; (3) membrane with 7DHC (with JDChol1 = −0.2 nm−1), Js = −0.084 nm−1, Jr = −0.030 nm−1; (4) membrane with 7DHC (with JDChol2 = −0.1 nm−1), Js = −0.072 nm−1, Jr = 0.002 nm−1. We chose the energy of flat undeformed raft bilayer as the reference energy level. According to computation results (data not shown), the energy is minimal when the boundaries of the upper domains in both rafts are shifted by the same distance, that is, when a1 = a2 = a; this is consistent with the results obtained earlier in the ref 54. Figure 3 illustrates comparison between the energy profiles of raft interaction for symmetric and antisymmetric configurations. These curves were obtained for soft rafts with zero spontaneous curvatures. For separation distances L > 0 the energy profiles of both configurations coincide. This allows us to restrict computations to the symmetric configuration only (Figure 2B).

(7)

These boundary conditions allow simplifying expressions in the first and the fifth zones (Figure 2) due to vanishing coefficients at polynomial in x terms and exponents, which do not decay far from the boundary. In the transition zones #2 and #4, the solutions are much more cumbersome because of different monolayer thicknesses and splay moduli for the upper and the lower monolayers and finite lengths a1 and a2 of these zones, which does not allow discarding polynomial and exponential terms. Spatial distributions of directors nu(x), nb(x) and shapes of neutral surfaces Hu(x), Hb(x) obtained in five zones are matched at the boundaries of the zones in the points x12 = −L/2 − a1, x23 = −L/2, x34 = L/2, and x45 = L/2 + a2 based on the conditions of continuity of director projections and neutral surfaces in each monolayer. Next, the total energy is minimized with respect to the undefined integration constants. All the results are obtained analytically; however, the final equations are too cumbersome to be presented here for any imaginable benefit. Instead, the results are illustrated graphically in the next section. Parameters of the System. For graphical illustration of the obtained results we use the following parameter values: tilt modulus (per monolayer) K = 40 mN/m = 10 kBT/nm2 (kBT ∼ 4 × 10−21 J),48 thickness of undeformed monolayer of the surrounding membrane hs = 2 nm, thickness of undeformed domain monolayer hr = 2.5 nm, and splay modulus of the surrounding membrane monolayer B = 10kBT.46 The values of splay modulus of the Lo phase reported by different authors range between about equal to the splay modulus of the surrounding membrane,49 and 2−5 times larger than that.50 We considered both cases, having run the calculations for B1 = 10 kBT (“soft” raft) and B2 = 20kBT (“rigid” raft) per monolayer. We use the lipid compositions and spontaneous curvatures of the monolayers of Lo and Ld phases as determined in the ref 51 for membranes of different average lipid compositions. Spontaneous curvature of 7DHC has not been accurately determined so far. However, from comparison of chemical structures of cholesterol and 7DHC (Figure 1), it can be seen that 7DHC has an extra double bond C7−C8, which leads to reduced conformational mobility of this part of the molecule, due to sp2hybridization of C7, C8 carbon atoms resulting in increased dihedral angle force constant for B and C rings. This should lead to reduction of effective volume of the hydrophobic part of the molecule, and thus to higher values of spontaneous curvature of 7DHC compared to cholesterol. However, for this to take place, the energy of variation of the dihedral angle in cholesterol should be small enough to allow thermal activation. The force constant could be obtained from the works focused on development and adjustment of force fields for molecular dynamics (MD) simulations. In the paper on force fields for molecular dynamics, ref 52, chemical structures of several sterols including cholesterol and ergosterol were compared. However, providing detailed information on MD simulation results of thermal fluctuation of torsion

■

RESULTS Domain Morphology in Ternary Lipid Mixtures of EPC/ ESM/Sterol. Generally, in cholesterol-containing membranes, domains have circular shape and usually merge upon approach into larger circular domains. The domain fusion process is 1595

DOI: 10.1021/acs.langmuir.5b03990 Langmuir 2016, 32, 1591−1600

Article

Langmuir

Figure 5. Time series of a rare event of Lo domains approaching (A,B), forming contact (C,D), and moving apart (E,F) without fusion in EPC/ESM/ cholesterol (45/45/10) GUV at 4 °C. The domains to be followed are marked by white arrow in panel (A). The duration of recording from (A) to (F) is about 2 s. The length of the bar shown in panel (A) is 20 μm.

Figure 6. Domain morphology in EPC/ESM/7DHC (45/45/10, 40/40/20, and 34/33/33) GUVs. Top panel: image sequence of concerted movement of agglutinated domains in EPC/ESM/7DHC (45/45/10) GUV (overall about 5 s). Bottom panel: snapshots of GUVs with different lipid ratios at the temperatures ranging from 25 to 4 °C. Bars 20 μm.

illustrated by Figure 4. The picture is representative of the processes occurring in EPC/ESM/cholesterol mixtures of all the compositions used (45/45/10, 40/40/20, and 34/33/33) in the entire investigated range of temperatures from 4 to 25 °C. Predictably enough, a few exceptions were observed at low temperature (4 °C), where cholesterol-based rafts failed to merge on contact. In these infrequent cases, they would again separate and continue moving independently of each other. An example of such behavior in a 45/45/10 EPC/ESM/cholesterol membrane is illustrated by Figure 5. Domain behavior in 7DHC-containing membranes differs significantly from that in cholesterol-containing membranes. In

this case, we did not observe merger of Lo domains; however, once having formed contact, these domains would agglutinate into necklace-like aggregates stable on the time scale of the experiments (Figure 6) The tendency to form necklace-like aggregates becomes progressively more pronounced at decreasing temperatures (see Figure 6, bottom panel). Raft Interaction Energy Profile. The experimentally observed features of domain morphology and interaction are consistent with the developed theoretical model of the raft boundary structure. Calculated dependence of raft interaction energy on the distance between their boundaries for different 1596

DOI: 10.1021/acs.langmuir.5b03990 Langmuir 2016, 32, 1591−1600

Article

Langmuir spontaneous curvatures at the temperature of 15 °C is presented on Figure 7. Figure 7A and B represents the cases of soft and rigid rafts, respectively.

In the case of nonzero spontaneous curvatures of raft and surrounding membrane monolayers, when Jr ∼ Js, the energy profile of approaching rafts remains almost the same: positions of barriers and local minima occur almost at the same separation distances L (Figure 7A, curve 2) as for Jr = Js = 0. However, in the case of positive difference of spontaneous curvatures of raft and surrounding membrane monolayers (Jr > Js), which appears when cholesterol is replaced by 7DHC, the energy profile changes qualitatively (Figure 7A, curves 3 and 4). When the difference of spontaneous curvatures is Jr − Js = 0.054 nm−1, which results from complete replacement of cholesterol by 7DHC with the spontaneous curvature JDChol1 = −0.2 nm−1, the depth of the local energy minimum M1 reaches 0.035kBT/nm (3.7kBT for two rafts of the diameter of 1 μm), and the height of the energy barrier of the lower domain fusion B1 increases to 0.135kBT/nm (14.3kBT for two rafts of the diameter of 1 μm) (Figure 7A, curve 3), which is almost two times larger than that for the cholesterol membranes (Figure 7A, curve 2). For the case of 7DHC with assumed spontaneous curvature of JDChol2 = −0.1 nm−1 (Figure 7A, curve 4), the depth of the local minimum M1 is almost the same, and height of the energy barrier B1 for fusion of the lower monolayers is larger than in the case of 7DHC spontaneous curvature of JDChol1 = −0.2 nm−1 (Figure 7A, curve 3). In both cases, the energy of the local minimum M1 becomes smaller than the energy of the local minimum M2 (Figure 7A, curves 3 and 4, compare with curve 2). This means that when the difference of spontaneous curvatures of raft and surrounding membrane is positive (Jr − Js > 0), rafts either merge completely with fusion of both monolayers, or do not merge at all  the state corresponding to the local minimum M2 should be least occupied. Now consider the case of a rigid raft. In the case of zero spontaneous curvatures (Figure 7B, curve 1), energy profile does not qualitatively differ from a corresponding curve for a soft raft, except for proportional increase of deformation energy in all points of the plot. In case of spontaneous curvatures calculated for the membrane with cholesterol (Figure 7B, curve 2), the energy profile becomes smoother. For this curve, the depth of the local minimum M1 as well as the heights of the barriers B1 and B2 are small enough to allow raft merger upon each collision. In case of positive difference of spontaneous curvatures of raft and surrounding membrane (Figure 7B, curves 3, 4), the energy profile takes form similar to the corresponding profile in the case of a soft raft. The depth of the local minimum M1 is approximately 0.055kBT/nm (5.8kBT for two rafts of the diameter of 1 μm), and the height of energy barrier B1 increases up to 0.170kBT/nm (18.0kBT for two rafts of the diameter of 1 μm) for the case of spontaneous curvature of 7DHC equal JDChol2 = −0.1 nm−1. Thus, from theoretical analysis, it follows that independently on the ratio of splay moduli of raft and the surrounding membrane, positive difference between their spontaneous curvatures leads to deeper local minimum M1 and higher energy barrier B1. For practical purposes, it means that after replacement of cholesterol by 7DHC rafts should energetically prefer to stay at a finite distance L ∼ 7 nm from each other, corresponding to the local minimum M1: both merger and large distances separation require overcoming a significant energy barrier. Thus, necklace-like aggregates of rafts should form, where rafts neither merge nor part. This result is fully supported by our experimental observations. The interaction energy profiles for the temperature of 4 °C are presented in Figure 8 for the case of rigid raft. To calculate the

Figure 7. Dependence of the raft interaction energy on the distance between their boundaries at 15 °C. (A) Splay moduli of raft and surrounding membrane are equal; (B) splay modulus of raft is two times larger than the splay modulus of the surrounding membrane. Curves 1− 4 correspond to different values of spontaneous curvatures of raft monolayers and surrounding membrane: curves 1 (dashed, red), Js = Jr = 0; curves 2 (green), Js = −0.122 nm−1, Jr = −0.117 nm−1; curves 3 (blue), Js = −0.084 nm−1, Jr = −0.030 nm−1; curves 4 (cyan), Js = −0.072 nm−1, Jr = 0.002 nm−1.

Let us consider the case of a soft raft first. At zero spontaneous curvature (Figure 7A, curve 1), for large distances L between domains, the energy as a function of L stabilizes at the value corresponding to the energy of boundaries of two isolated rafts. In order to achieve the local minimum M1 at L ∼ 7 nm, rafts first need to overcome a potential barrier B0 at L ∼ 11 nm having the height of 0.005kBT per 1 nm of the boundary. Local energy minimum M1 depth is 0.027kBT/nm relative to the peak of the barrier B0. Based on Derjaguin−Landau−Verwey−Overbeek theory, in order to obtain the absolute interaction energies (rather than the energy per unit length of the boundary) of circular rafts, the linear energy density should be multiplied by the specific length L DLVO = 2 2λR , where λ is characteristic length of the energy profile and R is domain radius. Thus, we estimate that, for two approaching rafts with equal diameters of 2R = 1 μm and characteristic length of decay of deformations λ ∼ h02/l ∼ 2.8 nm (see eq 6), the absolute values are 0.5kBT for the height of the barrier B0 and 2.8kBT for the depth of the local minimum M1. To achieve fusion of the lower domains, rafts need to overcome the energy barrier B1 at L ∼ 3−4 nm of 0.074kBT/nm height (7.8kBT for two rafts of diameter 1 μm), whereafter the system gets into a deep local minimum M2 at L ∼ − 1 nm (depth of ∼0.150kBT/nm with respect to the barrier B1 top, resulting in 15.9 kBT for two rafts of the diameter of 1 μm). For subsequent fusion of the upper domains, the energy barrier B2 at L ∼ −6 nm having the height of 0.105 kBT/nm (11.1kBT for two rafts of the diameter of 1 μm) has to be crossed. Thus, according to the analysis of the proposed model, merger of rafts occurs through consecutive fusion of their monolayer domains, in two stages. The first stage is fusion of lower monolayer domains (Figure 2B); the local minimum M2 corresponds to this stage on energy profiles. The second stage is fusion of domains in the second monolayer (the upper on Figure 2B). This stage is described by the left-most points of energy profiles, located approximately at L = −10 nm. 1597

DOI: 10.1021/acs.langmuir.5b03990 Langmuir 2016, 32, 1591−1600

Article

Langmuir

layers. Therefore, the area of Lo phase in each monolayer must be equal. The results of theoretical analysis in the framework of the suggested model are in good agreement with the experimental data. In order to maintain symmetry of the membrane when the monolayer domain boundaries are shifted with respect to each other in equilibrium, the difference of areas between the upper and lower monolayer domains in one raft has to be compensated by similar (but opposite) difference in other rafts. Since the energy profiles of the symmetric and asymmetric configurations of the interacting boundaries coincide up to the point of contact of the domains (Figure 3), the model adequately describes the experimental system. In cellular membranes, the inner monolayer composition differs greatly from that of the outer monolayer. According to ref 55, the outer monolayer is enriched with the saturated lipids promoting formation of the Lo phase. It is reasonable to assume that the area of Lo phase in the outer monolayer must exceed that of the inner monolayer. These conditions are difficult to achieve in GUV experiments; however, the asymmetry of Lo phase distribution between the monolayers is naturally accounted for in our model. In order to compensate the difference of the domain areas, the radii of the Lo domains on the outer monolayer must on the average exceed those of such domains on the inner monolayer. It is exactly the symmetric configuration corresponding to the free energy minimum in our model, i.e. this is the equilibrium configuration (see Figure 2B). It is for this configuration that the raft interaction energy profiles were calculated. Thus, the results of analysis can be readily extrapolated on cellular membranes. In the present paper, we demonstrate that a subtle difference in chemical structure of sterol significantly affects raft shape, size, and dynamics. We hypothesize that the induced changes in the domain interaction parameters disturb various raft-dependent or raft-associated processes, including protein sorting, membrane trafficking, and cell signaling. The influence of substances modifying raft properties upon cell signaling has been discussed by a number of authors.20−22 A marker of raft phase, ganglioside GM1, is known to notably alter raft morphology even at low concentrations (fractions of molar percent).56 It has also been found to be capable of inducing apoptotic signal transfer in Tlymphocytes in a raft-dependent manner.18−20 In our model, replacement of cholesterol with 7DHC alters the raft interaction energy profiles in a manner that should first of all affect raft fusion rates. It is therefore possible that 7DHC significantly affects cellular membrane organization. Indeed, 7DHC-containing model membranes differ radically from the cholesterolcontaining membranes of otherwise identical composition in terms of spatial arrangement and dynamics of Lo domains, which are in case of 7DHC membranes incapable of growing through fusion. This difference appears to be crucial for membrane lipid and protein distribution between raft and nonraft phases, as well as for ability of proteins and lipids localized in different rafts to interact. It is consistent with the previously reported facts that membrane domains formed by 7DHC differ with those formed by cholesterol in protein composition,57 packing,58 and stability.59 In vivo, 7DHC is also able to deteriorate raft stability, expel some proteins from the rafts, and alter their activity.60 Raft fusion being an important stage of signal transduction in the cell signaling system, it is natural to assume that 7DHC raftlike domains defective in their fusion rate can impair signal transduction across cellular membranes and other important biological functions, causing a complex symptomatic reaction of human organism known as SLOS.

Figure 8. Dependence of raft interaction energy on the distance between their boundaries at 4 °C for membranes containing (A) cholesterol and (B) 7-dehydrocholesterol. The splay modulus of raft is assumed two times larger than the splay modulus of the surrounding membrane. Solid curves correspond to 4 °C; dashed curves to 15 °C (the same as corresponding curves on the Figure 7). Spontaneous curvatures of rafts and surrounding membrane are Js = −0.105 nm−1, Jr = −0.086 nm−1 for panel (A); and Js = −0.055 nm−1, Jr = 0.033 nm−1 for panel (B).

profiles, we utilized the temperature dependencies of thickness mismatch and spontaneous curvatures of Lo and Ld phases experimentally determined in ref 51. Accordingly, for this figure, we assumed the thickness of undeformed monolayer of the surrounding membrane hs = 1.9 nm and the thickness of undeformed Lo domain monolayer hr = 2.6 nm. The following temperature coefficients of spontaneous curvatures were used: − 0.0011 (nm·°C)−1 for EPC, and −0.0035 (nm·°C)−1 for cholesterol, 7DHC, and ESM. From Figure 8, it follows that characteristic features of interaction energy profiles become more pronounced at lower temperatures. The depth of local minimum M1 increases to about 0.200kBT/nm (21.2kBT for two rafts of the diameter of 1 μm); the height of the energy barrier for the lower domain fusion, B1, increases to about 0.400kBT/nm (42.3kBT for two rafts of the diameter of 1 μm) for a membrane containing 7DHC with large spontaneous curvature (Figure 8B). Such high values of energies mean that, for the conditions of Figure 8B, rafts, once having formed contact, should neither merge nor separate by any significant distance on the time scale of the experiment, even for relatively small rafts. This is in perfect agreement with our experimental data (see Figure 6). For cholesterol-containing membrane, the height of the energy barrier B1 also increases to 0.110kBT/nm (11.6kBT for two rafts of the diameter of 1 μm) at low temperature (Figure 8A). Experimentally it should mean that fraction of raft that fail to merge after forming contact should increase at low temperatures. Indeed, rare events of rafts forming contact and then separating again without fusion were observed in cholesterol-containing membranes only at the low temperature of 4 °C (Figure 5). Small depth of the local minimum M1 for cholesterol-containing membrane at low temperature (about 0.030kBT/nm or 3.2kBT for two rafts of the diameter of 1 μm) (Figure 8A) is qualitatively consistent with relatively small time (less than 1 s) the two rafts spend close to each other before they separate in these events (Figure 5).

■

DISCUSSION Domain morphology was experimentally investigated in the model membrane with identical lipid composition of mono1598

DOI: 10.1021/acs.langmuir.5b03990 Langmuir 2016, 32, 1591−1600

Article

Langmuir

■

(17) Garcia-Saez, A. J.; Chiantia, S.; Schwille, P. Effect of line tension on the lateral organization of lipid membranes. J. Biol. Chem. 2007, 282, 33537−33544. (18) Hueber, A. O.; Bernard, A. M.; Herincs, Z.; Couzinet, A.; He, H. T. An essential role for membrane rafts in the initiation of Fas/CD95triggered cell death in mouse thymocytes. EMBO Rep. 2002, 3, 190− 196. (19) Grassme, H.; Jekle, A.; Riehle, A.; Schwarz, H.; Berger, J.; Sandhoff, K.; Kolesnick, R.; Gulbins, E. CD95 signaling via ceramide rich membrane rafts. J. Biol. Chem. 2001, 276, 20589−20596. (20) Burek, C.; Roth, J.; Koch, H. G.; Harzer, K.; Los, M.; ShlutzeOsthoff, K. The role of ceramide in receptor- and stress-induced apoptosis studied in acidic ceramidase Farber desease cells. Oncogene 2001, 20, 6493−6502. (21) Akimov, S. A.; Frolov, Vl.A.; Kuzmin, P. I.; Zimmerberg, J.; Chizmadzhev, Yu.A.; Cohen, F. S. Domain formation in membranes caused by lipid wetting of protein. Phys. Rev. E 2008, 77, 051901. (22) Dietschy, J. M.; Turley, S. D. Cholesterol metabolism in the brain. Curr. Opin. Lipidol. 2001, 12, 105−112. (23) Porter, F. D. Smith−Lemli−Opitz syndrome: pathogenesis, diagnosis and management. Eur. J. Hum. Genet. 2008, 16, 535−541. (24) The American Heritage Stedman’s Medical Dictionary. Houghton Mifflin Company: Boston, 2007. (25) Yu, H.; Patel, S. B. Recent insights into the Smith-Lemli-Opitz syndrome. Clin. Genet. 2005, 68, 383−391. (26) DeBarber, A. E.; Eroglu, Y.; Merkens, L. S.; Pappu, A. S.; Steiner, R. D. Smith-Lemli-Opitz syndrome. Expert Rev. Mol. Med. 2011, 13, e24. (27) Bianconi, S. E.; Cross, J. L.; Wassif, C. A.; Porter, F. D. Pathogenesis, epidemiology, diagnosis and clinical aspects of SmithLemli-Opitz syndrome. Expert Opin. Orphan Drugs 2015, 3, 267−280. (28) Singh, P.; Paila, Y. D.; Chattopadhyay, A. Differential effects of cholesterol and 7-dehydrocholesterol on the ligand binding activity of the hippocampal serotonin(1A) receptor: Implications in SLOS. Biochem. Biophys. Res. Commun. 2007, 358, 495−499. (29) Ehtesham, M.; Sarangi, A.; Valadez, J. G.; Chanthaphaychith, S.; Becher, M. W.; Abel, T. W.; Thompson, R. C.; Cooper, M. K. Liganddependent activation of the hedgehog pathway in glioma progenitor cells. Oncogene 2007, 26, 5752−5761. (30) Megha, O. B.; London, E. Cholesterol precursors stabilize ordinary and ceramide-rich ordered lipid domains (lipid rafts) to different degrees: Implications for the bloch hypothesis and sterol biosynthesis disorders. J. Biol. Chem. 2006, 281, 21903−21913. (31) Frolov, V. A. J.; Chizmadzhev, Y. A.; Cohen, F. S.; Zimmerberg, J. “Entropic traps” in the kinetics of phase separation in multicomponent membranes stabilize nanodomains. Biophys. J. 2006, 91, 189−205. (32) Kuzmin, P. I.; Akimov, S. A.; Chizmadzhev, Yu.A.; Zimmerberg, J.; Cohen, F. S. Line tension and interaction energies of membrane rafts calculated from lipid splay and tilt. Biophys. J. 2005, 88, 1120−1133. (33) Akimov, S. A.; Frolov, V. A.; Kuzmin, P. I. Line tension and size distribution function of nanorafts in bilayer lipid membranes. Biol. Membr. 2005, 22, 413−426. (34) Akimov, S. A.; Kuzmin, P. I. Line tension of raft/surround monolayer boundary calculated from splay, tilt and area compression/ stretching. Biol. Membr. 2005, 22, 137−146. (35) Hamm, M.; Kozlov, M. M. Elastic energy of tilt and bending of fluid membranes. Eur. Phys. J. E: Soft Matter Biol. Phys. 2000, 3, 323− 335. (36) Risselada, H. J.; Marrink, S. J. The molecular face of lipid rafts in model membranes. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 17367− 17372. (37) Schaefer, L. V.; Marrink, S. J. Partitioning of lipids at domain boundaries in model membranes. Biophys. J. 2010, 99, L91−L93. (38) Pantano, D. A.; Moore, P. B.; Klein, M. L.; Discher, D. E. Raft registration across bilayers in a molecularly detailed model. Soft Matter 2011, 7, 8182−8191. (39) Perlmutter, J. D.; Sachs, J. N. Interleaflet interaction and asymmetry in phase separated lipid bilayers: molecular dynamics simulations. J. Am. Chem. Soc. 2011, 133, 6563−6577.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax: +359 2 8723787. Tel: +359 2 9793686 (G.S.). *E-mail: [email protected]. Fax: +7-495-952-55-82. Tel: +7-495952-55-82 (S.A.A.). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Part of the experimental work was supported by Grant DFNI B02/23/2014 of National Science Fund, Bulgaria. The work was supported by Russian Science Foundation (Project # 15-1400060), by Russian Foundation for Basic Research (Project #1634-01203), and by the Ministry of Education and Science of the Russian Federation in the framework of Increase of Competitiveness Program of “MISiS”.

■

REFERENCES

(1) Brzustowicz, M. R.; Cherezov, V.; Caffrey, M.; Stillwell, W.; Wassell, S. R. Molecular organization of cholesterol in polyunsaturated membranes: microdomain formation. Biophys. J. 2002, 82, 285−298. (2) Rubenstein, J. L.; Smith, B. A.; McConnell, H. M. Lateral diffusion in binary mixtures of cholesterol and phosphatidylcholines. Proc. Natl. Acad. Sci. U. S. A. 1979, 76, 15−18. (3) de Almeida, R. F. M.; Fedorov, A.; Prieto, M. Sphingomyelin/ phosphatidylcholine/cholesterol phase diagram: boundaries and composition of lipid rafts. Biophys. J. 2003, 85, 2406−2416. (4) Veatch, S. L.; Keller, S. L. Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol. Biophys. J. 2003, 85, 3074−3083. (5) Baumgart, T.; Hess, S. T.; Webb, W. W. Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension. Nature 2003, 425, 821−824. (6) Samsonov, A. V.; Mihalyov, I.; Cohen, F. S. Characterization of cholesterol− sphingomyelin domains and their dynamics in bilayer membranes. Biophys. J. 2001, 81, 1486−1500. (7) Dietrich, C.; Bagatolli, L. A.; Volovyk, Z. N.; Thompson, N. L.; Levi, M.; Jacobson, K.; Gratton, E. Lipid rafts reconstituted in model membranes. Biophys. J. 2001, 80, 1417−1428. (8) Edidin, M. The state of lipid rafts: from model membranes to cells. Annu. Rev. Biophys. Biomol. Struct. 2003, 32, 257−283. (9) Anderson, R. G.; Jacobson, K. A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains. Science 2002, 296, 1821−1825. (10) Galimzyanov, T. R.; Molotkovsky, R. J.; Kuzmin, P. I.; Akimov, S. A. Stabilization of bilayer structure of raft due to elastic deformations of membrane. Biol. Membr. 2011, 28, 307−314. (11) Galimzyanov, T. R.; Molotkovsky, R. J.; Bozdaganyan, M. E.; Cohen, F. S.; Pohl, P.; Akimov, S. A. Elastic membrane deformations govern interleaflet coupling of lipid-ordered domains. Phys. Rev. Lett. 2015, 115, 088101. (12) Brown, D. A. Lipid rafts, detergent-resistant membranes, and raft targeting signals. Physiology 2006, 21, 430−439. (13) Edidin, M. Shrinking patches and slippery rafts: scales of domains in the plasma membrane. Trends Cell Biol. 2001, 11, 492−496. (14) Simons, K.; Ikonen, E. Functional rafts in cell membranes. Nature 1997, 387, 569−572. (15) McIntosh, T. J.; Vidal, A.; Simon, S. A. Sorting of lipids and transmembrane peptides between detergent-soluble bilayers and detergent resistant rafts. Biophys. J. 2003, 85, 1656−1666. (16) Heftberger, P.; Kollmitzer, B.; Rieder, A. A.; Amenitsch, H.; Pabst, G. In situ determination of structure and fluctuations of coexisting fluid membrane domains. Biophys. J. 2015, 108, 854−862. 1599

DOI: 10.1021/acs.langmuir.5b03990 Langmuir 2016, 32, 1591−1600

Article

Langmuir (40) Veatch, S. L.; Keller, S. L. Seeing spots: Complex phase behavior in simple membranes. Biochim. Biophys. Acta, Mol. Cell Res. 2005, 1746, 172−185. (41) Veatch, S. L.; Gawrisch, K.; Keller, S. L. Closed-loop miscibility gap and quantitative tie-lines in ternary membranes containing diphytanoyl PC. Biophys. J. 2006, 90, 4428−4436. (42) Angelova, M.; Dimitrov, D. Liposome electroformation. Faraday Discuss. Chem. Soc. 1986, 81, 303−311. (43) Ayuyan, A. G.; Cohen, F. S. Lipid peroxides promote large rafts: effects of excitation of probes in fluorescence microscopy and electrochemical reactions during vesicle formation. Biophys. J. 2006, 91, 2172−2183. (44) Zhao, J.; Wu, J.; Shao, H.; Kong, F.; Jain, N.; Hunt, G.; Feigenson, G. Phase studies of model biomembranes: macroscopic coexistence of La+Lb, with light-induced coexistence of La + Lo phases. Biochim. Biophys. Acta, Biomembr. 2007, 1768, 2777−2786. (45) Konyakhina, T. M.; Goh, S. L.; Amazon, J.; Heberle, F. A.; Wu, J.; Feigenson, G. W. Control of a nanoscopic-to-macroscopic transition: modulated phases in four-component DSPC/DOPC/POPC/Chol giant unilamellar vesicles. Biophys. J. 2011, 101, L8−L10. (46) Rawicz, W.; Olbrich, K. C.; McIntosh, T.; Needham, D.; Evans, E. Effect of chain length and unsaturation on elasticity of lipid bilayers. Biophys. J. 2000, 79, 328−339. (47) Leikin, S.; Kozlov, M. M.; Fuller, N. L.; Rand, R. P. Measured effects of diacylglycerol on structural and elastic properties of phospholipid membranes. Biophys. J. 1996, 71, 2623−2632. (48) Hamm, M.; Kozlov, M. M. Tilt model of inverted amphiphilic mesophases. Eur. Phys. J. B 1998, 6, 519−528. (49) Pan, J.; Tristram-Nagle, S.; Nagle, J. F. Effect of cholesterol on structural and mechanical properties of membranes depends on lipid chain saturation. Phys. Rev. E 2009, 80, 021931. (50) Baumgart, T.; Das, S.; Webb, W. W.; Jenkins, J. T. Membrane elasticity in giant vesicles with fluid phase coexistence. Biophys. J. 2005, 89, 1067−1080. (51) Kollmitzer, B.; Heftberger, P.; Rappolt, M.; Pabst, G. Monolayer spontaneous curvature of raft-forming membrane lipids. Soft Matter 2013, 9, 10877−10884. (52) Cournia, Z.; Smith, J. C.; Ullmann, G. M. A molecular mechanics force field for biologically important sterols. J. Comput. Chem. 2005, 26, 1383−1399. (53) Melo, M. N.; Ingolfsson, H. I.; Marrink, S. J. Parameters for Martini sterols and hopanoids based on a virtual-site description. J. Chem. Phys. 2015, 143, 243152. (54) Galimzyanov, T. R.; Molotkovsky, R. J.; Kheyfets, B. B.; Akimov, S. A. Energy of the interaction between membrane lipid domains calculated from splay and tilt deformations. JETP Lett. 2013, 96, 681− 686. (55) van Meer, G.; Voelker, D. R.; Feigenson, G. W. Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell Biol. 2008, 9, 112−124. (56) Bao, R.; Li, L.; Qiu, F.; Yang, Y. Atomic force microscopy study of ganglioside GM1 concentration effect on lateral phase separation of sphingomyelin/dioleoylphosphatidylcholine/cholesterol bilayers. J. Phys. Chem. B 2011, 115, 5923−5929. (57) Keller, R. K.; Arnold, T. P.; Fliesler, S. J. Formation of 7dehydrocholesterol-containing membrane rafts in vitro and in vivo, with relevance to the Smith-Lemli-Opitz syndrome. J. Lipid Res. 2004, 45, 347−355. (58) Berring, E. E.; Borrenpohl, K.; Fliesler, S. J.; Serfis, A. B. A comparison of the behavior of cholesterol and selected derivatives in mixed sterol-phospholipid Langmuir monolayers: a fluorescence microscopy study. Chem. Phys. Lipids 2005, 136, 1−12. (59) Wolf, C.; Chachaty, C. Compared effects of cholesterol and 7dehydrocholesterol on sphingomyelin-glycerophospholipid bilayers studied by ESR. Biophys. Chem. 2000, 84, 269−279. (60) Kovarova, M.; Wassif, C. A.; Odom, S.; Liao, K.; Porter, F. D.; Rivera, J. Cholesterol deficiency in a mouse model of Smith-Lemli-Opitz syndrome reveals increased mast cell responsiveness. J. Exp. Med. 2006, 203, 1161−1171. 1600

DOI: 10.1021/acs.langmuir.5b03990 Langmuir 2016, 32, 1591−1600