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LINE ACTIVITY OF GANGLIOSIDE GM1 REGULATES RAFT SIZE DISTRIBUTION IN A CHOLESTEROL-DEPENDENT MANNER Timur R. Galimzyanov, Anna S. Lyushnyak, Veronika V. Aleksandrova, Liudmila A. Shilova, Ilya I. Mikhalyov, Irina M. Molotkovskaya, Sergey A. Akimov, and Oleg V. Batishchev Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b00404 • Publication Date (Web): 21 Mar 2017 Downloaded from http://pubs.acs.org on March 24, 2017

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Abstract 45x29mm (300 x 300 DPI)

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Figure 1. Schematic cross-section of the raft and the surrounding membrane by the plane, which is perpendicular to the phase boundary. The raft is shown in grey color. Cartesian coordinate system is introduced: Ox axis is perpendicular to the raft boundary, Oy axis is parallel to the boundary, Oz axis is perpendicular to the membrane plane. L is the distance between the boundaries of the domain monolayer in the top and bottom membrane leaflets. The values related to the bottom monolayer we denote by the index “b”, and those for the top monolayer by the index “t”. The thickness of undeformed Lo monolayer is hr, the thickness of undeformed Ld monolayer is hs. In the zone I (surrounding membrane), thicknesses of monolayers coincide and are equal to hs. In the raft zone III, they are equal to hr. In the intermediate zone II, thicknesses of top and bottom monolayers are different: hr for the top monolayer and hs for the bottom one. 82x58mm (300 x 300 DPI)


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Figure 2. AFM topography images of membranes prepared from DOPC:eSM:Chol (1:1:1, by mole) mixture containing ganglioside GM1 with indicated concentrations in mol.%. 87x129mm (300 x 300 DPI)


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Figure 3. Dependence of average Lo domain size on GM1 content (in mole %) in the membrane from DOPC:eSM:Chol (1:1:1, by mole). 201x141mm (300 x 300 DPI)


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Figure 4. The dependence of the raft boundary line tension on the position of ganglioside-rich stripe. The following parameters were used: the equilibrium thickness of Lo monolayer hr = 1.8 nm and Ld monolayer hs = 1.3 nm; spontaneous curvatures of the raft and the surrounding membrane were assumed to be zero (Jr = 0, Js = 0); splay modulus of Lo and Ld monolayers were BR = 30 kBT and BS = 10 kBT, respectively; tilt modulus Kt = 10 kBT/nm2 = 40 mN/m; ganglioside-rich stripe width l = 1 nm, its spontaneous curvature Jp = 0.1 nm–1. 108x156mm (300 x 300 DPI)


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Figure 5. Dependence of the line tension of the raft boundary on the total ganglioside content for two sets of parameters: (A) our system, DOPC:eSM:Chol 1:1:1; (B) the system described in ref49, DOPC:eSM:Chol 2:2:1. 56x20mm (300 x 300 DPI)


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LINE ACTIVITY OF GANGLIOSIDE GM1 REGULATES RAFT SIZE DISTRIBUTION IN A CHOLESTEROL-DEPENDENT MANNER

T.R. Galimzyanov†,‡, A.S. Lyushnyak†,ǁ, V.V. Aleksandrova‡, L.A. Shilova†,ǁ, I.I. Mikhalyov§, I.M. Molotkovskaya§, S.A. Akimov†,‡, O.V. Batishchev†,ǁ*

†

A.N. Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of

Sciences, 31/4, Leninskii prospekt, Moscow, 119071 Russia ‡

National University of Science and Technology “MISiS”, 4, Leninskii prospekt, Moscow, 119049

Russia ǁ

Moscow Institute of Physics and Technology, 9, Institutskii per., Dolgoprudnyi, Moscow region,

141700 Russia §

M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of

Sciences, 16/10, Miklukho-Maklaya str., Moscow, 117997 Russia

* Corresponding author. E-mail: [email protected]; Fax: +7-495-952-55-82; Tel: +7-495-95486-73 (O.V.B.)

ABSTRACT

Liquid-ordered lipid domains, also called rafts, are assumed to be important players in different cellular processes, mainly signal transduction and membrane trafficking. They are thicker than the disordered part of the membrane and are thought to form for compensating the hydrophobic mismatch between transmembrane proteins and lipid environment. Despite the existence of such structures in vivo is still an open question, they are observed in model systems of multicomponent lipid bilayers. Moreover, the predictions obtained from model experiments allow explaining 1 ACS Paragon Plus Environment

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different physiological processes possibly involving rafts. Here we present the results of the study of regulation of raft size distribution by ganglioside GM1. Combining atomic force microscopy with theoretical considerations basing on the theory of membrane elasticity, we predict that this glycolipid should change the line tension of raft boundaries in two different ways, mainly depending on cholesterol content. These results explain the shedding of gangliosides from the surface of tumor cells and the following ganglioside-induced apoptosis of T-lymphocytes in a raftdependent manner. Moreover, the generality of the model allows predicting line activity of different membrane components based on their molecular geometry.

CARTOON FOR ABSTRACT

INTRODUCTION

Lipid matrix of cell membranes is highly heterogenic due to a large variety of lipid species1. Although the fluidity of biological membranes is one of the main properties determining their normal functionality, there is a possibility of formation of ordered lipid domains differing by structure and composition from the rest of the membrane2-4. These domains, also called rafts, are responsible for proper biological functions of certain membrane proteins5-8, involved in signal transduction in cells9,10, and play a significant role in processes of membrane trafficking11,12, cytoskeletal organization13,14, and even viral infection15. They are thicker than the other (liquid2 ACS Paragon Plus Environment

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disordered) part of the membrane16-18. Deformations arising to compensate the thickness mismatch determine an energy penalty per unit length of the boundary between raft and surrounding membrane called line tension19,20. Acting against entropy, line tension regulates the equilibrium size distribution of domains17,21. It is commonly believed that the mission of ordered lipid domains is to compensate hydrophobic mismatch between thick transmembrane part of the proteins and surrounding membrane22. That is why many authors contribute raft-forming function to the proteins23-25. Nevertheless, in the past two decades the role of lipids in emerging the domain formation has been established26-28. As Simons and Ikonen state5, rafts should be enriched in cholesterol and sphingolipids. There are also many evidences of formation of liquid-ordered lipid domains in ternary mixtures without sphingolipids, but containing other lipids with saturated hydrocarbon tails18,29. This means that lipids can form a large variety of domains, which, in turn, may influence protein clustering and cellular processes30. The regulating role of lipids in these events is still under debates, mainly due to difficulties in study of rafts in different systems. The well-known discrepancy exists between the results obtained in model systems and in cell membranes, since nobody sees micron-sized lipid domains in cell membranes, while they commonly exist in model ones31-33. The first problem is impossibility of observation of nanoscopic domains by optical techniques. Other methods, such as atomic force microscopy, requires specific model systems, e.g., supported lipid monolayers or bilayers16,34, while biochemical techniques give contradictory results35,36. Some authors describe rafts as ensemble of nanoscale domains, merging into micrometer-sized assemblies under certain stimulus19,24,37. This possibility of domain merging could be finely regulated by lipid chemical structure38 or membrane composition39. On the other hand, the size of domains is dictated by the line tension of their boundaries. This parameter and corresponding raft morphology strongly depend on the lipid composition38,39. Several lipid species, called line-active components40,41, can powerfully influence the line tension at raft boundaries in a concentration-dependent way. For example, gangliosides, having high affinity to the liquid-ordered phase of the membrane, vary the size of lipid domains42. They can also form gel-phase domains in 3 ACS Paragon Plus Environment

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the membrane by its own42,43, or in combination with other membrane components44. These glycosphingolipids are involved in a large variety of cellular processes, such as reception, neuronal differentiation, and the formation of axons, dendrites, and synapses45. Moreover, in refs46,47 authors show that these lipids shedded from cancer cells may trigger the apoptosis of T-lymphocytes. Monosialoganglioside GM1, the most studied representative of ganglioside family, demonstrates activity on domain behavior in different model systems, from lipid monolayers to giant unilamellar vesicles34,48-50. However, the question remains unanswered is the ability of GM1 to change size distribution of lipid domains dramatically, being in very small concentration, fractions of molar percent49. It means that this lipid should accumulate in a narrow zone near domain boundary, significantly influencing the line tension. Since GM1 hydrocarbon tails are fully saturated, we cannot associate this effect with the model of line activity suggested in ref41, which attributed the line activity to “hybrid” lipids possessing one saturated and one unsaturated tail only. Recently we have demonstrated that two leaflets of bilayer lipid domain should not be completely in register, so there is a narrow transition zone around the domain necessary to compensate elastic deformations arising from height mismatch at domain boundary51. Numerous molecular dynamics simulations52-54 support this result. In the present study, we conducted series of experiments on supported bilayer lipid membranes using atomic force microscopy and combined them with theoretical calculations to show the effect of small fractions of GM1 on size distribution of liquid-ordered domains depending on lipid composition of the membrane.

MATERIALS AND METHODS

A. Experimental section Materials. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), egg sphingomyelin (eSM), Npalmitoyl-D-erythro-sphingosylphosphorylcholine (pSM) and cholesterol (Chol) were purchased from Avanti Polar Lipids (USA). Monosialoganglioside (GM1), methanol (> 99.0%), chloroform (> 4 ACS Paragon Plus Environment

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99.0%), sodium chloride (NaCl) and HEPES were purchased from Sigma (USA). All chemicals were used without further purification. In experiments, we used Milli-Q water with a resistivity of 18.2 MΩ⋅cm. Preparation of supported lipid bilayer (sBLM). Liposomes were prepared as described in ref55. In brief, lipid solution containing DOPC, eSM (pSM in control experiments) and Chol dissolved in chloroform in a molar ratio 1:1:1 or 2:2:1 plus 0.25-2 mol. % of GM1 dissolved in chloroform : methanol (3:1 by volume) with a final lipid concentration of 1 mg/ml was rotary evaporated on the bottom of a glass flask and kept under vacuum for one hour. The resulting dried lipid film was resuspended in the working buffer solution (100 mM NaCl, 10 mM HEPES, pH 7.1). After 10 freeze-thaw cycles for 5 min each and vigorous vortexing the solution was extruded 19 times through two polycarbonate membranes with pore diameter of 100 nm (Avestin, Canada) to give unilamellar liposomes. The supported bilayer lipid membrane (sBLM) was created by deposing the liposomes on freshly cleaved mica (200 µl/~1.75 cm2) followed by incubation for 1 h at room temperature (RT) to allow lipid bilayer formation on the support. Then the sample was rinsed five times with working buffer solution and leaved covered by it to prevent bilayer dehydration. Atomic force microscopy (AFM). AFM experiments were performed using Multimode Nanoscope IV setup (Veeco Digital Instruments, USA) equipped with J type scanner and electrochemical fluid cell. All experiments were performed in tapping mode in working buffer solution. For scanning, we used Ultrasharp SiN3 cantilevers with nominal spring constant of 0.06 N/m and tip radius of approximately 2 nm (Bruker, USA). Z-axis resolution was 0.1 nm. After placing of a sample into the cell and filling of the cell with working buffer solution the sample was heated to 50°C with the rate of 2°C per 1 min using Thermal Applications Controller (Bruker, USA) and then incubated for 10 min at this temperature to allow all lipids to melt to liquid-disordered state. After that, the sample was cooled down to 18°C with the rate of 1°C per min, again incubated for 10 min and scanned. All images were scanned with dimensions of 3×3 µm2 and processed using WSxM software56 after scanning. 5 ACS Paragon Plus Environment

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B. Theoretical section We consider the bilayer lipid membrane as a continuous liquid-crystalline elastic medium in which liquid-ordered domains (rafts) form because of the phase separation19,20. For simplicity, we assume that the size of domains is large enough, so we can consider their boundaries locally as straight lines. Thus, the system possesses translational symmetry along the raft boundary, and we can treat it as effectively one-dimensional. This assumption is valid if the radius of the raft is considerably greater than the characteristic length of elastic deformations, which is about several nanometers51. Numerous studies report that thickness of raft is larger than the thickness of the surrounding membrane16-18. If raft monolayers are homogeneous and flat up to the boundary, jump in thickness of high energy will exist at the boundary. To prevent exposure of hydrophobic membrane interior to water raft monolayers should deform at the boundary51,57. We analyze the energy of membrane deformations at the domain boundary in the framework of theory of elasticity of liquid crystals adapted to lipid membranes58. We take into account two fundamental membrane deformations: splay and tilt. We minimize the elastic energy functional with respect to spatial distribution of deformations, obtain and solve Euler-Lagrange equations under appropriate boundary conditions. Substituting the deformations back into the energy functional and integrating over the monolayer surfaces, we obtain the energy of the boundary (per unit length), or line tension. As we show in refs51,57, the line tension of liquid ordered domain boundary is minimal when opposed monolayer domains are not completely in register, but relatively shifted by some equilibrium distance of about 2-4 nm (Fig. 1). In the framework of “elastic” approach, we consider the ganglioside, having bulk polar part and relatively small hydrophobic region, as the component possessing positive spontaneous curvature. Ganglioside can laterally distribute into liquid-ordered, Lo, and liquid disordered, Ld, phases, as well as into the intermediate zone between them (zone II in Fig. 1). However, GM1 is known to be the marker of Lo phase34. Thus, we assume that its content in Ld phase is negligible. It seems reasonable that to provide the line activity the gangliosides should be 6 ACS Paragon Plus Environment

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enriched in a narrow stripe-like zone close to the raft boundary. Only in this case boundary structure can be changed significantly by small amounts of GM1. The exact distribution of the GM1 between the stripe and the bulk raft phase, the width of the stripe, and its lateral position we find from the condition of minimal elastic energy of the system.

Figure 1. Schematic cross-section of the raft and the surrounding membrane by the plane, which is perpendicular to the phase boundary. The raft is shown in grey color. Cartesian coordinate system is introduced: Ox axis is perpendicular to the raft boundary, Oy axis is parallel to the boundary, Oz axis is perpendicular to the membrane plane. L is the distance between the boundaries of the domain monolayer in the top and bottom membrane leaflets. The values related to the bottom monolayer we denote by the index “b”, and those for the top monolayer by the index “t”. The thickness of undeformed Lo monolayer is hr, the thickness of undeformed Ld monolayer is hs. In the zone I (surrounding membrane), thicknesses of monolayers coincide and are equal to hs. In the raft zone III, they are equal to hr. In the intermediate zone II, thicknesses of top and bottom monolayers are different: hr for the top monolayer and hs for the bottom one.

The spontaneous curvature of homogeneous monolayer of non-raft phase, JS, does not depend on the coordinates and concentration of ganglioside. In Lo phase, the spontaneous curvature, JR, is assumed to be proportional to the concentration of ganglioside, cR:

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J R = J R 0 (1 − cR ) + J G cR ,

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(1)

where JG is the spontaneous curvature of pure GM1 monolayer, JR0 is the spontaneous curvature of Lo phase monolayer without ganglioside. The spontaneous curvature of the stripe with high content of ganglioside GM1 we determine similarly:

J st = J R 0 (1 − cst ) + J G cst ,

(2)

where cst is the ganglioside concentration in the stripe. GM1 concentration in the bulk raft phase and raft stripe in each monolayer should obey the condition of matter conservation:

S R cR + Lst d st cst = S0cG ,

(3)

where SR is the raft phase area, Lst is the length of the boundary of the raft phase, dst is the width of the stripe enriched by ganglioside GM1, S0 is the total area of the lipid monolayer surface, cG is the total concentration of ganglioside in the system. The location, width and composition of the stripes containing ganglioside are determined by minimizing the total elastic energy under the condition of Eq. (3). The resulting expressions are analytical, but very bulky, so we will illustrate the results graphically in the following section.

RESULTS AND DISCUSSION

AFM experiments. We performed AFM experiments for the membrane from “canonical” raftforming mixture of DOPC:SM:Chol (1:1:1) with and without ganglioside GM1. Resulting topography images are shown in Figure 2. In absence of GM1 we observed two clearly distinguishable lipid phases with the height difference of about 1 nm, meaning the formation of ordered domains in our system22-24. The average size of the higher phase domains and their fraction were in in good agreement with reported values for this system39. Although shape of domains was not perfectly round, it was also far from dendrite-like or fractal, seen in monolayers47. This fact, together with large average size of domains, allows concluding that: (i) domains were in liquid state; (ii) line tension of domain boundaries was rather high25. In ref39 authors demonstrate that Lo 8 ACS Paragon Plus Environment

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domains merge over time. It means that the area of the individual domains may change in time with the preserved conditions of the system. After such process, size of initial domains could be distinguished only near the merged domain boundary. Therefore, we overlapped circles of different diameter over domains of liquid-ordered phase near its boundary to find the best match (see Supporting Information, Fig. S1), and average diameter of such circles was set as the raft diameter for given lipid composition (Fig. 3). This phase pattern changed dramatically when the system contained GM1. For 0.25 mol. % of GM1 we observed fragmentation of the domains to smaller ones, with average diameter near 60 nm, and the formation of circular domains with the size of approximately 40 nm in diameter for 0.5 mol. % of GM1 (Fig. 3). Such abrupt reduction of average size of Lo domains indicates the sharp decrease of line tension of phase boundary21. It is generally accepted that GM1 preferentially redistributes to the liquid-ordered part of the membrane34. Note, that the distance between rafts was of the same order of magnitude as their size (tens of nanometers), so such pattern of rafts in membrane cannot be observed using conventional optical fluorescent microscopy50, resulting in artificial treatment of such pattern as homogeneous lipid membrane. This observation supports the idea that only part of real phase diagram for multicomponent lipid system can be detected optically50. At 0.75 mol. % of GM1 rafts became bigger again, and for 1 mol. % of GM1 in the membrane we observed the Lo domains similar in shape and size to the case of GM1-free system (Fig. 3). Some clusters of height near 2.5 nm and average diameter of about several nanometers appeared for 0.75 mol. % and higher content of GM1 (see Fig. 2). According to ref49, such clusters could be GM1 domains since this glycolipid forms domains by its own, or gel-phase clusters of GM1 with SM and Chol44. Another possibility is that the clusters resulted from long-tail fraction of egg SM, since this lipid extract from Avanti contains 3 mol. % of 22:0 fraction and 3 mol. % of 24:1 fraction. To exclude the case we performed the same experiments with synthetic pSM and obtained similar pattern of phase separation (see Supporting Information, Fig. S2), so long-tail fractions of egg SM are not responsible for formation of the above mentioned clusters. The increased average size of domains indicates the growth of line 9 ACS Paragon Plus Environment

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tension at their boundaries. For 2 mol. % of GM1 the average domain size and the morphology of domains almost totally coincide with that for the ganglioside-free raft-forming mixture (Fig. 2), with exception of some higher clusters staying near the domain boundaries. Therefore, line tension, being decreased at 0.25 mol. % and 0.5 mol. % of GM1, started to grow up for higher amounts of GM1 in the system. Increase of line tension for high GM1 content (up to 10 mol. %) is observed by fluorescent microscopy in giant unilamellar vesicles48. At the same time, this fact contradicts the results of the ref49, where authors show monotonic decrease of domain size with increasing molar fraction of GM1 in the membrane. However, the authors used different raft-forming mixture of lipids (DOPC:eSM:Chol ratio is 2:2:1 by mole). For clarity reasons we repeated their experiments for 0, 0.5 and 1 mol. % of GM1 and obtained the same results as in the ref. 49 (see Supporting Information, Fig. S3). In the next section, we analyzed the dependence of domain line tension on GM1 concentration basing on thickness mismatch model of raft boundary structure51,57.


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Figure 2. AFM topography images of membranes prepared from DOPC:eSM:Chol (1:1:1, by mole) mixture containing ganglioside GM1 with indicated concentrations in mol.%.


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Figure 3. Dependence of average Lo domain size on GM1 content (in mole %) in the membrane from DOPC:eSM:Chol (1:1:1, by mole).

Theoretical model. To clarify the non-linear effect of GM1 on the line tension of raft boundary observed in AFM experiments, we studied the distribution of ganglioside in the raft phase using the continuum model51,57. In this model, we assumed the energy of the raft boundary to be determined by deformations arising in order to compensate the thickness mismatch between raft and surrounding membrane. We calculated the energy of the raft boundary as a function of the ganglioside position in the membrane. A typical dependence of the line tension, γ, on the location of GM1 is shown in Fig. 4. We obtained that the optimal location of ganglioside is a narrow stripe is the top (Lo) monolayer of the intermediate zone II (see Fig. 1). It correlates with the fact that the transition zone of raft boundary is the most deformed region of lipid bilayer and ganglioside allows partial relaxing of the elastic stress. Thus, in further calculations we considered only one ganglioside-rich stripe located in the top monolayer of the transitional zone II (Fig. 1).


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Figure 4. The dependence of the raft boundary line tension on the position of ganglioside-rich stripe. The following parameters were used: the equilibrium thickness of Lo monolayer hr = 1.8 nm and Ld monolayer hs = 1.3 nm; spontaneous curvatures of the raft and the surrounding membrane were assumed to be zero (Jr = 0, Js = 0); splay modulus of Lo and Ld monolayers were BR = 30 kBT and BS = 10 kBT, respectively; tilt modulus Kt = 10 kBT/nm2 = 40 mN/m; ganglioside-rich stripe width l = 1 nm, its spontaneous curvature Jp = 0.1 nm–1.

We calculated the dependence of line tension of the raft boundary on the overall content of GM1 in the membrane (Fig. 5) for two sets of parameters. The first set (Fig. 5A) corresponded to our experimental system (DOPC:eSM:Chol 1:1:1): BR = 30 kBT (kBT ∼ 4×10–21 J), BS = 10 kBT, Kt = 10 kBT/nm2 = 40 mN/m, JG = 0.25 nm–1, hs = 1.3 nm, hr = 1.8 nm, JS = –0.14 nm–1, JR = –0.15 nm–1. The second set of parameters (Fig. 5B) corresponded to the experimental conditions of the work by R. Bao et al.49 for the DOPC:eSM:Chol 2:2:1 system: BR = 30 kBT, BS = 10 kBT, Kt = 10 kBT/nm2, JG 13 ACS Paragon Plus Environment

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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= 0.25 nm–1, hS = 1.3 nm, hR = 1.8 nm, JS = –0.09 nm–1, JR = –0.06 nm–1. Values of monolayer elastic moduli were taken from the refs59,60, spontaneous curvature of GM1 JG = 0.25 nm–1 was taken from the ref61, spontaneous curvature of all other components were taken from refs62,63: Js(Chol) = –0.44 nm-1, Js(DOPC) = –0.11 nm-1, Js(SM) = 0.12 nm-1. Spontaneous curvatures of the phases were set as concentration-weighted sum of spontaneous curvatures of their components. Phase composition is taken from DOPC:eSM:Chol ternary phase diagram measured in ref39 and summarized in table 1.

Table 1. Phase compositions and spontaneous curvatures. Component ratio (DOPC:SM:Chol) 1:1:1 2:2:1

Liquid-ordered phase Composition Spontaneous curvature 5% DOPC, –0.15 nm–1 48% eSM, 47% Chol 2% DOPC, –0.06 nm–1 67% eSM, 31% Chol

Liquid-disordered phase Composition Spontaneous curvature 72% DOPC, –0.14 nm–1 11% eSM, 17% Chol 75% DOPC, –0.09 nm–1 18% eSM, 7% Chol

Figure 5. Dependence of the line tension of the raft boundary on the total ganglioside content for two sets of parameters: (A) our system, DOPC:eSM:Chol 1:1:1; (B) the system described in ref49, DOPC:eSM:Chol 2:2:1.

In our experiments, small fraction of ganglioside significantly decreased the line tension of the raft boundary (see Fig. 2). It means that up to the concentration of about 0.5 mol. % this line-active 14 ACS Paragon Plus Environment

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component distributed exclusively to the raft boundary region. It resulted in increase of the width of the ganglioside-rich stripe and its spontaneous curvature. Although the total concentration of GM1 in the membrane is relatively small, its local concentration in the stripe near the domain boundary may reach tens of molar percent. This results in the formation of “gel”-phase clusters of ganglioside with eSM and Chol44 near the domain boundary already at 0.75 mol. % of GM1. Since the total area of such clusters is rather small (see Fig. 2), we assumed that GM1 mostly distributes between raft boundary and domain bulk region. However, the line tension trend for GM1 content higher than 0.5 mol. % depends on the initial spontaneous curvature of monolayer of raft and non-raft phases, i.e. on composition of the membrane (Fig. 5). Composition of phases in 1:1:1 and 2:2:1 systems differs mainly in SM and Chol amount (see Table 1). Absolute value of cholesterol spontaneous curvature (Js = –0.44 nm-1) is about four times larger than for SM (Js = 0.12 nm-1). Thus, cholesterol content generally determines the overall spontaneous curvature of the phase. After the threshold concentration of GM1 in the stripe, larger negative spontaneous curvature of phases in 1:1:1 system leads to upward trend of line tension (Fig. 5A). Relatively low spontaneous curvature in 2:2:1 membrane leads to reverse trend of line tension (Fig. 5B) described in the ref49 as decrease of average size of Lo domains.

CONCLUSIONS

In the present work, we developed the analytical model describing the behavior of liquid-ordered domains in the presence of line-active components such as ganglioside GM1. In the framework of the model, the major property responsible for line activity of the component was its non-zero spontaneous curvature, i.e. conical or inverted-conical effective shape of the molecule. Results of calculations predict that the influence of GM1 on the pattern of domain size distribution in lipid membrane greatly depends on the initial composition of the system described in terms of spontaneous curvature of its components. Cholesterol possesses significantly more pronounced 15 ACS Paragon Plus Environment

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spontaneous curvature of all the components used in raft-forming lipid mixtures, thus its content generally determines the overall spontaneous curvature of a phase. Therefore, we can conclude that in two cases presented here, the main factor determining the behavior of the system was amount of cholesterol: depending on its content, GM1 at certain concentration can either decrease or increase the line tension of the Lo domain boundary. The former case is observed together with the high content of sphingomyelin typical for outer leaflet of plasma membrane. This lipid forms gel-like domains even at physiological temperature (37°C), and this effect is the most pronounced in the absence of macroscopic Lo domains, as it is shown by raft disruption by cholesterol depletion64. The coexistence of gel and liquid-disordered lipid phases is deadly for cells since this leads to increase of ion permeability of plasma membrane65. On the other hand, for cells with high content of cholesterol, such as tumor cells66, the effect of gangliosides is reverse, as for the case of our DOPC:eSM:Chol 1:1:1. For concentration of ganglioside higher than 0.5 mol. % we should obtain almost linear increase of the line tension of the raft boundary leading to budding of raft vesicles at concentrations of GM1 higher than 2 mol. %48. The interplay of the observed two distinct dependencies of raft line tension on GM1 content one can see for the tumor-induced apoptosis in cytotoxic T-lymphocytes67. It is well established that tumor cells shed ganglioside and gangliosidecontaining microvesicles from their surface68, and we could explain this effect in the frameworks of the proposed model by high line tension of Lo domains. Incorporation of shedded gangliosides into normal cell membranes, such as plasma membranes of activated T-lymphocyte, should reduce the line tension of raft boundaries, for DOPC:eSM:Chol 2:2:1 leading to vanishing of Lo domains and following formation of gel-like domains of sphingomyelin64 or GM1-eSM heterodimers49. This process, acting similar to cholesterol extraction from cell membranes, will increase ion permeability of plasma membrane65, calcium mobilization in T-lymphocytes69, and disrupt raft-associated signal pathways necessary for normal functioning of T-cell70. All of the observed results can lead to cell death.


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The last thing noteworthy is the fact that we treat the ganglioside in our system just as a component with given spontaneous curvature. Thus, the proposed model is not limited exclusively to GM1, and applicable to the wide range of non-bilayer components of lipid membrane, which could also influence the raft size distribution and interaction.

ACKNOWLEDGEMENTS

Authors thank Professor Sarah L. Keller for fruitful suggestions for the work and Dr. V.I. Zolotarevskiy for great technical assistance. The work was supported by the Russian Foundation for Basic Research (projects #16-34-01203, #15-54-15006), by the Ministry of Education and Science of the Russian Federation in the framework of Increase of Competitiveness Program of “MISiS”, and by grant of the President of the Russian Federation MK-6058.2016.4.

SUPPORTING INFORMATION AVAILABLE Figures S1-S3. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org/.

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