Cholesterol and Sphingomyelin-Containing Model Condensed Lipid

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Cholesterol and Sphingomyelin-Containing Model Condensed Lipid Monolayers: Heterogeneities Involving Ordered Microdomains Assessed by Two Cholesterol Derivatives Marie-France Lecompte,†,# Gérald Gaibelet,‡,§,∇ Chantal Lebrun,∥ François Tercé,⊥ Xavier Collet,⊥ and Stéphane Orlowski*,‡,§ †

INSERM U563, Faculté de Médecine de Rangueil, 31062 Toulouse, France INSERM U563, CHU Purpan, 31024 Toulouse cedex 3, France § SB2SM and UMR8221/9198 CNRS, IBiTec-Saclay, CEA, 91191 Gif-sur-Yvette cedex, France ∥ INRA UMR 1331, 31027 Toulouse cedex 3, France ⊥ INSERM U1048, Université Toulouse III, UMR 1048, 31400 Toulouse, France ‡

ABSTRACT: Lipid monolayers are often considered as model membranes, but they are also the physiologic lipid part of the peripheral envelope of lipoproteins and cytosolic lipid bodies. However, their structural organization is still rather elusive, in particular when both cholesterol and sphingomyelin are present. To investigate such structural organization of hemimembranes, we measured, using alternative current voltammetry, the differential capacitance of condensed phosphatidylcholine-based monolayers as a function of applied potential, which is sensitive to their lipid composition and molecular arrangement. Especially, monolayers containing both sphingomyelin and cholesterol, at 15% w/ w, presented specific characteristics of the differential capacitance versus potential curves recorded, which was indicative of specific interactions between these two lipid components. We then compared the behavior of two cholesterol derivatives (at 15% w/w), 21methylpyrenyl-cholesterol (Pyr-met-Chol) and 22-nitrobenzoxadiazole-cholesterol (NBD-Chol), with that of cholesterol when present in model monolayers. Indeed, these two probes were chosen because of previous findings reporting opposite behaviors within bilayer membranes regarding their interaction with ordered lipids, with only Pyr-met-Chol mimicking cholesterol well. Remarkably, in monolayers containing sphingomyelin or not, Pyr-met-Chol and NBD-Chol presented contrasting behaviors, and Pyr-met-Chol mimicked cholesterol only in the presence of sphingomyelin. These two observations (i.e., optimal amounts of sphingomyelin and cholesterol, and the ability to discriminate between Pyr-met-Chol and NBD-Chol) can be interpreted by the existence of heterogeneities including ordered patches in sphingomyelin- and cholesterol-containing monolayers. Since such monolayer lipid arrangement shares some properties with the raft-type lipid microdomains well-described in sphingomyelin- and cholesterol-containing bilayer membranes, our data thus strongly suggest the existence of compact and ordered microdomains in model lipid monolayers.

1. INTRODUCTION Biological membranes are very generally formed by an assembly of amphiphilic lipids (mainly phospholipids and cholesterol) arranged in bilayers, in which various membrane proteins are embedded. These membranes are of key importance to realize compartmentalization between two aqueous media, which is crucial for a number of cellular functions. However, in a few cases, such as cytosolic lipid bodies/droplets1 (intracellular organels that store lipid energetic and metabolic reserves) and circulating lipoproteins2 (systemic shuttles that allow exchanges of lipids between tissues), it is necessary to wrap a hydrophobic phase (typically made of triglycerides and cholesterol esters). This last situation is suitably realized by a monolayer membrane (“hemimembrane”) made of amphiphilic lipids. In particular, the study of lipoproteins (belonging to different classes with different lipid compositions) is of importance since they are © XXXX American Chemical Society

involved in the pathophysiology of hypercholesterolemia and various cardiovascular diseases. Although the structural organization of their peripheral membrane is relevant for their specific interaction with their cognate receptors born by the target cells, which hence determines their various physiological actions, it still remains elusive. A model lipid monolayer provides a simplified (no curvature, no proteins, and defined lipid composition) yet valuable approach for addressing this question of the lipoprotein hemimembrane structure. In addition, such model lipid monolayers can also represent an approach for studying bilayer membrane aspects for which they can be assumed to present two rather independent leaflets.3 Received: July 17, 2015 Revised: September 22, 2015

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DOI: 10.1021/acs.langmuir.5b02646 Langmuir XXXX, XXX, XXX−XXX

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and 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) have been previously shown on model bilayer membranes to present contrasting behaviors. Indeed, Pyr-met-Chol can get inserted into different, more or less ordered, model bilayers like cholesterol with respect to its intramembrane distribution,19 whereas NBD-Chol insertion differs from that of cholesterol.20−22 This can be explained for Pyr-met-Chol by its wellsuited hydrolipophilic balance (better than a previously synthesized Pyr-O-Chol derivative),19 whereas NBD-Chol presents a constrained conformation due to the hydrophilicity and size of the NBD moiety grafted onto the alkyl chain that cannot remain in the hydrophobic core of the membrane as the cholesterol alkyl chain can.22 Since these two cholesterol derivatives are thus expected to differentially interact with ordered, raft-type membrane microdomains, we investigated their respective behaviors, and compared them with cholesterol, when included in phosphatidylcholine-based model monolayers of various compositions, with or without SM, in order to address the lipid arrangement of these monolayers, in particular regarding possible lateral heterogeneities. In this work, we observed clear-cut differences in the behaviors of cholesterol and the two cholesterol derivatives, Pyr-met-Chol and NBD-Chol, when included in model lipid monolayers, pointing out the importance of the amount of SM present in these monolayer membranes when considering their molecular arrangement, as addressed by the capacitance measurements. Furthermore, our data strongly suggest, based on observed analogies, the existence of ordered lipid (micro)domain-type lateral heterogeneities in SM- and cholesterolcontaining monolayer membranes that are reminiscent of the lipid raft microdomains well described in bilayer membranes.

A major advance in the molecular description of bilayer lipid membranes, including both model and physiological ones, has been the description of their lateral heterogeneities, witnessed by various types of microdomains, and especially with the emergence of the concept of lipid domains called “lipid rafts”.4−6 The existence of these raft domains is essentially dependent on the simultaneous presence of sufficient amounts of cholesterol and sphingomyelin (SM) (or other sphingolipids), and favored by specific interactions involving H-bonds between them.4 Due to a higher richness of saturated acyl chains, these domains are characterized by a more compact and ordered structural organization than other regions of the membrane on which they are “floating”. They have size smaller than optical microscopy resolution, and thus have been more or less indirectly evidenced using various biochemical (such as differential solubilization) and biophysical (such as single particle tracking) approaches. Their biological importance relies on their involvement in various cellular processes, in particular providing aggregation platforms for some membrane receptors and enzymes that regulate their functionning. However, the relationships between the two leaflets constituting a raft domain are still under debate.7 In this context, the question of whether related lipid domains could also exist in monolayer membranes of similar composition has been raised, and is here addressed, using a classical electrochemical interfacial approach that fruitfully completed the few other techniques already reporting on model monolayers and addressing the role of SM and cholesterol. We performed these studies on suitable model lipid monolayers with alternative current (AC) voltammetry, used under conditions allowing measurement of the electrical capacitance of a condensed lipid monolayer membrane constituting the interface between an electrolytic medium and a mercury electrode, and submitted to a variable applied potential.8,9 Since this interfacial capacitance of the monolayer mainly depends on the nature and the arrangement of its constituting molecules, this approach has been previously successfully applied to gain information about the effects of various lipid compositions on the interfacial properties of a stable monolayer,8,10−13 as well as for monitoring interactions of such a monolayer with exogenous small molecules11,12 or with amphipatic proteins8,10,13−16 added to the bulk aqueous electrolytic medium. Indeed, both interfacial capacitance10 and surface plasmon resonance studies17 have shown a two-step interaction of apolipoprotein A-I with monolayer membranes as a process relevant for initial HDL formation. Furthermore, association of capacitance measurements with Brewster angle microscopy and infrared reflection−absorption spectroscopy has allowed the showing of cluster formation in cardiolipincontaining monolayers, induced by mitochondrial creatine kinase and depending on the acyl chain composition.13,15 Here, we measured capacitance variations under different conditions as an operational parameter to investigate the molecular arrangement of a monolayer (in its initial, nonperturbed state) that includes cholesterol and/or SM, especially addressing its degree of order and homogeneity/heterogeneity. We used model lipid monolayers with the aim of finally addressing questions about lipid molecule arrangement in lipoprotein peripheral membranes, in connection with SM content since, besides cholesterol, it is low in HDL but high in LDL.18 Cholesterol derivatives are usefull molecular tools for investigating the structure of biological lipid membranes. Among them, 21-methylpyrenyl-cholesterol (Pyr-met-Chol)

2. MATERIALS AND METHODS 2.1. Chemicals. Pyr-met-Chol was a kind gift from Dr. A. Lopez (CNRS Toulouse, France ; patent WO/2006/100388), and stock solution was solubilized in chloroform/methanol (9:1); NBD-Chol (22-nitrobenzoxadiazole-cholesterol) was purchased from SigmaAldrich, and stock solution was solubilized in chloroform/methanol (2:1). Chromatographically pure egg lecithin (phosphatidylcholine, PC, grade I) and bovine brain SM were purchased from Lipid Products (Nutfield, United Kingdom) and supplied in chloroform− methanol (2:1) solution. 2.2. Electrochemical Method and Procedures. AC voltammetry consists of electrical admittance measurements that allow (at sufficiently low AC frequency) the determination of the differential capacitance (C, expressed per area unit, i.e., μF/cm2) of the interface between a hanging mercury drop electrode (HMDE) and an aqueous electrolytic solution, measured as a function of the electrical potential E applied between them. Here, the interface consists of a selfassembled lipid monolayer, and this lipid assembly essentially controls C due to its insulating property.16 By saturating the argon−solution interface and hence the electrode surface with lipids, the obtained selfassembled monolayer is considered “condensed”. It becomes a valuable model of a biological hemimembrane since it presents a defined molecular arrangement, dependent on its lipid composition. Since C is a measure of the accumulation of mobile charges at the close vicinity of the electrode, it reflects the accessibility of the electrolyte to the electrode. Thus, the C versus E curve recorded for a monolayer with a given lipid composition is sensitive to changes in its molecular arrangement that lead to various degrees of structural defects, hence providing some clues about the initial state of the lipid molecules. Initially, the hydrophobic side of the monolayer is in contact with the HMDE, and the hydrophilic one with the aqueous electrolytic medium (Scheme 1, top). We used a conventional three-electrode Metrohm polarographic cell. The working electrode, under electrochemical control, was the B


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C was stabilized at its lowest value and remained practically constant over a fairly wide potential range around the PZC (potential of zero charge) of the electrode (about −0.45 V), as described previously.11 Such a “saturation” of the C value was the indicator that the selfassembled lipid monolayer was obtained in a condensed state, allowing then recording of C vs E curves with good reproducibility: accuracy of less than 0.05 V for peak potentials and of about 10% for capacitance values. The potential range around PZC reveals the stability domain of this monolayer.

Scheme 1. Top: Schematic Representation of the Arrangement of the Lipid Monolayer Submitted to Electrical Differential Capacitance Measurements by AC Voltammetry;a Bottom: Proposed Molecular Organization, around the PZC, of Model Monolayers of Various Lipid Compositions (Depicted in the Inset)b

3. RESULTS 3.1. Differential Capacitance versus Applied Potential Curves for a Condensed PC-Based Lipid Monolayer Including (or Not) SM and Cholesterol. We first measured variations in the differential capacitance C in response to different applied potential E for a lipid monolayer made of PC alone in contact with the mercury electrode (Figure 1). When

a

The self-assembled condensed lipid monolayer is present at the interface between the electrolytic medium and the HMDE, and submitted to an applied, slowly varying negative potential E. For low values of the applied potential, around the PZC, the hydrophobic chains of the lipid molecules are in contact with mercury while their polar headgroups are surrounded by the electrolytic medium (the lipid molecules are not in scale with the Hg drop). bIn (a) and (b), the monolayer is in liquid-disordered state. In (c), the monolayer is in more compact, liquid-ordered state. In (d) and (e), the monolayer presents seggregated domains in liquid-ordered state (violet ellipse). Figure 1. Differential capacitance C as a function of the applied electrical potential E for PC-based monolayers. The C vs E curves were recorded for a PC monolayer, either alone (dotted line) or containing 5% (w/w) of both cholesterol and SM (full line), and for the aqueous electrolytic medium alone (dashed line). The sections (i), (ii), (iii), and (iv) depict the respective stages of the C vs E curve for the PC alone monolayer, as described in the text.

HMDE, formed at the extremity of a thin capillary tip and positioned in contact with the liquid phase, its surface covered by the phospholipid monolayer. Mercury was purified and twice distilled under vacuum. The Ag/AgCl saturated KCl reference electrode was used to measure and control the potential E applied at the HMDE. The AC was circulating between the HMDE and the platinum gauge counter-electrode. The equipment was a homemade device, according to what was described previously,23 in which the selective amplifier has been replaced by a lock-in amplifier. Electrochemical measurements were carried out as described previously.8,16 For C measurement, the frequency and amplitude of the sinusoidal signal were respectively 80 Hz and 10 mV. The differential capacitance versus applied potential response curves (“C vs E curves”) were recorded by applying a slow scanning of E over time (50 mV/s), this scan rate being chosen to maintain the system close to the equilibrium. The starting potential was set in the stable domain of the monolayer, namely at −0.2 V, while the curves were recorded by varying E toward increasingly negative values. Measurements were performed at 22 °C. Oxygen was displaced from the solution by argon bubbling before lipids were spread at the surface; then the experiments were carried out in streaming argon atmosphere. Dried lipid samples (PC and mixtures with SM and/or cholesterol or analogues) were dissolved in hexane, and spread at the surface of the aqueous electrolytic solution (0.15 M NaCl, 20 mM Tris-HCl, pH 7.4, 55 Ω.cm, in deionized water (Millipore ∼18 MΩ.cm)). After each addition of the lipid mixture studied with a new mercury drop, the state of the monolayer was checked by monitoring the C vs E curve. Additions were repeated until

scanning the potential from −0.2 V toward increasingly negative values, the C vs E curve recorded for such a monolayer exhibited four successive domains: (i) for E less negative than −0.7 V, a C value much lower than that of the electrolyte alone; (ii) for E ranging between −0.7 and −0.95 V, a rather thin double peak of C; (iii) for E more negative than −0.95 V, an intermediate C value clearly higher than in (i) (approximately half of the electrolyte alone); and (iv) for E around −1.25 V, a sharp peak of C, leading, for increasingly more negative E, to a C value similar to that of the electrolyte alone.10 Actually, this C vs E curve presents a specific profile, in particular including the flat phase (i) and the well-discernible peaks (ii) and (iv). The E range of the phase (i), around PZC = −0.45 V where C is the lowest, appears to be the stability domain of the molecular assembly in the monolayer since the wider this potential ranges, the more “resistance” to applied E is exhibited by the monolayer structure, hence bringing more stability.11 The peak (ii), whose steepness indicated a critical alteration of the C


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Langmuir lipid molecular assembly, is called the “transition peak”.24 It should be noted that such peak is only observed for condensed monolayers,24 which reinforces their propensity as exhibiting a defined structural organization. The peak (iv) is attributed to the desorption of the lipids from the electrode, and is called the “desorption peak”.25 Both peaks have been previously reported and studied for various lipid monolayers.9,26 The presence of significant amounts of other lipids in the PC monolayer induces modifications of the C vs E curve recorded, considering the potential values at which the transitions appear and the shape of the corresponding peaks, or even the presence or absence of these peaks. Indeed, as previously reported,10,12 the presence in a PC monolayer with either 10% cholesterol or 30% (w/w) SM induced alterations of the transition peaks, which were clearly broadened, with a decreased height and a less negative value of the onset potential. 3.2. Influence of Variable Amounts of SM and Cholesterol on the C vs E Curve of a PC-Based Monolayer. When the PC monolayer contained small amounts of both cholesterol and SM (5% w/w for each), forming a ternary monolayer, the C vs E curve presented the same clear transition and desorption peaks as for PC alone, but with a significant shift by 0.1 V toward less negative values of E for both of them (Figure 1). We then recorded the C vs E curve for a PC-based monolayer containing higher amounts of SM and cholesterol, 15% w/w each (Figure 2). Interestingly, we

We then checked the influence of variable relative amounts of SM and cholesterol, keeping constant the PC amount. For a monolayer made of PC70/SM10/Chol20 (% w/w), a broad transition was observed (Figure 2), starting at less negative E values (−0.35 V) than for a PC alone monolayer (see Figure 1). For a monolayer with inverted SM/Chol ratio, PC70/SM20/ Chol10 (% w/w), a steeper transition than for the SM10/ Chol20 monolayer was observed, although with the same onset potential (Figure 2). This indicated that a relative excess of either SM or cholesterol has a “destabilizing” effect (meaning reduced resistance against E) on the monolayer when compared to the SM15/Chol15 (% w/w) mixture, with a relative excess of cholesterol inducing a smoother transition. Futhermore, for the potential whole range (up to −1.1 V), the C value for the SM10/Chol20 mixture was lower than for the SM20/Chol10 one, which is compatible with the fact that it has been shown that monolayers containing a high fraction of cholesterol (a noncharged lipid) do not present a transition peak.27 Furthermore, both SM20/Chol10 and SM10/Chol20 monolayers exhibited a shift in their desorption peak toward less negative E values similarly to SM15/Chol15 (Figure 2), indicating a facilitated lipid desorption. Finally, a monolayer containing higher amounts yet a similar ratio of SM and cholesterol, PC40/SM30/Chol30 (% w/w), shows a quasiplateau profile of the C vs E curve (data not shown) similar to the SM15/Chol15 monolayer, but different than the monolayer PC90/SM5/Chol5 (% w/w), for which a clear double transition peak was observed (Figure 1). These data show the critical importance of a defined relationship between cholesterol and SM contents for lipid assembly stability of a PC-based monolayer. Further experiments were thus focused on monolayers with the 70/15/15 lipid composition. 3.3. Influences of Two Cholesterol Derivatives on the C vs E Curve of a SM-Containing PC-Based Monolayer. We first tested the effects of two cholesterol derivatives, Pyrmet-Chol or NBD-Chol, by including low amounts (i.e., substituting 10% of cholesterol) of either of them in the ternary monolayer 70/15/15 taken as reference. In both cases, the C vs E curve of the monolayer composed of PC70/SM15/ Chol13.5/Pyr-met-Chol1.5 or PC70/SM15/Chol13.5/NBDChol1.5 (% w/w) showed a similar profile (a quasi-plateau instead of a clear peak) when compared to the C vs E curve of the control monolayer PC70/SM15/Chol15 (data not shown). This indicated that these two cholesterol derivatives show no clear detectable effects when present in rather small amounts in a lipid monolayer. They will thus be further used as molecular tools for testing their ability to be substituted for all cholesterol molecules in the considered model monolayers. We then tested the effect of higher ratio of 15% (w/w) of cholesterol derivative, either NBD-Chol or Pyr-met-Chol, instead of cholesterol, in a PC-based monolayer containing 15% w/w SM. In the presence of NBD-Chol, the C vs E curve was very different from the pattern observed with cholesterol: (i) the C value around the PZC, before the onset of transition, was clearly higher, (ii) the potential for onset of transition (around −0.45 V) was largely less negative, (iii) a peak at −0.85 V within the transition domain was clearly detected, and (iv) the desorption peak was almost completely erased (Figure 3). By contrast, in the presence of Pyr-met-Chol, the C vs E curve was quite indistinguishable from what was observed with cholesterol (Figure 3). These data show that these two cholesterol derivatives exhibit clearly distinct effects when included in an SM-containing PC-based monolayer.

Figure 2. Differential capacitance C as a function of the applied electrical potential E for PC-based monolayers containing various relative amounts of SM and cholesterol. The C vs E curves were recorded for a PC-based monolayer made of 70% (w/w) of PC and of either SM 10/Chol 20 (dotted line), or SM 15/Chol 15 (full line), or SM 20/Chol 10 (dashed line).

observed an important depression of the double transition peak observed in the curves displayed in Figure 1, and it was replaced by a quasi-plateau between −0.7 V and −1.15 V, i.e., the onset of the desorption peak that still culminated at −1.25 V. D


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Figure 3. Differential capacitance C as a function of the applied electrical potential E for ternary, SM-containing, PC-based monolayers including cholesterol derivatives. The C vs E curves were recorded for a PC-based monolayer made of PC 70%/SM 15% (w/w) and containing 15% (w/w) of either cholesterol (solid line) or NBD-Chol (dot-dashed line) or Pyr-met-Chol (dotted line).

Figure 4. Differential capacitance C as a function of the applied electrical potential E for binary PC-based monolayers including cholesterol derivatives. The C vs E curves were recorded for a PC monolayer containing 15% (w/w) of either cholesterol (full line) or NBD-Chol (mixed line) or Pyr-met-Chol (dotted line).

domain can be schematically represented by using the three following independent parameters: (i) the onset potential of the first peak, (ii) the maximal capacitance corresponding to the peak height (Cmax; in case of a double transition peak, we considered the highest one) or the plateau level (Cplateau), and (iii) the transition potential range (the “peak width”, measured at half-maximum of peaks; in case of a plateau, we considered the whole potential range of the transition until the onset of the desorption peak). In order to conveniently compare and analyze the C vs E curves recorded for monolayers of various lipid compositions, we chose a graphic representation in a dispersion plot of the two last parameters, considering it best suited for visualization and analysis (Figure 5), also compared to the two other pairs of parameters (not shown). This dispersion plot showed that all experimental dots, representing data extracted from the curves obtained with the various monolayers tested, were not uniformly distributed, but their dispersion showed two dot clouds, reflecting two very distinct behaviors, which were related to lipid composition. Specifically, the left uppermost group (“group I”) is composed of monolayers made of either PC alone (or very rich), or binary mixtures with SM or cholesterol or NBD-Chol, or the ternary mixture PC/SM/NBD-Chol, while the right lowermost group (“group II”) is composed by monolayers made of the ternary lipid mixtures, with SM and cholesterol or Pyr-met-Chol and the binary mixture with Pyr-met-Chol.

3.4. Influences of Two Cholesterol Derivatives on the C vs E Curve of a PC-Based Monolayer without SM. In order to further characterize the influence of SM, we then considered binary PC-based monolayers (i.e., in the absence of SM) containing either cholesterol or a derivative. First, in the presence of 15% (w/w) cholesterol, we observed a double transition peak, starting at −0.6 V (Figure 4), that shifted to less negative values by approximately 0.1 V when compared to a PC-alone monolayer (see Figure 1). In addition, the desorption peak also positively shifted by about 0.05 V. The shift of the transition potential shows that the potential range of stable monolayer is restrained, which indicates perturbations in the molecular arrangement of PC-based monolayer in the presence of cholesterol. Then, in the presence of 15% (w/w) NBD-Chol, the double transition peak was fairly similar to that observed with cholesterol (Figure 4). In contrast, in the presence of 15% (w/w) Pyr-met-Chol, the double peak was clearly depressed in the range −0.7 to −0.9 V, with a steep increase of C at −0.65/ −0.70 V slightly shifted by −0.05 V (at its maximum) with respect to the first peak observed for Chol and NBD-Chol (Figure 4). In all three cases, the desorption peaks occurred at the same potential. These data confirmed the very distinctive properties of these two cholesterol derivatives when considering the monolayer interfacial electrical properties, and they bring further evidence that these properties strongly depend on the presence of SM (by comparison with Figure 3). 3.5. Dispersion Plot for the Various Collected C vs E Curves. In a C vs E curve, the “transition domain”, revealed by a C increase generally hallmarked by a capacitance peak but sometimes by a plateau instead of a peak, is expected to give insight into some characteristics of the initial, nonelectrically perturbed lipid arrangement of a monolayer. This transition

4. DISCUSSION For a given lipid composition of a tested condensed monolayer, electrical capacitance measurements lead to C vs E curves that, when analyzed, uncovers information on the lipid molecular arrangement, e.g., degree of organization, initially present in the unperturbed monolayer considered. More specifically, we demonstrated the importance of the respective amounts of E


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domain indicates that a 15/15% ratio of cholesterol/SM mixture in PC appears to be especially well suited to support a specific lipid arrangement in a monolayer. This stability was in agreement with our observation that the C vs E profile was not perturbed by the inclusion of a small amount (1.5%) of NBDChol in such ternary monolayer (taking into account that NBD-Chol is known to destabilize ordered bilayers). This requirement for the simultaneous presence of sufficient and defined amounts of SM and cholesterol for supporting such a particularly stable molecular arrangement in the monolayer can be interpreted as an indication of the existence of specific interactions between these two lipid components. Moreover, it is consistent with the presence of cholesterol- and SMcontaining (micro)domains reminiscent of the “lipid rafts” present in lipid bilayers.4−6 In order to test this possibility, we thus used two cholesterol derivatives, NBD-Chol and Pyr-metChol, in the absence or presence of SM, to take advantage of their differential properties reported in lipid bilayers. 4.2. Differential Effects of Inclusion of Pyr-met-Chol or NBD-Chol in Model Lipid Monolayers: Importance of SM. 4.2.1. Global Analysis. Interestingly, the dots illustrating various experimental C vs E curves in the dispersion plot between Cmax (or Cplateau) values and potential ranges of the transition domain segregated into two well-separated groups (Figure 5), showing two qualitatively different behaviors representative of two types of lipid monolayers. In group I, characterized by a clear transition peak, are found most of the binary mixtures and the ternary one PC/SM/NBD-Chol (Scheme 1a,b); these monolayers harbor a rather disordered homogeneous (i.e., “fluid”) state (see Section 4.2.2). In group II, characterized by a depressed transition peak, are found the binary mixture PC/Pyr-met-Chol and the ternary ones PC/ SM/Chol and PC/SM/Pyr-met-Chol (Scheme 1c,d,e); these monolayers harbor a more ordered, more or less heterogeneous state (see Section 4.2.3), as correlated with the respective presence of the two cholesterol derivatives. These data globally emphasize the importance of SM for lipid monolayer molecular arrangement, especially in the presence of cholesterol. 4.2.2. Binary Monolayers. In a binary PC-based model monolayer (i.e., without SM), inclusion of 15% either NBDChol or Pyr-met-Chol led to clearly different behaviors, when respectively compared to the physiologic component cholesterol (Figure 4). It should be noticed that, in that specific case, the two cholesterol derivatives do not appear to behave similarly in monolayers or in bilayers when respectively compared to cholesterol (although model bilayers considered in the literature are not made of PC alone). Indeed, NBD-Chol, similarly to cholesterol, induced a limited alteration of the C vs E curve with respect to a PC-alone monolayer, whereas Pyrmet-Chol induced a smoothing and broadening effect on the transition peak. It has been previously shown that a condensed phospholipid monolayer with highly fluid alkyl chains (e.g., unsaturated chains) led to a C vs E curve with a thin and wellmarked transition peak occurring at a rather highly negative applied potential,24 which is indicative of a stable structural organization for such monolayer, fairly suited to “resist” an electrical perturbation. By inference, our data can thus be interpreted by considering that, since the PC-alone monolayer used presents a rather high fluidity, inclusion of either cholesterol or NBD-Chol induces an alteration of fluidity that is rather limited, due to the presence of either the flexible cholesterol alkyl chain or the NBD moiety (as illustrated in Scheme 1a, left, and b, left: these binary monolayers belong to

Figure 5. Dispersion plot between the Cmax values and the transition potential ranges observed in the various C vs E curves presented for the model monolayers considered. The various lipid compositions of the monolayers studied are (1) PC, (2) PC + SM 30%, (3) PC + SM 5% + Chol 5%, (4) PC + SM 15% + Chol 15%, (5) PC + SM 10% + Chol 20%, (6) PC + SM 20% + Chol 10%, (7) PC + SM 30% + Chol 30%, (8) PC + SM 15% + NBD-Chol 15%, (9) PC + SM 15% + Pyrmet-Chol 15%, (10) PC + Chol 15%, (11) PC + NBD-Chol 15%, (12) PC + Pyr-met-Chol 15%. The dashed lines illustrate the separation between the two groups of experimental point clouds, corresponding to qualitatively distinguishable behaviors for model monolayers that present a different molecular organization, based on either liquiddisordered (left upper group, “group I”) or liquid-ordered arrangements (right lower group, “group II”).

SM and cholesterol, and we further highlighted clear-cut differences between the behaviors of two cholesterol derivatives, NBD-Chol and Pyr-met-Chol, used as “lipid disorder/ order sensors” (from their known properties in bilayers), when respectively compared to cholesterol. In the case of cholesteroland SM-containing monolayers, analysis of our data converged to indicate the existence of lateral heterogeneities that presented some similarities with the widely described lipid rafts in bilayer membranes. 4.1. Importance of Cholesterol and SM Content and SM/Cholesterol Ratio for the Molecular Arrangement of Model Lipid Monolayers. Since, for a given monolayer lipid composition and an applied electrical potential E, the measured C value reports on the accessibility of the electrolyte to the electrode; analysis of the C vs E curve profiles provides insight into perturbations of the lipid monolayer, leading to variable degrees of defects induced by the applied potential, which depend on its initial state. In particular, in a model monolayer containing SM (15%), the presence of 15% cholesterol induced a specific profile for the C vs E curve, with a new feature represented by the observation of an unusual quasi-plateau in the range −0.7 to −1.15 V instead of transition peaks recorded in other cases (Figure 2 compared to Figure 1). Remarkably, the presence of this quasi-plateau was dependent on both (i) relative amounts of cholesterol and SM (by comparison with the 20/10% and 10/20% ratios) and (ii) total amounts of cholesterol and SM (by comparison with the 30/30% and 5/5% ratios). These data provide evidence of a stoichiometric constraint on relative amounts of SM and cholesterol required for exhibiting a specific response to the applied potential E. In addition, the use of a ternary mixture containing cholesterol and SM at 15/15% exhibited the largest range of applied E below the onset potential of the transition peak, i.e., corresponding to a stable monolayer structure (as compared to ratios of 20/10% and 10/20%). This optimal stability F


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Langmuir “group I” in Figure 5). By contrast, inclusion of Pyr-met-Chol appears to induce a somehow decreasing effect on monolayer fluidity, which can be attributed to a “packing effect” of its rigid pyrene moiety inserted in the hydrophobic part of the monolayer (Scheme 1c; this monolayer belongs to “group II” in the dispersion plot displayed in Figure 5). Such an ordering effect of pyrene moiety is consistent with reported data on the insertion of a pyrene molecule in the core of model bilayer membranes28 (even if Pyr-met-Chol and pyrene may have slightly different positionings in the membrane), as well as with the in-silico behavior of pyrene-labeled phospholipids in membranes.29 More generally, this behavior of Pyr-met-Chol is also in accordance with reported preferential partitioning of polycyclic aromatic hydrocarbons, structurally related to pyrene, in the ordered lipid domains of model bilayers.20 4.2.3. Ternary Monolayers. In a ternary PC-based monolayer containing SM, 15% NBD-Chol or Pyr-met-Chol also led to different behaviors. However, when compared to cholesterol, each of them had an opposite effect to that observed in the absence of SM (Figure 3, as compared to Figure 4). Indeed, NBD-Chol led to a C vs E curve quite different from cholesterol, with a decreased potential range of stability, indicating a decreased stability of the monolayer structure, as well as an increased C value in this stability domain that suggested a significant electrolyte penetration in the monolayer, probably due to somehow disordered lipid molecules (Scheme 1b, right; this monolayer belongs to “group I” in Figure 5). In contrast, Pyr-met-Chol induced a C vs E curve virtually equivalent to the one obtained with cholesterol, in particular with a low C value in the monolayer stability domain, which indicated a good impermeability to the electrolyte. Comparison of these data with those obtained with binary monolayers (without SM) clearly shows that it is only in the presence of SM that Pyr-met-Chol displays the same effects as cholesterol, and thus behaves like it regarding a rather ordered environment of the molecular arrangement of the lipid monolayer (Scheme 1d,e; these monolayers belong to “group II” in Figure 5). Actually, our results demonstrating a pivotal respective influence of SM and cholesterol are in line with reports analyzing cholesterol- and SM-containing monolayer properties using various techniques, all indicating specific relationships between cholesterol and SM: (i) voltammetry experiments allowed to show that the redox properties of two hydrophobic molecules30,31 and of a lipophilic peptide32 were clearly different when included in a ternary monolayer containing simultaneously SM and cholesterol, as compared to monolayers of other compositions, which revealed the existence of a specific lipid environment; (ii) thermodynamic analyses of the effect of SM on the compressibility properties of a monolayer containing PC and/or cholesterol demonstrated that SM markedly alters its chemico-physical properties,33,34 which led to suggest existence of local heterogeneities, called “condensed complexes”;35,36 (iii) determination of cholesterol accessibility to either cholesterol oxidase or methyl-β-cyclodextrin showed that cholesterol displays preferential interactions with SM than with PC.37,38 As a whole, our results point out the existence of a monolayer lipid molecules arrangement (i) that requires a simultaneous presence of sufficient and defined amounts of SM and cholesterol, and otherwise (ii) that has the ability to differentiate between the two cholesterol derivatives, Pyr-metChol versus NBD-Chol, as compared to cholesterol. The

characteristics of such monolayer lipid arrangement are thus shared with those of the ordered microdomains described in bilayer membranes,4,5 where Pyr-met-Chol, similarly to cholesterol but in contrast to NBD-Chol, can get inserted within ordered lipids. Our observations are therefore in accordance with the existence of lateral heterogeneities, such as ordered (micro)domains, in SM- and cholesterol-containing monolayers. 4.3. Insights on the Molecular Arrangement of Model Lipid Monolayers Provided by the C vs E Curve Profiles: Indication of Lipid “Patches” for Cholesterol- and SMContaining Monolayers. The electrical potential E applied to lipid monolayers perturbs their lipid arrangement. In particular, the presence in the C vs E curves of transition peaks of various heights and widths, or even their absence, is a signature that could be associated with certain characteristics of the initial molecular organization of the lipid monolayers tested. The question is then to evaluate the consequences on the recorded C vs E curve of the presence of lateral heterogeneities within a lipid monolayer, as compared to a “simple” reference monolayer made of PC alone. 4.3.1. Molecular Significance of a Thin Transition Peak. When an homogeneous PC-based phospholipid monolayer is submitted to a low E, typically around the PZC (part (i) of the C vs E curve in Figure 1), phospholipids are submitted to dominating hydrophobic forces, and their polar headgroups are present in the aqueous phase while their alkyl chains are in contact with the Hg electrode (hence efficiently shielded from the electrolytic solution, giving a low C value: yellow lipid phase in Scheme 2a). By contrast, when E reaches sufficiently negative values (part (iii) of the C vs E curve), the electrical forces sensed by the phospholipids are dominating over the hydrophobic forces, and the polar headgroups are strongly attracted by the negative electrode, leading to an ill-defined molecular arrangement presenting a rather moderately increased C value (when remaining below the desorption peak: pink lipid phase in Scheme 2a). Thus, when following the C vs E curve between these two extreme situations, there is an obvious tendency for inversion of the orientation of the lipid molecules on the Hg electrode that progressively occurs within the transition peak (part (ii) of the C vs E curve).9,24,39−42 However, when the peak is reached, the opposite electrical and hydrophobic forces should somehow compensate their effect on the lipid molecules when becoming of similar strength. In simple cases, such as a PC alone monolayer, phospholipid molecules are expected to be independent from each other, and thus can individually respond to an applied E. Hence, when they are submitted to these dually acting forces, only a fraction of molecules (within a simplified electrostatic model, this corresponded to half39) will be randomly inverted when the peak reaches its maximum, which will lead to a “highly disorganized intermediate molecular arrangement”. Such a highly perturbed arrangement of lipid molecules on the electrode, whatever its molecular organization, should obviously lead to a large access of mobile charges in the electrolytic interface to the Hg electrode through the numerous defects likely present between the hydrophobic parts of these highly disordered (and probably highly dynamic) lipid molecules. Such a disruption of “continuity” of a lipid monolayer would well explain the high Cmax value measured at the peak26,27,39 (white lipid phase in Scheme 2a), as well as the unexpectedly very low mechanical resistance recently reported by measuring AFM tip threshold penetration force (≈10 pN), dramatically G


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Langmuir Scheme 2. Top Inset: Schematic C vs E curvesa; Bottom: Corresponding State of the Condensed Lipid Monolayer Self-Assembled at the Interface between the Electrolytic Solution and the Mercury Electrodeb

sense the applied potential and randomly respond to it to contribute to an intermediate structural disorganization. 4.3.2. Influence of Molecular Interactions on the Transition Domain. However, in various cases, monolayers lead to C vs E curves exhibiting a smoothened and broadened transition peak. This is typically observed for various lipid mixtures, as well as in the case of cytochrome c specifically interacting with a cardiolipin monolayer.16 In all those situations, this observation likely reflects an alteration of the “local homogeneity”,40 due to molecular interactions, and leading to a partial loss of the “one-by-one” behavior of the lipid molecules in the monolayer during its structural transition. Furthermore, in the specific case of a lipid monolayer structured in “patches” (or “domains/microdomains”), it can be expected that each of these patches (of not too large size) will undergo on its own (“bloc-by-bloc” behavior) an electrically induced inversion of lipid orientation, while preserving molecular cohesion within each of them. As a consequence, during this transition, the access of electrolytes to the electrode should be rather limited since lipid defects would only exist at the frontiers of, and/or between, these patches, so the Cmax value would be much lower than for a transition occurring within a PC alone monolayer (see Scheme 2b), hence leading to a depression in the transition peak. In addition, broadening over a large potential range of the lipid structural transition (giving a “quasi-plateau”) would be explained by the fact that different (micro)domains and interdomains would likely be submitted to an orientation inversion under different values of applied potential E because of different sizes and/or local lipid compositions (Scheme 2b). In the simultaneous presence of SM and cholesterol (in adequate relative amounts), the disappearance of any observable transition peak in the C vs E curve, replaced by a broad quasi-plateau with intermediate C values above the destabilizing threshold potential (Figure 2), would thus be an indication for the existence of compact and cohesive lipid microdomains, which appeared as clearly more plausible than an homogeneous monolayer. The necessary simultaneous presence of the two partners, SM and cholesterol, for this specific behavior as revealed by the C vs E curves, strongly supports that they are inherently involved in the formation of these microdomains, likely by a specific association that is reminiscent of that underlying the lipid rafts existing in bilayer membranes. 4.4. Specific Molecular Arrangement of Cholesteroland SM-Containing Model Lipid Monolayers: Possible Application to Lipoprotein Periphery. Altogether, our observations provide strong convergent indication that cholesterol- and SM-containing model monolayers present a molecular arrangement with lateral heterogeneities forming compact and cohesive ordered (micro)domains. Indeed, such heterogeneities (i) are expected to require a precise ratio between cholesterol and SM amounts for an optimal stability of the monolayer arrangement in response to the applied potential E, (ii) they should be characterized by a markedly different behavior between Pyr-met-Chol (presenting a good mimicking ability) and NBD-Chol (clearly contrasting) within monolayers, and (iii) they finally allow interpretation of a specific profile (i.e., pseudoplateau) observed in the transition region of the C vs E curve. These results thus reinforce previous reports on specific relationships between cholesterol and SM in lipid monolayers,30−38 and they even extend them by indicating a possible

a

Et is the potential of the transition peak, Ed is the potential of the desorption peak; E0 < E1 < Et < E2 < Ed. b(a) A “simple” monolayer (e.g., made of PC alone) composed of identical lipid molecules without lateral heterogeneities (in yellow). In the absence of electrical perturbation (E0), the monolayer limits electrolyte accessibility to the electrode, as illustrated by very few small dots depicting the mobile charges. When the applied electrical potential E increases from E1 to E2, the lipid molecules are submitted to a structural transition (yellow to pink lipid phase). At the intermediate state (Et), there is an even greater excess of electrolyte accessibility signing the transition, depicted by the presence of numerous small dots in the highly perturbed lipid phase (in white). Above the desorption peak (Ed), the electrolytic solution (blue phase) regains a quite full access to the electrode. (b) A monolayer presenting compact and cohesive lipid microdomains (in orange). At E0, their compactness makes them even more impermeant to the electrolyte than in (a). In the range between E1 and E2, due to their cohesion, each microdomain is submitted to a “one block” transition (orange to red domains), with no electrolyte penetration during the transition. In addition, since various domains and interdomains are submitted to the structural transition at different E values, the global excess of electrolyte accessibility in the monolayer remains very limited.

much lower than for a condensed monolayer and a stable bilayer (≈450 and ≈1500 pN, respectively).41 The observation of sharp transition peaks in a C vs E curve (e.g., for a PC alone monolayer) therefore indicates that, for the characteristic potential value corresponding to that peak, all the lipid molecules are able to identically, but individually (“one by one”, i.e., each one being independent from its neighbors), H


Langmuir

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existence of lateral domains. With regard to this aspect, our data are consistent with certain findings using two types of imaging experiments on model monolayers, and reporting apparent heterogeneities, although performed under somehow different experimental conditions than ours: (i) atomic force microscopy showed domains with specific morphologies in monolayers composed of lipid components of bilayer lipid rafts, i.e., cholesterol and sulfatides, which form “small granule structures” for mixtures around equimolarity,43 or PC/cholesterol and ganglioside GM1 (in low amounts),44 or PC/cholesterol and SM,45 but in all cases the size of these domains depended on the lateral pressure applied to the monolayer (which questions their compactness); (ii) fluorescence microscopy, using a phase-specific fluorescent probe (but limited by the optical resolution), allowed to observe micrometer-range domains for PC/SM/Chol/GM1 monolayers.35,46 The observation, in one case, of certain biochemical characteristics typical of bilayer rafts, i.e., inclusion of GM1, sensitivity to cholesterol-chelating agents and partitioning of a GPI-anchored protein,46 gave some confidence for a possible analogy between microdomains in monolayers and bilayers. Indeed, since some monolayers present behavioral similarities, regarding relationships between SM, cholesterol, and cholesterol derivatives, to bilayers harboring well-described lipid rafts,4−6 the analysis of our data suggests the existence of ordered (micro)domains, envisioned as “hemirafts”, in model monolayers. Finally, this model of microdomains in lipid monolayers could be considered as a basis for addressing questions on the molecular arrangement of peripheral hemimembrane in the lipoproteins, HDL and LDL, which is still rather elusive. Indeed, HDL periphery have been described to be rather highly disordered,47 whereas LDL, with hemimembrane containing much more SM compared to HDL, has been reported to present two phospholipid populations.48 Actually, we recently showed that Pyr-met-Chol is able to form excimers when inserted in the peripheral membrane of LDL, but not in that of HDL,49 and this property revealed an ordered environment in the LDL envelope that is consistent with the presence of “hemirafts”, which is possibly of physiological importance.

Article

AUTHOR INFORMATION

Corresponding Author

*Address: SB2SM and UMR9198 CNRS, I2BC/IBiTecS, CEA, Centre de Saclay, 91191 Gif-sur-Yvette cedex, France. Fax: 33 1 69 08 87 17. E-mail: [email protected]. Present Addresses #

SIMAD LU50, Université Paul Sabatier de Toulouse, 31062 Toulouse cedex 9, France. ∇ CNRS UMR 5203, Institut de Génomique Fonctionnelle, 34094 Montpellier cedex 05, France. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge Dr. André Lopez for providing Pyrmet-Chol, and Dr. Nawel Mahrour for manuscript reading and linguistic revisions. We give many thanks to Philippe Tauzin for curve processing. This work was supported by grants from INSERM, from ANR (PNRA 5.34 “Absinte”), and from VLM (FC0906). X.C. was a recipient of a “Contrat d’Interface” from CHU Toulouse; G.G. was a recipient of a “Prix de Recherche” from SFN.

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ABBREVIATIONS: Chol, cholesterol; NBD, nitrobenzoxadiazole; Pyr, pyrene; met, methyl; AC, alternative current; HMDE, hanging mercury drop electrode; PZC, potential of zero charge; PC, phosphatidylcholine; SM, sphingomyelin; HDL, high-density lipoprotein; LDL, low-density lipoprotein

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REFERENCES

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5. CONCLUSION Using AC voltammetry to study condensed model monolayers of various lipid compositions, we observed convergent evidence that characterize the simultaneous presence of SM and cholesterol. Indeed, in the presence of SM, Pyr-met-Chol, but not NBD-Chol, can mimick cholesterol as well as in model bilayers harboring ordered (micro)phases. Such a good correlation involving these two cholesterol derivatives, along with the fact that SM and cholesterol appear to display specific interactions between them, is strongly indicative of the presence of lateral heterogeneities in SM- and cholesterolcontaining monolayers, reminiscent of the widely described raft-type lipid microdomains in bilayers, which can thus be envisioned as “hemi-rafts”. Since the peripheral hemimembrane of LDL is rich in SM and cholesterol (more than HDL), and its structural organization is still rather elusive, our findings on condensed model monolayers are of paramount importance, since they allow us to propose a new structural organization model involving the presence of ordered lipid microdomains, “hemirafts”. I


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Langmuir cholesterol stably and specifically associates with lipoprotein peripheral hemi-membrane: a new labelling tool. Biochem. Biophys. Res. Commun. 2013, 440 (4), 533−8.

K


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