Milk Sphingomyelin Domains in Biomimetic Membranes and the Role

Article pubs.acs.org/Langmuir

Milk Sphingomyelin Domains in Biomimetic Membranes and the Role of Cholesterol: Morphology and Nanomechanical Properties Investigated Using AFM and Force Spectroscopy Fanny Guyomarc’h,*,†,‡ Shan Zou,§ Maohui Chen,§ Pierre-Emmanuel Milhiet,∥ Cédric Godefroy,∥ Véronique Vié,⊥ and Christelle Lopez†,‡ †

INRA, UMR1253 STLO, 35042 Rennes, France AGROCAMPUS OUEST, UMR1253 STLO, 35042 Rennes, France § Measurement Science and Standards, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada ∥ Centre de Biochimie Structurale, UMR5048 CNRS, U1054 INSERM, Université Sud de France, 34090 Montpellier, France ⊥ Université de Rennes 1, Institut de physique de Rennes, UMR6251, CNRS, 35042 Rennes, France ‡

ABSTRACT: Milk sphingomyelin (MSM) and cholesterol segregate into domains in the outer bilayer membrane surrounding milk fat globules. To elucidate the morphology and mechanical properties of theses domains, supported lipid bilayers with controlled molar proportions of MSM, dioleoylphosphatidylcholine (DOPC) and cholesterol were produced in buffer mimicking conditions of the milk aqueous phase. Atomic force microscopy imaging showed that (i) for T < 35 °C MSM segregated in gel phase domains protruding above the fluid phase, (ii) the addition of 20 mol % cholesterol resulted in smaller and more elongated lo phase domains than in equimolar MSM/DOPC membranes, (iii) the MSM/cholesterol-enriched lo phase domains were less salient than the MSM gel phase domains. Force spectroscopy measurements furthermore showed that cholesterol reduced the resistance of MSM/DOPC membrane to perforation. The results are discussed with respect to the effect of cholesterol on the biophysical properties of lipid membranes. The combination of AFM imaging and force mapping provides unprecedented insight into the structural and mechanical properties of milk lipid membranes, and opens perspectives for investigation of the functional properties of MSM domains during milk fat processing or digestion.

1. INTRODUCTION In milk, dietary lipids and liposoluble bioactive molecules are conveyed in the form of milk fat globules, i.e., soft matter assemblies of triacylglycerol droplets surrounded by the milk fat globule membrane (MFGM) organized as a trilayer of polar lipids and proteins.1,2 Recent observations by confocal laser scanning microscopy (CLSM) evidenced the presence of domains on the surface of milk fat globules.3−8 These domains were interpreted as the lateral segregation of milk sphingomyelin (MSM) and cholesterol into lipid rafts5 in analogy with rafts in mammalian plasma membranes.9 In mammalian cells, the rafts have been associated with trafficking and signaling functions, partially mediated by the ability of sphingolipidcholesterol platforms to control membrane curvature and fusion.10,11 The external bilayer of the MFGM derives from the © 2014 American Chemical Society

apical plasma membrane during secretion of the fat globule by the mammary cell.12 The major polar lipids found in the MFGM are phosphatidylethanolamine (PE), phosphatidylcholine (PC), MSM, phosphatidylserine (PS), and phosphatidylinositol (PI). MSM weights for 20−45 wt % of milk polar lipids1,6 while cholesterol, which exhibits a high affinity toward SM,13,14 is ∼30 wt % of the MFGM lipid fraction.15 SM and cholesterol are preferably located on the external leaflet of plasma membranes,16,17 hence probably on that of the MFGM. MSM contains several molecular species with long chain saturated fatty acids (16:0, 20:0, 22:0, 24:0) leading to Received: February 27, 2014 Revised: May 15, 2014 Published: May 16, 2014 6516

dx.doi.org/10.1021/la501640y | Langmuir 2014, 30, 6516−6524

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≥99%; Sigma-Aldrich, Milwaukee, WI, USA), NaCl 50 mM (Sigma), and CaCl2 5 mM (Sigma) were dissolved in Milli-Q water and adjusted to pH 6.7 using NaOH 5 M. Samples were prepared by dissolving appropriate stock solutions of MSM, DOPC, and cholesterol in chloroform/methanol (2:1 v/v) and mixing them in the following molar proportions MSM/DOPC: 50/50 and MSM/ DOPC/cholesterol: 40/40/20. The organic solvent was then evaporated under a stream of dry nitrogen at 50 °C. 2.2. Methods. For Differential Scanning Calorimetry (DSC) measurements, the dried mixtures were hydrated with PIPES buffer at 65 °C to reach a final concentration of 20% wt lipids. The dispersions were heated above the chain melting transition temperature of MSM and thoroughly mixed in a vortex stirrer to form large multilamellar vesicles. DSC measurements were performed with a DSC Q1000 (TA Instruments, Newcastle, DE). An aliquot of the samples (∼10 mg) was loaded into DSC hermetically sealed aluminum pans (TA Instruments). An empty, hermetically sealed, aluminum pan was used as reference. The calorimeter was calibrated with indium (ΔH = 28.41 J/ g; melting point = 156.66 °C). The samples were heated up to 70 °C and then cooled down to −5 °C at 5 °C/min. The temperature of MSM phase transition from the fluid phase to the gel phase (noted Tc) was determined as the intersection between the baseline and the first main exothermic peak recorded on cooling. For AFM experiments, both monolayers and bilayers were prepared. For monolayer preparation, ∼1 mM lipid mixtures in chloroform/ methanol (2:1 v/v) were gently deposited at the air/liquid interface of PIPES buffer at 20 °C in a 7 × 15 cm2 Langmuir trough (Nima Technology, Cambridge, UK). After 10 min evaporation of the solvent, lipid films were compressed by moving barriers at 5 cm2/min to 30 mN/m. The Langmuir film was transferred onto freshly cleaved mica plates at this constant surface pressure by raising the mica vertically through the lipid/liquid interface at 2 mm/min. The transfer ratio was typically 1. AFM imaging of Langmuir−Blodgett films (LB films) was performed in contact mode using a Pico-plus AFM (Agilent Technologies, Phoenix, AZ) at 20 °C using silicon nitride tips (nominal spring constant k ≈ 0.06 N/m). For bilayer preparation, the dried mixtures were hydrated with PIPES buffer at 65 °C to a final concentration of 0.1% wt lipids and then thoroughly vortexed. Small unilamellar vesicles (SUV) were produced at ∼65 °C by bath sonication30 or by extrusion through polycarbonate 0.1 μm-cutoff membranes (Avanti). Hot lipid solution (10 μg) was deposited onto freshly cleaved mica in an homemade brass liquid cell or in a humidity chamber, then incubated at ∼65 °C for 30 min. Slow cooling of the samples was performed using either inertia of the humidity chamber (∼50 mL water at 70 °C), a programmed incubator or a Peltier stage at rates sequentially decreasing from ∼1 to ∼0.1 °C/min. Once equilibrated at room temperature, the bilayers were extensively rinsed with PIPES buffer. AFM imaging was performed in contact mode using a Nanowizard II Bio AFM (JPK Instruments, Berlin, Germany), a Multimode 8 AFM controller (Bruker Nano Surfaces Division, Santa Barbara, CA, USA) or an MFP-3D AFM (Asylum Research, Santa Barbara, USA) and silicon MSNL probes (Bruker) with k ranging from 0.03 to 0.1 N/m (thermal noise method) and loading forces typically below ∼2 nN. Force mapping was performed at 20 ± 1 °C over a 3 × 3 μm2 (32 × 32 or 64 × 64 pixels) bilayer area using the Nanowizard instrument and MSNL probes. Typically, an applied load with a range of 2−12 nN, a piezo speed of 1 μm/s, and a data rate of 4096 Hz were used. The collected force curves were batch-analyzed using a self-developed algorithm.30,31 AFM images were typically planefitted at order 0 and then flattened at order 1 using the respective constructor softwares of the above AFM equipment. Sections were drawn across images to measure the height difference between features of the images. Image analysis was performed using ImageJ software (National Institute of Health, USA) to measure the area and perimeter of features in the images and to calculate circularity as 4π*area/(perimeter)2. Statistical analysis of the data was performed using Excel’s Student t test (Microsoft, Redmond, WA, USA), yielding a p-value, p.

tendency to interdigitation18 or to specific ordering.19,20 This indicates that the complex chemical composition of MSM in comparison to brain SM (∼50% 18:0-SM) or egg SM (∼85% 16:0-SM) could lead to specific physical properties. On milk ejection, dairy cows release about 600 m2 of MFGM per min, but little is known of how homeostasis of the plasma membrane is maintained.12 Dietary sphingolipids have moreover been associated with specific nutritional properties, like reduction of cholesterol absorption or diversion of pathogenic bacteria during digestion.2 Whether or not the MSM-enriched domains mediate biological functions and/or help control fat globule digestion by the host neonate is largely unknown but would help progress in, e.g., manufacture of infant formula milk.6,7 In recent years, atomic force microscopy (AFM) has proven to be a decisive technique to investigate the structure and mechanical properties of biological objects at the nanoscale.21 However, only a few authors have investigated the organization of milk polar lipids or of the fat globule using AFM. Balasuriya et al.22 observed changes in roughness and elasticity of native and processed fat globules in relation to physical damage or to interaction of the MFGM with milk proteins during processing. The presence of membrane proteins prevented closer resolution of the MFGM. Authors23−25 have reported that Langmuir−Blodgett monolayers of polar lipids isolated from milk fractions or processed milk show domains at various temperatures and lateral surface pressures. However, this approach may account neither for the respective roles of the lipid components nor for the hydrated multilayer structure of the MFGM. The preparation of supported lipid bilayers (SLB) has proven a well-adapted alternative to model cellular biomembranes.26−29 Therefore, the objective of the present study was to evaluate the effects of lipid composition and temperature on the formation, morphology, and mechanical properties of MSM domains, using SLB as a biomimetic model of the MFGM.

2. EXPERIMENTAL SECTION 2.1. Materials. The lipids, sphingomyelin from bovine milk (MSM; >99%), cholesterol (from ovine wool, >98%) and 1,2dioleoyl-sn-glycero-3-phosphocholine (DOPC; 18:1; >99%) were purchased from Avanti Polar Lipids (Alabaster, AL) and used as received. The N-acyl chains distribution of MSM is shown in Figure 1. PIPES (1,4-piperazinediethanesulfonic acid) buffer with an ionic strength and pH similar to milk was used: PIPES 10 mM (purity

Figure 1. Composition of bovine milk sphingomyelin given on Avanti Polar Lipids Web site. 6517


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grams of the pure MSM exhibited a clear, 10 °C-broad, exothermic transition at Tc = 35 °C (Figure 2) indicating crystalline ordering into a gel phase.32 Solubilization of MSM into 50 mol % DOPC broadened and delayed the phase transition to Tc = 23 °C. Cooling of the ternary mixture MSM/ DOPC/cholesterol showed that the partitioning of 20 mol % cholesterol decreased the phase transition of MSM, indicating a preferential interaction of cholesterol with MSM molecules. However, 20 mol % cholesterol did not totally abolish the phase transition of MSM. Fritzsching et al.33 also reported a phase transition on heating DOPC/eggSM/cholesterol in ternary mixtures with SM/cholesterol at 2/1 molar ratio. In spite of the larger affinity of cholesterol toward SM than DOPC,14 they showed that cholesterol partitions between SM and DOPC, thus reducing the proportion of cholesterol actually interacting with SM molecules. In conclusion, all systems exhibited order at ∼20 °C, albeit binary or ternary systems may not be fully stabilized. AFM imaging of MSM/DOPC (Figure 3) and MSM/ DOPC/cholesterol (results not shown) were performed upon cooling of the bilayers. In a first approach, stepwise plateaus at constant temperature allowed stable imaging of the bilayers at decreasing temperatures (Figure 3A,E). Alternatively, imaging was performed directly upon cooling at 1 °C/min while constantly trimming the loading force to correct the upward thermal drift (i.e., decreasing force) of the AFM probe while imaging (e.g., Figure 3B−D). In the latter case, only partial images could be obtained due to the limited range of the AFM photodiode, but they provided direct insight of transitional events occurring in a single sample. Since bilayers in the solid phase are thicker than bilayers in the fluid phase, higher

3. RESULTS AND DISCUSSION 3.1. Formation of Lipid Domains on Cooling of Bilayers. Both DSC and AFM were used to seek evidence of lipid ordering. DSC experiments performed on cooling of multilamellar vesicles allowed the determination of the temperatures of phase transition of MSM alone or in the presence of DOPC and cholesterol (Figure 2). DSC thermo-

Figure 2. Differential scanning calorimetry thermograms of multilamellar vesicules containing pure milk sphingomyelin (MSM 100), 50/50 mol % MSM and dioleoylphosphatidylcholine (MSM/DOPC 50/50) or 40/40/20 mol % MSM, DOPC, and cholesterol (MSM/ DOPC/cholesterol 40/40/20). The samples were prepared in PIPES/ NaCl/CaCl2 buffer, pH 6.7. The thermograms were recorded on cooling at 5 °C/min from 55 °C down to −5 °C.

Figure 3. Effect of temperature on the phase separation of supported lipid bilayers composed of milk sphingomyelin (MSM) and dioleoylphosphatidylcholine (DOPC) 50/50 mol %, as observed by AFM height imaging. The images were obtained at different temperatures during cooling from 60 °C at 1 °C/min. The 5 × 5 μm2 AFM height images were recorded at 45 °C (A) and 25 °C (E); the other images with x = 5 μm and various y scales were recorded upon cooling (B−D; see text). The color scale bars to the right of the images provide height references for the images and reveal transition temperature (Tc) of the MSM domains (in green) dispersed in the DOPC fluid phase (in orange), as presented in the bottom sketch (F). 6518


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Figure 4. AFM height images (5 × 5 μm2) of supported lipid bilayers containing 50/50 mol % milk sphingomyelin (MSM) and dioleoylphosphatidylcholine (DOPC, left column) or of 40/40/20 mol % MSM, DOPC, and cholesterol (right column) prepared as indicated in the left legend (A, B, C). Bilayers were imaged in PIPES/NaCl/CaCl2 buffer, pH 6.7 at room temperature. The color scale bars to the right of the images provide height references for the images (z-range). Cross-sectional line profiles are shown below each AFM height image.

systems.19,34 Presence of the mica substrate has also been reported to lower the phase transition temperature of POPE/ POPG by 3 °C when observed by AFM as compared to DSC.35 3.2. Changes in Morphology of the MSM Domains in the Presence of Cholesterol. AFM height imaging was employed to visualize MSM domains in the binary bilayers of MSM/DOPC and to visualize how cholesterol influences the lateral organization of SLB. AFM topographical images of hydrated MSM/DOPC (50/50 mol %) and MSM/DOPC/

domains consisting of MSM can be seen for T < Tc of MSM (Figure 3). Phase separation was clearly visible in SLB at temperatures ranging 30−35 °C (arrow in Figure 3), i.e., near the enthalpic transition of pure MSM bilayers rather than that of MSM/DOPC mixtures (Figure 2). AFM imaging displays surface topography of the sample and detects segregation of the MSM into salient features upon phase transition (Figure 3F), while DSC thermograms report on an ensemble of enthalpydriven events, such as phase separation in DOPC/SM 6519


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Figure 5. AFM height images (5 × 5 μm2) of Langmuir−Blodgett (LB) monolayers composed of 50/50 mol % milk sphingomyelin (MSM) and dioleoylphosphatidylcholine (DOPC, left column) or of 40/40/20 mol % MSM, DOPC, and cholesterol (right column) prepared as indicated in the left legend. Monolayers were imaged in air. The color scale bar to the right of the images provide height references for the images (z-range). Crosssectional line profiles are shown below each image.

domains when cholesterol was present in the SLB. In certain experimental conditions, the domains were connected to the extent that phase contrast was very low39,40 (AFM images in Figure 4C). In the LB monolayers (Figure 5), surface coverage by the domains did not seem to vary significantly depending on cholesterol addition, in contrary to SLB systems. The area of the domains only slightly increased from ∼0.05 to ∼0.07 μm2 upon cholesterol addition, but their circularity decreased from 0.6 ± 0.2 to 0.5 ± 0.2, indicating more contorted shapes (p < 0.001). Overall, the AFM height images indicated that the presence of cholesterol yielded narrow and more elongated MSM/cholesterol lo domains compared to those formed by the gel-phase MSM in the binary MSM/DOPC system (Figures 4 and 5). Similar results have been reported using other SM sources on LB monolayers28,36,39 or on bilayers.38,40 Rinia et al.37 found branched but larger domains on addition of cholesterol to DOPC/SM bilayers. Size and shape of the microdomains result from opposing forces from repulsion between the MSM molecules and from line tension at the phase boundary.14,41 When egg SM or 18:0-SM is used, transition of the domains into branched shape occurs upon addition of ∼30 mol % cholesterol37,38 rather than 20% as in the present study using MSM, and phase separation is no longer visible at about 50 mol % cholesterol. With brain SM, lower values of ∼20 and 33 mol % cholesterol were reported.40 In the cited reports, the cholesterol effect therefore becomes significant at SM/ cholesterol 3/1 to 1/1 mol/mol. In these conditions, the gelphase SM domains are transformed into an lo phase upon cholesterol addition and the continuous DOPC ld phase also tends toward lo ordering by excess cholesterol.34 Under these conditions of converging physical states of the two phases, line tension is reduced, allowing formation of irregular-shaped domains. However, phase diagrams may differ depending on the SM species, as suggested by the literature.

cholesterol (40/40/20 mol %) bilayers prepared following different procedures are shown in Figure 4, as well as images of LB monolayers (Figure 5). In the MSM/DOPC bilayers, the AFM height images showed the phase separation of MSM gel phase (lighter regions) and DOPC liquid-disordered (ld) phase (darker matrix) with the protruding of MSM domains from their ld environment. In the ternary bilayers of MSM/DOPC/ cholesterol, the AFM height images show the coexistence of the MSM/cholesterol-enriched liquid-ordered (lo) phase (lighter regions) and DOPC-enriched ld phase (darker matrix). This result is similar to the previous studies reporting phase separation in model membranes.36−38 Also, residual lipid vesicles, which did not fuse with the bilayer, appeared as brighter dots in the AFM images (see example in Figure 4C). In spite of the different preparation and cooling methods, the results showed robust trends when comparing the morphology of the bilayers, e.g., size and shape of the MSM domains in the gel phase (left panels in Figures 4 and 5) and the MSM/ cholesterol-enriched lo phase domains formed in the presence of 20 mol % cholesterol (right panels of Figures 4 and 5). In all three types of SLB, large μm-size MSM gel phase domains with corrugated contours occupied ∼35% of the total area of AFM images (left panels in Figure 4). These data demonstrate that a proportion of MSM molecules was dispersed in the ld phase at room temperature (MSM phase transition spreading below room temperature in the presence of DOPC, as showed Figure 2). In the presence of cholesterol, the AFM images showed narrower and contorted features, interpreted as branched and/ or networks of connected domains in the lo phase that occupied ∼60% of the total area of images (right panels in Figure 4). The circularity was 0.7 ± 0.2 for domains of MSM in the gel phase and 0.5 ± 0.2 for lo phase domains enriched in MSM and cholesterol (p < 0.001; circularity ranges to 0 for elongated object to 1 for perfect circle). These quantitative results confirmed the significant elongation of the shape of the 6520


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Figure 6. Typical force−distance curves (A: n = 10; C: n = 9) and the histograms of the breakthrough forces (B: n = 1675; D: n = 1024) respectively obtained on the 50/50 mol % milk sphingomyelin (MSM) and dioleoylphosphatidylcholine (DOPC; A and B) or on the 40/40/20 mol %. MSM, DOPC, and cholesterol (C and D) bilayers in PIPES buffer. The analysis was based on the single rupture events, and there was no obvious phase separation found on the rupture force maps. Gaussian distributions in dotted lines are drawn to guide the eye.

observed in egg-SM/DOPC (1/1 mol/mol) bilayers, and interpreted as an asymmetry in the monolayer composition (e.g., bilayer composed by a monolayer of solid SM and the other monolayer of fluid DOPC37). The existence of gel−gel separation has already been reported for bovine brain SM.42 The presence of subdomains in bilayers can be revealed by varying the imaging parameters and is indicative of a complex organization of the lipids in the domains.31,37 On Langmuir−Blodgett monolayers, significant average height difference between the two lipid phases have also been characterized on AFM images, with a decrease in H values from 1.4 ± 0.2 nm for the gel phase MSM domains down to 1.1 ± 0.1 nm for the lo phase domains formed in the presence of 20 mol % cholesterol (p < 0.001; Figure 5). The H values characterized in this work for MSM bilayers are in agreement with previous reports on other SM species.36−38,43,44 For monolayers, the measured H was somewhat higher than those previously reported on other SM species.23,36,39,45 Preferred partitioning of cholesterol into MSM domains results in the gel-to-lo phase transition of these domains, which reduces their thickness (thinning). As cholesterol increases, the DOPC l d phase could also progressively turn into lo phase (thickening) and could further contribute to reduce H values.14,34,38 3.3. Changes in Nanomechanical Properties of the MSM/DOPC Bilayers in the Presence of Cholesterol. AFM height images do not provide quantitative mechanical information on the bilayers. Hence, the nanomechanical properties of hydrated MSM/DOPC 50/50 mol % and MSM/DOPC/cholesterol 40/40/20 mol % bilayers were investigated at 20 °C (e.g., for T < Tc MSM; Figure 2) using force spectroscopy. Typical force curves in Figure 6 (top row) show that when applying near-normal load against the membrane using the AFM tip, a jump occurs in the force curve that indicates breakthrough and allows measurement of

Measurements of average height difference between the two lipid phases on AFM images (noted H in sketch of Figure 3F) was performed on various sections drawn across 2 to 3 different AFM images (see examples of sections in Figure 4). The results consistently showed that MSM/cholesterol-enriched lo phase domains were less salient than gel-phase MSM domains (Hlo < Hgel), although specific experimental procedures yielded differences in the average height values. More precisely, introduction of 20 mol % cholesterol in the bilayers induced a significant (p < 0.001) decrease of H from Hgel = 1.1 ± 0.3 nm to Hlo = 0.4 ± 0.1 nm in the set of AFM images obtained after cooling of the SLB using programmed incubator (Figure 4 A); from Hgel = 0.9 ± 0.1 to Hlo = 0.6 ± 0.1 nm after cooling of SLB on Peltier stage (Figure 4B) and from about Hgel = 0.8 ± 0.1 to Hlo = 0.6 ± 0.1 nm (Figure 4C). Interestingly, two levels of H could be characterized in the gel-phase MSM domains (left image in Figure 4A; slow cooling in an incubator), as indicated by arrows in the AFM height image and in the corresponding cross-sectional line profile of Figure 4A. This demonstrated the existence of gel−gel phase separation between the SM molecular species present in MSM. The lowest Hgel level of MSM gel phase domains was 0.8 ± 0.1 nm and the highest Hgel level was 1.3 ± 0.2 nm above the fluid phase (Figure 4A, left panel). These two levels of gel phase domains could correspond to the segregation of individual MSM species composed by different N-acyl chain length (see Figure 1). Indeed, 16:0-SM molecules could form a gel phase with a lower thickness of the bilayer than 24:0-SM molecules. Mismatch of the N-acyl chain in the gel phase could also explain the differences in H observed for MSM. These different H levels could be specific to natural mixtures of SM composed by individual molecular species with various chain lengths, such as MSM. Indeed, these two levels of gel domains do not exist in studies performed with SM with a single N-acyl chain length (e.g., 18:0-SM38). These intermediate levels were previously 6521


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Figure 7. Molecular scheme of the biomimetic bilayer of 50/50 mol % milk sphingomyelin (MSM) and dioleoylphosphatidylcholine (DOPC) at (A) temperature T exceeding Tc ≈ 34 °C in the presence (not represented) or in the absence of cholesterol, (B) at temperature T < Tc and in the absence of cholesterol and (C) at temperature T < Tc and in the presence of 20 mol %. cholesterol. ld: liquid disordered phase, lo: liquid ordered phase. Hgel and Hlo are the average height difference between the two phases. (B and C) The width of the arrows is proportional to the mean values of the breakthrough forces (FB) determined by force mapping on the bilayers.

the required force (breakthrough force).27,30 The breakthrough force is the maximum force that a bilayer withstands before its rupture and is used as a measure of the bilayer’s mechanical stability. When performing force mapping, up to ∼4000 values of breakthrough forces may be calculated across the 64 × 64 pixels of the scanned area, resulting in frequency histograms of breakthrough force values. Hence, a material with homogeneous nanomechanical properties should yield a Gaussian distribution centered on its mean breakthrough force value. During force mapping of lipid membranes, the high lateral resolution of the AFM can allow discrimination of two (or more) Gaussian distributions, indicative of separated phases with distinct nanomechanical properties.31 In the present study, the histogram of breakthrough forces calculated for the MSM/ DOPC 50/50 mol % bilayers was somewhat bimodal, with maxima at ∼1.7 and ∼3 nN (Figure 6B). A tail peak could also be determined at ∼5.5 nN (Figure 6B). This indicated heterogeneity in the nanomechanical properties of the MSM/ DOPC bilayer, albeit clear phase separation was not observed as expected.31,38,46 The correlated AFM height images and breakthrough force maps showed that the maxima of breakthrough forces at ∼1.7 nN could correspond to the shorter DOPC ld phase and that the breakthrough forces at ∼3 nN and ∼5.5 nN could correspond to the taller MSM gel phase (with the lower and higher Hgel levels, respectively). The histogram obtained for MSM/DOPC/cholesterol (40/40/20 mol %) bilayers showed two overlapped peaks of breakthrough forces with a mean value at ∼0.3 and ∼0.5 nN (see double Gaussian in Figure 6D) that could correspond to the DOPCenriched liquid-disordered (ld) phase and to the MSM/ cholesterol-enriched lo phase domains, respectively. The

histogram also included force curves without rupture events (Figure 6D). While lipid phase separation in the presence of 20 mol % cholesterol was visible in AFM topographical images (Figure 4), the lo and ld phases did not clearly exhibit distinct nanomechanical properties, which yielded an histogram where breakthrough forces were too close together to be well resolved (Figure 6D). Sullan et al.38 reported that the two distributions of the histograms started to be convoluted when cholesterol concentration increased in the bilayers, which coincided with the cholesterol-induced fluidization. Furthermore, breakthrough force mapping is sensitive to the mechanical stability of the bilayers.38 Hence, we related the two overlapped breakthrough force peaks to the low mechanical stability of the MSM/DOPC/cholesterol bilayers, as compared with stable bilayers of 18:0-SM/DOPC/cholesterol (2/2/1).38 The low stability of the bilayers could first be due to the temperature, since phase transition was not fully completed at ∼20 °C (Figure 2). It could also be due to the mixture of MSM molecular species with various chain lengths (Figure 1), and to the ionic strength of the samples (addition of NaCl and CaCl2 to mimic the aqueous phase of milk). Instead of force mapping, force curves can be separatedly collected on the lo and the ld phases of the bilayer sample, which helps discrimination of phases with closed nanomechanical properties, as reported on 18:0-SM/DOPC/cholesterol (37/37/26 mol %) bilayers.46 In this study, breakthrough forces were regarded as fingerprints of the bilayer mechanical stability determined at the nanometer scale. As compared to the MSM/DOPC bilayers, the addition of 20 mol % cholesterol significantly lowered breakthrough forces distribution from 1 to 7 nN to less 6522


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better understand the behavior of the MFGM domains during cold storage or processing of milk, or during milk fat globule digestion.

than 1 nN (p < 0.0001; Figure 6B,D). We showed that cholesterol significantly decreased resistance of the MSM/ DOPC bilayer to perforation and revealed the fluidizing effect of cholesterol in MSM/DOPC bilayers. This is in agreement with previous observations using pure 18:0-SM, where the breakthrough force shifted to lower values when the molar proportion of cholesterol increased.38 When gel phase SM is turned into the lo phase in the presence of cholesterol, lateral mobility of the molecules increases.14 Furthermore, cholesterol was also shown to decrease the interfacial elasticity47 and to increase deformability of SM membranes.48 Hence, molecules in a MSM/DOPC/cholesterol bilayer are expected to part more easily when the AFM tip pushes against the membrane. The fact that morphological changes in the MSM/cholesterol domains (Figure 4) occurred at lower proportion of cholesterol as those reported in studies using 18:0-SM or egg-SM31,38 further suggested that bilayers containing MSM-rich domains may be more sensitive to the fluidizing effect of cholesterol than other SM species. The range of measured breakthrough force values, 0 to 1 and 0 to 7 nN in the presence and in the absence of cholesterol, respectively (Figure 6), was lower than other measurements with respect to loading rate (3 to 5 nN on DOPC/18:0-SM/cholesterol38) or ionic strength of the buffer phase (4 to 14 nN on DOPC/SM/cholesterol27,46). Under some conditions, interfacial elasticity of the membranes was reduced by cholesterol to a larger extent when SM is 24:0 or 26:049 as in MSM; and may partially explain this difference. Figure 7 proposes a scheme of MSM/DOPC bilayer arrangement as a function of temperature and in the presence of cholesterol. The average height difference between the MSM gel phase and the fluid ld phase DOPC is noted Hgel, while the average height difference between the MSM/cholesterol enriched lo phase and the DOPC-rich ld fluid phase is noted Hlo ; with Hgel > Hlo. Also, Figure 7 integrates the information provided by force mapping and histograms of breakthrough forces recorded at 20 °C, highlighting the differences in the nanomechanical properties between the MSM/DOPC and MSM/DOPC/cholesterol bilayers and between the domains and the surrounding phase within each type of bilayer.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The Asylum Research MFP3B-BIO atomic force microscope was funded by the European Union (FEDER), the French Ministry of Education and Research, INRA, Conseil Général 35 and Rennes Métropole. The authors also gratefully thank the INRA division CEPIA for funding the project “Action Nouvelle Soutenue: Sphingolait”. S.Z. and M.C. thank Drs. L. J. Johnston and Z. Lu for discussions, and NRC Measurement for Emerging Technology program for the imaging facility support. F.G. thanks the COST action TD 1002 AFM4Nanomed&Bio for training and networking.

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REFERENCES

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4. CONCLUSIONS In this study, the influence of temperature on the formation of MSM domains and the role of cholesterol on the morphology, lateral organization, and nanomechanical stability of lipid bilayers with compositions that simulate the milk fat globule membrane were examined. Kinetics in temperature allowed us to observe by AFM the formation of MSM domains for Tc ≈ 35 °C. The presented results suggest the immiscibility of the MSM gel phase or the MSM/cholesterol lo phase domains with the fluid DOPC ld phase and highlight the roles of temperature and cholesterol in the physical properties of MSM domains in supported bilayer membranes. The use of AFM force mapping is a valuable complement to AFM imaging, as it provides unprecedented insight into lipid membrane mechanical properties that will contribute in a better understanding of their functions. These results are important both to better understand the functions of lipid rafts in the apical plasma membrane of mammary epithelial cells, and in the MFGM. Change in the structure and/or the physical properties of MSM domains on the surface of the milk fat globule may indeed bear important consequences in terms of, e.g., liability of the membrane to rupture upon deformation or to lipolysis by the digestive enzymes. This study therefore opens interesting perspectives to 6523


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Milk Sphingomyelin Domains in Biomimetic Membranes and the Role

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