Phospholipid Architecture of the Bovine Milk Fat Globule Membrane

Article pubs.acs.org/JAFC

Phospholipid Architecture of the Bovine Milk Fat Globule Membrane Using Giant Unilamellar Vesicles as a Model Haotian Zheng,*,†,‡,∥ Rafael Jiménez-Flores,∥ Derek Gragson,§ and David W. Everett†,‡ †

Riddet Institute, Palmerston North, 4442 Manawatu, New Zealand Department of Food Science, University of Otago, Dunedin, 9054 Otago, New Zealand ∥ Dairy Products Technology Center, California Polytechnic State University, San Luis Obispo, 93407 California, United States § Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, 93407 California, United States ‡

ABSTRACT: Giant unilamellar vesicles (GUVs) were constructed using an electroformation technique to mimic the morphology of the native milk fat globule membrane (MFGM) for the purpose of structural investigation. Bovine milk derived phospholipids were selected to manufacture GUVs which were characterized by confocal laser scanning microscopy after fluorescent staining. Circular nonfluorescent dark regions were observed in a 3/7 (mol/mol) surface mixture of 1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3 phosphoethanolamine. Linear shaped dark lipid domains were found in GUVs containing sphingomyelin (SM) in the absence of cholesterol. The dark regions were interpreted as a gel phase formed by a high gel−liquid phase transition temperature (Tm) of DPPC and SM. This study provides a strategy for investigating the lipid structural organization within the native MFGM using a model lipid bilayer system and reveals that a SM and cholesterol association network is not the only requirement for nonfluorescent lipid domain formation and that PE is preferably located in the inner leaflet of the phospholipid bilayer. KEYWORDS: milk fat globule membrane, giant unilamellar vesicles, confocal laser scanning microscopy, phospholipid bilayer

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ment.12 Argov et al. (2008) extended the discussion on the importance of MFG structure and pointed out that the effects associated with MFGs are only expected to retain nutritional and structural functionality without altering the unique macrostructure of MFGs.13 According to current and widely accepted topological models,4,5 the MFGM is a trilayer system with an outer phospholipid bilayer (containing cholesterol and membrane proteins), with a cytoplasm and phospholipid layer forming an inner layer covering the triacylglyceride core. MFGM components are not randomly distributed, as specific locations for different phospholipids have been reported,14 and lateral segregation of phospholipids has been observed by microscopic techniques.15 Nevertheless, as cellular membranes, the MFGM has a highly intricate architecture, and it is difficult to reveal the structure in terms of lipid and protein localization and lipid− protein interactions in its native state. It is still difficult to trace the specific functions in space and time of both membrane lipid−protein network and specific membrane domains which had been observed under confocal laser scanning microscopy (CLSM).15−17 In recent studies, a fluorescent headgroup labeled phospholipid analogue 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (RdDOPE) was used to label MFGs in which lateral segregation and lipid domains were characterized for both bovine and

INTRODUCTION Fat typically accounts for 3.5−4.7% of bovine milk in the form of milk fat globules (MFGs) varying in size (mostly between 0.2 and 15 μm in diameter), suspended in the aqueous phase of milk as an emulsion system.1,2 The MFGs are stabilized by a physiological functional milk fat globule membrane (MFGM). This membrane is mainly constructed of glycerolphospholipids and sphingolipids (with a small amount of cholesterol) as the backbone in which many different proteins, including glycosylated proteins, cytoplasm proteins, and enzymes, are associated.3−5 Although the MFGM accounts for 2−6% of the mass of MFGs, phospholipids and cholesterol together only make up about 27% of the mass of MFGM materials.1 These components are significantly important for both physical and physiological processes.6−8 Biomembranes act as barriers between different biological compartments that govern communication between living cells and are involved with the passage of signals.9 As a consequence, the MFGM in its native and integral form plays an important role in physiological processes including nutrient adsorption, digestion, and signal exchange, especially for neonates who use milk as essential food. Michalski and Januel (2006) reviewed the controversial and largely positive effects of homogenization of bovine milk on human health in which the macrostructure of MFGs was altered and the MFGM was broken.10 Coverage of MFGs with the native MFGM in human milk results in more efficient gastric lipolysis than for homogenized lipid droplets in infant formula;11 other recent work has shown that both the size and the interfacial region of MFGs may determine triglyceride bioavailability in a simulated digestive environ© 2014 American Chemical Society

Received: Revised: Accepted: Published: 3236

January 7, 2014 March 18, 2014 March 18, 2014 March 18, 2014 dx.doi.org/10.1021/jf500093p | J. Agric. Food Chem. 2014, 62, 3236−3243

Journal of Agricultural and Food Chemistry

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human MFGs.16,18,19 These studies discussed the possibility that the nonfluorescent dark domains are lipid rafts (liquidordered phase, Lo) enriched in SM and cholesterol15,16 as RdDOPE is preferably portioned into liquid-disordered phase (Ld).20 This proposal remains controversial as it has been demonstrated that the coexistence of phase regions [for instance, gel (Lβ) + liquid crystalline (Lα) and Ld + Lo)] may not always distinguish between Lo and gel regions, thus giving misleading information about lipid surfaces when using fluorescent imaging techniques.21 MFGM contains relatively high Lβ−Lα phase transition temperature (Tm) phospholipids [for instance, 16:0-PC (DPPC) makes up ∼11.1% (w/w) and SM makes up ∼29.6% (w/w) of total polar lipids in bovine milk]2 which remain in a gel-phase state at ambient temperature, and the Lβ may contribute to the dark regions on MFG surfaces by CLSM. Although the Lβ phase may be transformed to the Lo phase by the inclusion of cholesterol,21 the molar amount of cholesterol is less than the molar amounts of the high Tm phospholipids in the MFGM,1 consequently, the existence of an Lβ phase in the MFGM at ambient temperature is expected. A supported monolayer model system was prepared from milk-derived phospholipids to examine lipid−lipid interactions and phase coexistence.22 However, nonsupported phospholipid bilayer systems are rarely used to model the MFGM. Giant unilamellar vesicles as an artificial bilayer membrane system constructed from phospholipids with/without cholesterol have been used to mimic the morphology of native biomembranes and to investigate interactions between proteins and lipids and specific functions of lipid domains.9,23 Techniques such as fluorescent imaging24,25 have provided fundamental knowledge of GUV formation. In the current study, we have constructed GUVs from milk-derived phospholipids and use this as a model to examine the lipid morphology of the MFGM.

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the lipid domain formation. Set 1: DPPC, DPPC + DOPE (7/3, 1/1, 3/7, mol/mol), and DOPE; set 2: SM, SM + DOPE (7/3, 1/1, 3/7, mol/mol), and DOPE. In simulating the MFGM composition, an extra sample containing DPPC, SM, DOPE, and cholesterol (8/8/8/4, mol/ mol or 28.6 mol % for polar lipids and 14.3 mol % for cholesterol) was prepared to mimic the morphology of the native MFGM. It has been reported that the weight ratio between phospholipid and cholesterol in the MFGM is 25:2, w/w (∼25:4, mol/mol, calculated based on the mean molecular weight) in which phosphatidylethanolamine (PE), phosphatidylcholine (PC), and sphingomyelin (SM) are the major phospholipid constituents (>90% w/w of total phospholipid);1 moreover, DOPE (47.0% PE, w/w) and DPPC (32.2% PC, w/w) are the major subspecies.34 DPPC, DOPE, and SM were chosen in this study as they are the major phospholipid species in bovine milk,2 and they have a broad range of Lβ/Lα phase transition temperatures.26,27,35,36 Phospholipids were dissolved in chloroform to yield 10 mg/mL sample solutions which contained Rd-DOPE or NBD-PC (0.25 mol %) as a fluorescent dye during the electroformation of GUVs. Electroformation of GUVs. Briefly, 2.5 μL lipid formula sample solutions were placed on the surface of ITO slides. After completely evaporating any residual moisture in a desiccator (room temperature, 1 h), the samples were rehydrated in 100 mM sucrose solution. GUVs were formed under AC field conditions (3 V, 10 Hz, ∼55 °C) in a custom designed chamber for 45 min.24,32 The prepared GUVs were taken from the electroformation chamber and placed into 100 mM glucose solutions (1/4, v/v) using a wide-mouth pipet (>1 mm diameter), at which point the newly formed GUVs were ready for CLSM observation. It was reported (supported by our own observations) that aging or insufficient cleaning of ITO slides may affect the GUV electroformation process resulting in a low yield of GUVs; therefore, the ITO slides were handled by a standard cleaning procedure following a mild annealing process.31 The fluorescent intensities at the boundary of vesicles (liquid crystalline phases) were measured to ensure that these exhibited a bilayer structure.29 Electroformation was carried out in duplicate for each lipid combination. CLSM Observation of GUVs. GUV preparations were pipetted onto observation slides containing a small well (∼3 mm depth) for holding GUVs without extrinsic pressure from the coverslip. An Olympus FV1000 inverted confocal laser scanning microscope (Olympus America Inc., Center Valley, PA) with oil-immersion objective lenses (40× and 60 ×) was used to observe the structural properties and morphology of GUVs. Fluorescent signal collection (for rhodamine B and NBD) and the CLSM configuration were adopted from our previous work,16 with modification. Rd-DOPE and NBD-DLPC were excited by a diode laser (559 nm) and Ar + laser (488 nm), respectively, and the emitted spectrum was measured between 570 and 670 nm and 500−600 nm, respectively. 3D images of GUVs were constructed from a group of individual 2D images taken across a defined z-axis section. Differential interfacial contrast illumination was used in the transmitted light channel for determination of the positioning of GUVs. All of the image processing was carried out at 22 ± 1 °C. The operating parameters of the CLSM were not necessarily kept constant for each image as the parameters were optimized to obtain the clearest images of GUVs. This was deemed to be acceptable as relative quantification of phospholipids was not the focus of the study.

MATERIALS AND METHODS

Materials. Indium tin oxide (ITO)-coated glass slides (surface resistivity 30−60 Ω/cm2) were purchased from Sigma-Aldrich (Saint Louis, MO). The fluorescent fatty acid-labeled phospholipid analogue 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}sn-glycero-3-phosphocholine (16:0−12:0, NBD-PC, 1 mg/mL in chloroform) and the fluorescent headgroup-labeled phospholipid analogue 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) ammonium salt (18:1, Rd-DOPE, 1 mg/mL in chloroform) were chosen as fluorescent probes; 1,2-dipalmitoyl-snglycero-3-phosphocholine (16:0-PC, DPPC in powder form, Lβ/Lα phase transition temperature 42 to 52 °C),26 1,2-dioleoyl-sn-glycero-3phosphoethanolamine (18:1-PE, DOPE in powder form, Lβ/Lα phase transition temperature −9.9 to −2.9 °C),27 and sphingomyelin (SM, fractionated from bovine milk and 16:0, Lβ/Lα phase transition temperature 35 to 82 °C)28 were all provided by Avanti Polar Lipids, Inc. (Alabaster, AL). Sucrose and glucose (both anhydrous) and chloroform, acetone, methanol, and water (HPLC grade) were all sourced from Fisher Scientific (Fair Lawn, NJ). A function generator (model 72-7710) was purchased from TENMA test equipment (Springboro, OH), and a digital phosphor oscilloscope (model TDS 3012, 100 MHz, 1.25 GS/s, 10K point) was provided from Tektronix (Beaverton, OR). Electroformation of Giant Unilamellar Vesicles (GUVs). GUVs were generated from combinations of phospholipids under an alternating current (AC) electrical field24,29−33 with modifications. The detailed sample plan and operation are explained as follows. Sample Plan and Preparation. GUVs were formed from unitary, binary (SM is considered as unitary), and quaternary systems including DPPC, DOPE, SM, and/or cholesterol. Two sets of sample groups were prepared for studying the effects of DPPC and SM on regulating

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RESULTS AND DISCUSSION 1. Fluorescent Staining and Electroformation of GUVs. To test the staining ability of the headgroup dye (RdDOPE) and fatty acid-group dye (NBD-DLPC) for fluorescent labeling of different phospholipids and sphingolipids in the model membrane system, the two dyes were applied to two unitary GUV systems (DPPC and SM), respectively. The zwitterionic glycerophospholipid PC and the zwitterionic phosphosphingolipid SM are more enriched in the outer leaflet 3237

dx.doi.org/10.1021/jf500093p | J. Agric. Food Chem. 2014, 62, 3236−3243


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the surfaces of GUVs. Nonfluorescent regions were found on the surfaces of GUVs made from a DPPC-DOPE (3/7, mol/ mol) binary system (Figure 2).

monolayer in the membranes of human and animal cells; moreover, in the case of MFGM, it has also been reported that PC and SM are located on the outside layer of the MFGM.14,37 Therefore PC and SM unitary systems were used in the preliminary experiments. The headgroup-labeled DOPE phospholipid analogue appeared to stain the surfaces of DPPC- and SM-induced GUVs systems well; the fatty acid group-labeled DLPC phospholipid analogue stained the DPPC GUV system well, whereas GUVs could not be successfully constructed from the NBD-DLPC-labeled SM system under the same GUV generation conditions. The reason for this unsuccessful construction is uncertain; it may be due to the current electroformation conditions not being capable of generating GUVs from this specific lipid combination (NBDDLPC + SM). Consequently, headgroup-labeled Rd-DOPE, a widely used fluorescent probe in MFG studies15,38 was used as the primary dye for probing the surface morphology of constructed GUV systems derived from milk phospholipids. No noticeable differences were apparent between headgrouplabeling and fatty acid-labeling when comparing DPPC bilayer systems (images not shown). In previous work, Rd-DOPE was first used for labeling the surface layer of native MFGs: the dark regions, which were not stained by Rd-DOPE, were interpreted as Lo (lipid rafts) domains thought to be enriched in SM and cholesterol.15 A possible hypothesis is that the PE-coupled headgroup fluorescent dye (Rd-DOPE) may not stain the outer monolayer components properly, which mainly consists of PC and SM. This would create unstained or heterogeneously stained regions on the surface of MFGs where dark regions occur through the absence of Rd-DOPE, and these regions may not necessarily be liquid disordered (Lo) regions. Consequently, a GUV system in which the membrane composition can be controlled is an ideal tool for testing the staining capability of fluorescent labels in different bilayer systems (such as Rd-DOPE). Electroformation of GUVs was carried out successfully where the Rd-DOPE probe was located at the periphery of the GUV, and the yield of GUVs was sufficient for further analysis. 2. GUVs Constructed From DPPC and/or DOPE. The combinations of DPPC (Tm: 42 to 52 °C) and DOPE (Tm: −9.9 to −2.9 °C) in the formulation of GUVs was based on the range of Lβ/Lα phase transition temperatures. No dark regions (nonfluorescent regions) could be found in the DPPC unitary and two DPPC-DOPE binary (7/3, 1/1, mol/mol) GUVs systems (Figure 1). There were no characteristic differences in surface morphology of these samples. The 3D images (Figure 1A), that were built from 2D cross-sectional images, show that the Rd-DOPE fluorescent probe was homogenously spread on

Figure 2. GUVs formed from DPPC.DOPE (3/7, mol/mol). A: 2D image taken at the top/bottom section of GUV, regular circular shape of dark regions (DRs) are shown by white arrows; B: 2D image taken in the middle section of a GUV, DR is shown by white arrow. C: 3D image showing apparent morphology of GUV in mimicking the morphology of native MFG. Scale bar: 5 μm.

Lissamine rhodamine B is a well-characterized fluorescent probe used for studying phase coexistence on GUV surfaces.29,39 Bagatolli and Gratton29 reported that Rd-DPPE molecules are segregated from the gel domains and locate in the liquid (crystalline) disordered domains in a DPPE-DPPC GUV system, which supports the current results where the liquid disordered domains are characterized as the location of Lissamine rhodamine B-labeled PE. Rd-DOPE, as a primary fluorescent dye, has been also used to probe the lateral segregation and morphology of the native MFGM.4,15,16 Lopez and co-workers (2010) applied Rd-DOPE as a probe to determine the location of milk phospholipids in the MFGM and found that Rd-DOPE did not partition into the triacylglycerols interior of MFGs but was distributed heterogeneously on the surfaces of MFGs.15 Consequently, the black interior regions of GUVs with no fluorescent emission are not considered in the current research.

Figure 1. Comparison of GUVs formed from DPPC and/or + DOPE (unitary and binary systems). A: DPPC, 3D image; B: DPPC/DOPE, 7/3 (mol/mol), 2D image; C: DPPC/DOPE, 1/1 (mol/mol), 2D image. Scale bar: 20 μm. (Dark regions on the surface of GUVs were not commonly observed.) 3238



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In previous work,15 the sizes of liquid-ordered or gel domains ranged from 0.6 to 2.7 μm depending on the size of the MFGs, and this is comparable with our current results as shown in Figure 2A and B from an artificial phospholipid membrane system containing only DPPC-DOPE (3/7, mol/mol). Also, the apparent morphology of the nonfluorescent emission domains, which correspond to the circular shapes on the GUV surface (Figure 2A), is in close agreement with that reported for MFGM by Lopez and co-workers.15 Evers et al. (2008)40 observed heterogeneity on the MFGM using different fluorescent probes and concluded that the lack of MFGM fluorescent emission was due to the absence of an outer bilayer. Lopez et al. (2010)15 challenged this hypothesis and suggested that the triacylglycerol inner core of the MFGs is unlikely to be directly in contact with the aqueous phase without surface coverage. Nevertheless, considering the current and widely accepted trilayer topology of the MFGM,4,5,15,41,42 it is reasonable to expect that the primary monolayer containing glycerolphospholipids and membrane proteins located underneath the outer bilayer may still maintain the stability of MFGs. An exposed protein inner monolayer on the surface of MFGs has been observed after centrifugal mechanical treatments;43 therefore, the explanation of the bare surfaces on MFGs, as reported by Evers et al. (2008),40 can be rationalized. However, this theory cannot explain the appearance of dark domains in the current GUV system containing only phospholipids, as a trilayer system is not present. Several CLSM studies4,15,16,18,19 suggest that the dark domains on Rd-DOPE fluorescently labeled MFGM are lipid rafts44−46 (analogous to Lo), comprising of saturated phospho/sphingo-lipids (mainly SM) and cholesterol. Dark domains, however, were generated in our artificial nonsupported model membrane system (Figures 2), containing only DPPC and DOPE (3/7, mol/mol). This phenomenon, to some extent, supports our hypothesis that the presence of nonfluorescent dark domains on the MFGM depends upon the proportion of the liquid phase: nonfluorescent regions found for DPPC/DOPE 3/7 mol/mol but not for DPPC/DOPE 7/3 nor 1/1 mol/mol. Consequently, the Lo phases (lipid rafts), which are mainly formed from SM and cholesterol,47 and the gel phases formed by the high Tm phospholipids (DPPC ∼11.1%, w/w in total bovine milk polar lipids,2 in this case) may both contribute to the formation of dark regions. In previous research4,15,16 and in our current work, CLSM characterization was carried out at ambient temperature (19− 22 °C), which is lower than the Tm of DPPC required to induce gel structure formation. Bagatolli et al. (2000)29 characterized the phase state of the lipid domain by using an excitation generalized polarization (GP) function at different temperatures. The CSLM GP images clearly show gel−liquid crystalline phase separation (DPPE/DPPC, 7/3, mol/mol) at the solid and fluid phase coexistence temperature region, 58− 42 °C.29 This is in close agreement with our results where ambient temperature is within the range of the phase coexistence temperatures (Figure 2). The 3D image (DPPC/DOPE, 3/7, mol/mol, Figure 2C) shows more details about the gel-phase (DPPC) segregation from the liquid-phase (DOPE) in the GUV model, where irregular dendritic, flower shapes, and regular circular shapes were found. Whether the irregular shapes were formed spontaneously and individually or were formed due to the aggregation of regular circular gel-phases induced by the fluidity of liquid-phases is not known. The dark regions on GUVs

(Figure 2C) are similar to the dark regions that were found on the surface of native MFGs.15,16,18,19 It was difficult to observe gel−liquid crystalline phase separation in the GUVs systems as presented in Figure 1. This may be related to the preferred molecular arrangement of different phospholipids (DPPC and DOPE) in the nonsupported model membrane systems, as shown in Figure 3. According to the critical packing parameter

Figure 3. Highly schematic illustration of proposed molecular arrangement of GUV (bilayer) systems formed from different species of phospholipids. A: DPPC/DOPE (7/3, mol/mol); B: DPPC/DOPE (1/1, mol/mol); C: DPPC/DOPE (3/7, mol/mol). Black (dashed): DPPC; Red (solid): DOPE.

and critical packing shape of phospholipid molecules48 it is proposed that PE is more favorably packed in the inner leaflet, with the bilayer system containing PC and/or SM, as the fatty acid tails of PE form cylindrical or inverted truncated cone shapes which favor planar bilayers or inverted micelles structures. Moreover, in this current work, the GUVs could not be constructed from the Rd-DOPE + DOPE sample under the same electroformation conditions which were used for the other samples. This negative result indirectly suggests that unlike PC and SM, PE molecules are difficult to form a flexible spherical bilayer structure, implying localization in the inner leaflet of the MFGM bilayer or GUV systems when PC and SM are present. Consequently, the outer leaflets of GUV bilayers made from DPPC and specific DPPC+DOPE mixtures (7/3 and 1/1, mol/mol) may be constructed from only DPPC (Figure 3). It is necessary to point out that CLSM is an appropriate technique for characterizing the homogeneity of the surface of fluorescently labeled phospholipid bilayers. The characterization depends upon the instrumental configuration; by setting up a correct configuration on the CLSM, Rd-DOPE-labeled DPPC concentrated GUVs (Figure 1) may be visible. Therefore, in the current work, CLSM was applied for characterizing the “immiscible phases” on the surfaces of GUVs rather than characterizing the individual absolute ordered or disordered phases. 3. GUVs Constructed From SM and/or DOPE. In GUV systems containing only bovine milk-derived SM, the nonfluorescent regions were observed with long elongated shapes (Figure 4A and B) where the images were taken at the top and bottom section of selected GUVs. The 2D GUV image taken from the middle section (Figure 4C) is similar to the MFGM images from previous reports;4,15,16 the tiny dark dots on the fluorescent GUV surface may contribute to the linear shaped microdomains in constructed 3D images. Unlike DPPC, SM derived from bovine milk is a mixture of lipid molecules with different lengths of fatty acids and headgroups which gave three major peaks for the purchased sample (Avanti Polar Lipids) in HPLC-ELSD analysis. The presence of other minor SM species are also expected. This is 3239



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milk SM molecules in GUVs may be expected. The hosting capacity of fluorescent probes in phospholipid membranes depends on the phospholipid molecular packing,16 consequently, the observation of phase separation, which could be induced by heterogeneous distribution of fluorescent probes in the GUV system containing different types of SM, is rationalized. Shipley et al. (1974)49 characterized a single lamellar bilayer structure in a SM (bovine brain) bilayer system and found phase separation for a higher degree of hydration in a SM bilayer system (>42% water, v/v) occurred at a lower phase transition temperature (25 °C). This is in close agreement with the current study where bovine milk SM is highly hydrated and characterized at ambient temperature; pulsed-field gradient NMR has shown that the lateral diffusional behavior is the same in a bilayer comprised of SM of either brain and milk origin.50 The dark regions only account for a small proportion of GUV (SM unitary system) surfaces as discrete linear/dot shapes and larger regular shapes are not seen (Figure 4, containing only milk SM). This unique structure and morphology is probably due to the specific individual molecular profile of SMs derived from bovine milk and their packing structure in the bilayer. Filippov et al. (2006)50 found that SM derived from brain and egg may induce larger phase coexistence areas in phospholipid bilayers than milk SM, probably due to the heterogeneous hydrocarbon chain in SM which gives a less tightly packed molecular structure. Therefore, the small dark regions could be formed by a small amount of tightly packed homogeneous hydrocarbon chains of specific SM molecules. Bagatolli et al. (2000)29 found similar elongated microdomains in GUV systems (DLPC/DPPC, 1/1, mol/mol) which were stained by different probes (including Rd-DPPE). Based on a previously proposed model,51 the elongated domains were formed due to a particular topology (unlike a pure gel phase) of specific phospholipid molecules (DPPC in this case), presented as filaments of gel in a particular liquid crystalline phase. Moreover, in a good agreement with our current results shown in Figure 6 (SM/DOPE, 1/1, mol/mol), elongated and polygonal domains have also been found in other GUV systems containing only pure phospholipids, where it has been pointed out the GUV radius of curvature and fluid line defects formed by liquid crystalline domains are important for forming and shaping lipid domains.29,52 Therefore, we can conclude that both intrinsic factors (composition, chemical and physicochemical nature of phospholipids, including hydrocarbon chain, Tm, and molecular packing shape) and extrinsic factors (size of the bilayer, environmental temperature, and physical property of liquid-domains) are important for forming lipid domains with specific morphology. 4. GUVs Mimicking the Morphology of Native MFGs. Lateral mobility of lipid domains (dark regions) with regular circular shapes was observed (Figure 7, DPPC/DOPE, 3/7, mol/mol), in good agreement with previous work where mobility of the MFGM constituents was found.15 The composition of the mobile nonfluorescent regions is difficult to ascertain due to the complexity of the composition and structure of the MFGM; however, in artificial membrane systems with controlled composition (Figure 7), it can be concluded that the mobile microdomain was formed from a gel phase and flowed into the liquid phase. The shape and the size of nonfluorescent microdomains did not change as a function of time, as reported before in the bovine MFGM.15 The specific shapes of nonfluorescent microdomains on the surface of

Figure 4. Dark regions (DRs, shown by white arrows) presented on GUVs formed from SM. A: 2D image, top/bottom and middle section of two GUVs, DRs were observed as elongated and dot shapes; B: 2D image, top/bottom section (in white circle), DRs were observed as irregular shapes; C: 2D image, middle section. Scale bar: 5 μm.

the same three-peak profile as our extracted bovine milk phospholipid mixture (results not shown); therefore, the proposed surface arrangement shown in Figure 3, which only represents two types of pure lipid molecules, does not apply in this case, and dark domains can occur in all SM containing samples (Figures 4, 5, and 6). These dark domains were

Figure 5. DRs (shown by white arrows) were found in GUVs formed from binary systems containing SM and DOPE. A: SM/DOPE, 7/3 (mol/mol); B: SM/DOPE, 1/1 (mol/mol); C: SM/DOPE, 3/7 (mol/ mol). Scale bar: 5 μm.

Figure 6. Apparent morphology of a GUV (SM/DOPE, 1/1, mol/ mol) in a 3D image modeling part of the morphology of a native MFG. White arrow: irregular dendritic shape; green arrow: linear shape. Scale bar: 10 μm.

probably induced by heterogeneous distribution of Rd-DOPE on the surfaces of GUVs which contained different types of milk SM. The composition of milk SM in terms of fatty acid and sphingoloid population varies considerably,2 and it was proposed that physical behavior (melting) of SM depends on both the sphingosine chain and the fatty acid distribution;49 therefore, different physical behaviors and molecular packing of 3240



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formed between the external (100 mM glucose) and internal solutions (100 mM sucrose). Cholesterol has a role in inducing domain formation54,55 and is dependent on the amount present.53 Scherfeld et al. (2003) reported that phase separation may be reduced by adding 10 mol % cholesterol into the phospholipid binary system (DOPC/DPPC. 0.5/0.5, mol/mol) and can increase by adding 20 mol %.53 Cholesterol accounts for ∼14 mol % in our current formulation for mimicking the lipid molar composition of MFGM, and phase separation can readily be observed with CLSM (Figure 8). Based on CLSM experimental evidence, a model of lateral topology of MFGM lipid organization can be deduced in which PC and SM are located in the outer leaflet of the bilayer and specific phospholipids having high gel−liquid crystalline phase transition temperatures contribute to lateral segregation in the MFGM. This suggests that association of SM and cholesterol is not the only contributing factor to ordered domain formation.

Figure 7. Monitoring mobility of DRs (pointed by arrows) on the surface of specific GUVs (DPPC/DOPE, 3/7, mol/mol). 2D images were taken at the top/bottom section of GUVs. A−I were obtained individually along a fixed time interval (20 s). Scale bar: 5 μm.

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GUVs, as shown in Figures 2 and 6 (without SM, and without a combination of SM and cholesterol), shows good agreement with the previously published surface morphology of native MFG.16 Based on the current results, we may conclude that the nonfluorescent microdomains on the surface of MFGs are not necessarily represented by the Lo phase, enriched in SM and cholesterol, but may also be formed from the gel phase containing the major MFGM phospholipid species DPPC and SM. A lipid formulation of DOPE/DPPC/SM/cholesterol (8/8/ 8/4, mol/mol) was used for mimicking the composition of the native MFGM. A large integrated dark region is shown in the 3D micrograph (Figure 8). The surface morphology is in good

AUTHOR INFORMATION

Corresponding Author

*Phone: +86 13716006816. Fax: +64 34797567. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Earle Food Research Fund from the Riddet Institute, New Zealand. We also gratefully acknowledge Dr. Jakob Schweizer from Department of Biochemistry, University of Oxford, United Kingdom for the discussion in GUVs construction. We acknowledge Andrea Laubscher and other members from the Dairy Products Technology Center at the California Polytechnic State University for technical support during the course of this research.

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ABBREVIATIONS USED MFGM, milk fat globule membrane; SM, sphingomyelin; PE, phosphatidylethanolamine; PC, phosphatidylcholine; GUVs, giant unilamellar vesicles; DPPC, 1,2-dipalmitoyl-sn-glycero-3phosphocholine; MFGs, milk fat globules; CLSM, confocal laser scanning microscope; Rd-DOPE, 1,2-dioleoyl-sn-glycero3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl); Lo, liquid ordered; Ld, liquid disordered; ITO, indium tin oxide; DIC, differential interfacial contrast; NBD-PC, 1-palmitoyl-2{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino] hexanoyl}-snglycero-3-phosphocholine; DOPE, 1,2-dioleoyl-sn-glycero-3phosphoethanolamine

Figure 8. 3D image of a GUV formed from a lipid formula (DOPE/ DPPC/SM/cholesterol, 8/8/8/4, mol/mol) mimicking the lipid composition of the native MFGM. DR is shown by the arrow. Scale bar: 5 μm.

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REFERENCES

(1) Huppertz, T.; Kelly, A. L. Physical Chemistry of Milk Fat Globules. In Advanced Dairy Chemistry Vol. 2 Lipids; Fox, P. F., McSweeney, P. L. H., Eds.; Springer US: New York, 2006; pp 173− 212. (2) MacGibbon, A. K. H.; Taylor, M. W. Composition and Structure of Bovine Milk Lipids. In Advanced Dairy Chemistry Vol. 2 Lipids; Fox, P. F., McSweeney, P. L. H., Eds.; Springer US: New York, 2006; pp 1− 42. (3) Fong, B. Y.; Norris, C. S.; MacGibbon, A. K. H. Protein and lipid composition of bovine milk-fat-globule membrane. Int. Dairy J. 2007, 17, 275−288. (4) Lopez, C.; Briard-Bion, V.; Ménard, O.; Beaucher, E.; Rousseau, F.; Fauquant, J.; Leconte, N.; Robert, B. Fat globules selected from whole milk according to their size: Different compositions and

agreement with previous GUV systems containing cholesterol where the existence of microdomains and domain patterns was regulated by the amount of cholesterol.53 Morales-Penningston et al. (2010)24 showed a similar pattern of microdomains on the surface of GUVs (composed from 18:0-SM/DOPC/ cholesterol in the molar ratio of 0.56/0.24/0.20), where the larger and regular circular microdomains were formed by the merging of smaller separated microdomains, as induced by osmotic swelling. This osmotic swelling mechanism may explain the apparent morphology on our GUV containing cholesterol (Figure 8) as an osmotic pressure gradient is 3241



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structure of the biomembrane, revealing sphingomyelin-rich domains. Food Chem. 2011, 125, 355−368. (5) Mather, I. H. Milk Lipids | Milk Fat Globule Membrane. In Encyclopedia of Dairy Sciences, 2nd ed.; Fuquay, J. W., Ed.; Academic Press: San Diego, CA, 2011; pp 680−690. (6) Dewettinck, K.; Rombaut, R.; Thienpont, N.; Le, T.; Messens, K.; Vancamp, J. Nutritional and technological aspects of milk fat globule membrane material. Int. Dairy J. 2008, 18, 436−457. (7) Jimenez-Flores, R.; Brisson, G. The milk fat globule membrane as an ingredient: why, how, when? Dairy Sci. Technol. 2008, 88, 5−18. (8) Spitsberg, V. L. Invited review: Bovine milk fat globule membrane as a potential nutraceutical. J. Dairy Sci. 2005, 88, 2289−2294. (9) Kahya, N. Protein-protein and protein-lipid interactions in domain-assembly: Lessons from giant unilamellar vesicles. BBA, Biochim. Biophys. Acta, Biomembr. 2010, 1798, 1392−1398. (10) Michalski, M.-C.; Januel, C. Does homogenization affect the human health properties of cow’s milk? Trends Food Sci. Technol. 2006, 17, 423−437. (11) Favé, G.; Coste, T. C.; Armand, M. Physicochemical properties of lipids: New strategies to manage fatty acid bioavailability. Cell. Mol. Biol. (Noisy-le-grand) 2004, 50, 815−831. (12) Garcia, C.; Antona, C.; Robert, B.; Lopez, C.; Armand, M. The size and interfacial composition of milk fat globules are key factors controlling triglycerides bioavailability in simulated human gastroduodenal digestion. Food Hydrocolloids 2014, 35, 494−504. (13) Argov, N.; Lemay, D. G.; German, J. B. Milk fat globule structure and function: nanoscience comes to milk production. Trends Food Sci. Technol. 2008, 19, 617−623. (14) Deeth, H. C. The role of phospholipids in the stability of milk fat globules. Aust. J. Dairy Technol. 1997, 52, 44−46. (15) Lopez, C.; Madec, M.-N.; Jimenez-Flores, R. Lipid rafts in the bovine milk fat globule membrane revealed by the lateral segregation of phospholipids and heterogeneous distribution of glycoproteins. Food Chem. 2010, 120, 22−33. (16) Gallier, S.; Gragson, D.; Jiménez-Flores, R.; Everett, D. Using confocal laser scanning microscopy to probe the milk fat globule membrane and associated proteins. J. Agric. Food Chem. 2010, 58, 4250−4257. (17) Evers, J. M.; Haverkamp, R. G.; Holroyd, S. E.; Jameson, G. B.; Mackenzie, D. D. S.; McCarthy, O. J. Fluorescent probes for studying the milk fat globule (membrane). Milchwissenschaft 2008, 63, 402− 405. (18) Lopez, C.; Ménard, O. Human milk fat globules: Polar lipid composition and in situ structural investigations revealing the heterogeneous distribution of proteins and the lateral segregation of sphingomyelin in the biological membrane. Colloids Surf., B 2011, 83, 29−41. (19) Zou, X.-Q.; Guo, Z.; Huang, J.-H.; Jin, Q.-Z.; Cheong, L.-Z.; Wang, X.-G.; Xu, X.-B. Human milk fat globules from different stages of lactation: A lipid composition analysis and microstructure characterization. J. Agric. Food Chem. 2012, 60, 7158−7167. (20) Sengupta, P.; Jovanovic-Talisman, T.; Skoko, D.; Renz, M.; Veatch, S. L. Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis. Nat. Methods 2011, 8, 969− 975. (21) Feigenson, G. W. Phase diagrams and lipid domains in multicomponent lipid bilayer mixtures. BBA, Biochim. Biophys. Acta, Biomembr. 2009, 1788, 47−52. (22) Gallier, S.; Gragson, D.; Jiménez-Flores, R.; Everett, D. W. Surface characterization of bovine milk phospholipid monolayers by Langmuir isotherms and microscopic techniques. J. Agric. Food Chem. 2010, 58, 12275−12285. (23) Menger, F. M.; Keiper, J. S. Chemistry and physics of giant vesicles as biomembrane models. Curr. Opin. Chem. Biol. 1998, 2, 726−732. (24) Morales-Penningston, N. F.; Wu, J.; Farkas, E. R.; Goh, S. L.; Konyakhina, T. M.; Zheng, J. Y.; Webb, W. W.; Feigenson, G. W. GUV preparation and imaging: Minimizing artifacts. BBA, Biochim. Biophys. Acta, Biomembr. 2010, 1798, 1324−1332.

(25) Politano, T. J.; Froude, V. E.; Jing, B.; Zhu, Y. AC-electric field dependent electroformation of giant lipid vesicles. Colloids Surf., B 2010, 79, 75−82. (26) Leonenko, Z. V.; Finot, E.; Ma, H.; Dahms, T. E. S.; Cramb, D. T. Investigation of temperature-induced phase transitions in DOPC and DPPC phospholipid bilayers using temperature-controlled scanning force microscopy. Biophys. J. 2004, 86, 3783−3793. (27) Shalaev, E. Y.; Steponkus, P. L. Phase diagram of 1,2dioleoylphosphatidylethanolamine (DOPE):water system at subzero temperatures and at low water contents. BBA, Biochim. Biophys. Acta, Biomembr. 1999, 1419, 229−247. (28) Malmsten, M.; Bergenstahl, B.; Nyberg, L.; Odham, G. Sphingomyelin from milk - characterization of liquid-crystalline, liposome and emulsion properties. J. Am. Oil Chem. Soc. 1994, 71, 1021−1026. (29) Bagatolli, L. A.; Gratton, E. Two photon fluorescence microscopy of coexisting lipid domains in giant unilamellar vesicles of binary phospholipid mixtures. Biophys. J. 2000, 78, 290−305. (30) Dimitrov, D. S.; Angelova, M. I. Lipid swelling and liposome formation on solid surfaces in external electric fields. In New Trends in Colloid Science; Hoffmann, H., Ed.; Steinkopff: Dresden, Germany, 1987; Vol. 73, pp 48−56. (31) Herold, C.; Chwastek, G.; Schwille, P.; Petrov, E. P. Efficient electroformation of supergiant unilamellar vesicles containing cationic lipids on ITO-coated electrodes. Langmuir 2012, 28, 5518−5521. (32) Veatch, S. L. Electro-formation and fluorescence microscopy of giant vesicles with coexisting liquid phases. Methods Mol. Biol. 2007, 398, 59−72. (33) Angelova, M. I.; Dimitrov, D. S. Liposome electroformation. Faraday Discuss. Chem. Soc. 1986, 81, 303−311. (34) Bitman, J.; Wood, D. L. Changes in milk fat phospholipids during lactation. J. Dairy Sci. 1990, 73, 1208−1216. (35) Barenholz, Y.; Thompson, T. E. Sphingomyelins in bilayers and biological membranes. BBA, Biochim. Biophys. Acta, Biomembr. 1980, 604 (C), 129−158. (36) Contreras, F. X.; Sot, J.; Ruiz-Argüello, M. B.; Alonso, A.; Goñi, F. M. Cholesterol modulation of sphingomyelinase activity at physiological temperatures. Chem. Phys. Lipids 2004, 130, 127−134. (37) Danthine, S.; Blecker, C.; Paquot, M.; Innocente, N.; Deroanne, C. Progress in milk fat globule membrane research: a review. Lait 2000, 80, 209−222. (38) Zheng, H.; Jiménez-Flores, R.; Everett, D. W. Bovine milk fat globule membrane proteins are affected by centrifugal washing processes. J. Agric. Food Chem. 2013, 61, 8403−8411. (39) Sun, Y.; Lee, C.-C.; Huang, H. W. Adhesion and merging of lipid bilayers: A method for measuring the free energy of adhesion and hemifusion. Biophys. J. 2011, 100, 987−995. (40) Evers, J. M.; Haverkamp, R. G.; Holroyd, S. E.; Jameson, G. B.; Mackenzie, D. D. S.; McCarthy, O. J. Heterogeneity of milk fat globule membrane structure and composition as observed using fluorescence microscopy techniques. Int. Dairy J. 2008, 18, 1081−1089. (41) Evers, J. M. The milkfat globule membranecompositional and structural changes post secretion by the mammary secretory cell. Int. Dairy J. 2004, 14, 661−674. (42) Michalski, M.-C.; Michel, F.; Sainmont, D.; Briard, V. Apparent [zeta]-potential as a tool to assess mechanical damages to the milk fat globule membrane. Colloids Surf., B 2002, 23, 23−30. (43) Zheng, H.; Gordon, K. C.; Everett, D. W. Innovative application of confocal Raman microscopy to investigate the interaction between trans-2-hexenal and bovine milk fat globules. Int. Dairy J. 2013, 32, 68−70. (44) Bacia, K.; Schwille, P.; Kurzchalia, T. Sterol structure determines the separation of phases and the curvature of the liquid-ordered phase in model membranes. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 3272− 3277. (45) Baumgart, T.; Hess, S. T.; Webb, W. W. Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension. Nature 2003, 425, 821−824. 3242



Article

(46) Veatch, S. L.; Keller, S. L. Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol. Biophys. J. 2003, 85, 3074−3083. (47) Simons, K.; Ikonen, E. Functional rafts in cell membranes. Nature 1997, 387, 569−572. (48) Israelachvili, J. N. 20 - Soft and Biological Structures. In Intermolecular and Surface Forces, 3rd ed.; Academic Press: San Diego, CA, 2011; pp 535−576. (49) Shipley, G. G.; Avecilla, L. S.; Small, D. M. Phase behavior and structure of aqueous dispersions of sphingomyelin. J. Lipid Res. 1974, 15, 124−131. (50) Filippov, A.; Orädd, G.; Lindblom, G. Sphingomyelin structure influences the lateral diffusion and raft formation in lipid bilayers. Biophys. J. 2006, 90, 2086−2092. (51) Parasassi, T.; Ravagnan, G.; Rusch, R. M.; Gratton, E. Modulation and dynamics of phase properties in phospholipid mixtures detected by laurdan fluorescence. Photochem. Photobiol. 1993, 57, 403−410. (52) Bagatolli, L. A.; Gratton, E. Two-photon fluorescence microscopy observation of shape changes at the phase transition in phospholipid giant unilamellar vesicles. Biophys. J. 1999, 77, 2090− 2101. (53) Scherfeld, D.; Kahya, N.; Schwille, P. Lipid dynamics and domain formation in model membranes composed of ternary mixtures of unsaturated and saturated phosphatidylcholines and cholesterol. Biophys. J. 2003, 85, 3758−3768. (54) Bacia, K.; Scherfeld, D.; Kahya, N.; Schwille, P. Fluorescence correlation spectroscopy relates rafts in model and native membranes. Biophys. J. 2004, 87, 1034−1043. (55) Wesolowska, O.; Michalak, K.; Maniewska, J.; Hendrich, A. B. Giant unilamellar vesicles - a perfect tool to visualize phase separation and lipid rafts in model systems. Acta Biochim. Pol. 2009, 56, 33−39.

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Phospholipid Architecture of the Bovine Milk Fat Globule Membrane

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