Palmitoyl ceramide promotes milk sphingomyelin gel phase domains formation and affects the mechanical properties of the fluid phase in milk-SM/DOPC supported membranes

Ceramides are minor structural components of membranes involved in biological functions. In the milk fatglobule membrane (MFGM), ceramides are susceptible to affect the lateral packing of polar lipids, especially themilk sphingomyelin (MSM). To investigate this, palmitoylceramide (PCer) was added to MSM/DOPC (dioleoylphosphatidylcholine)in order to form hydrated lipid bilayers. Differential scanning calorimetry evidencedinteractions of PCer with the MSM in the solid-ordered phase to form MSM/PCer structures with a higherthermostability than MSM. Atomic force microscopy revealed that PCer modified lipid packing in both theliquid-disordered DOPC phase where it increased thickness and mechanical stability, and the solid-ordered MSMphase where it recruited MSM molecules yet initially in the liquid phase at 26 °C and then increased the area ofthe MSM/PCer domains. The effect of PCer on the mechanical properties of the MSM-rich domains remains to beelucidated. These results bring new insights on the role of ceramides in the control of biophysical and biologicalproperties of the MFGM. They also open perspectives for the design of emulsions and liposomes, using milk polarlipids as food-grade ingredients

Sphingomyelin-rich domains in bilayer models of the milk fat globule membrane: temperature governs structural and mechanical heterogeneity.

The biological membrane enveloping the milk fat globules (MFGM) supplies consumers, especially suckling infants, with bioactive lipids and proteins, and constitutes the interface to digestion or immunity. Bioactive mechanisms depend on biological structure, which itself highly depend on temperature. However, the polar lipid assembly and biophysical properties of the MFGM are yet poorly known, especially in connection with the temperature history that milk can experience upon storage or processing and up to consumption, e.g. in human milk banks or in dietary intake of bovine milk. Noteworthy, in all mammalian milks, polar lipids of the MFGM include a significant proportion of species with a high phase transition temperature (Tm), especially milk sphingomyelin (MSM; ~30% w/w of the total polar lipids; Tm = 34°C – Murthy et al., 2015), that is held responsible for the formation of lipid domains at the surface of the milk fat globule [br/]The objective of this study was therefore to investigate the polar lipid packing in hydrated bilayers prepared with a MFGM extract, and to follow cooling-induced changes at inter-molecular level using a combination of differential scanning calorimetry (DSC), wide-angle X-ray diffraction (XRD), atomic force microscopy (AFM) imaging and force spectroscopy. On cooling, a liquid disordered (ld) to solid ordered (so) phase transition of MSM (and other high-Tm polar lipids) in MFGM bilayers started at ~40°C and reached its maximum at 30.3°C. Using temperature-controlled AFM, phase separation was observed for temperatures below 35°C, with formation of so phase domains with a height difference H 1 nm from the continuous ld phase (Fig.2). In complement to AFM topographical images, indentation measurements to map the yield (or breakthrough) force over the imaged surfaces showed that the mechanical stability was significantly higher for the so phase domains than for the ld phase. Also, the mechanical stability of both the domains and the fluid phase increased with decreasing temperature, probably as a result of lower molecular agitation and increased ordering. However, lipid packing, integrity and stability of the bilayers were adversely affected by fast cooling to 6°C or by cooling-rewarming cycle, which may have important consequences in handling milk samples in neonatal or food applications[br/]For the first time, AFM was successfully used to report correlated structural and mechanical changes in hydrated multi-component bilayer models of a complex biological membrane: that of the milk fat globule. AFM is therefore a promising tool to investigate temperature-induced changes in intermolecular forces within biologically relevant membranes, with nanoscale resolution

Interleaflet Decoupling in a Lipid Bilayer at Excess Cholesterol Probed by Spectroscopic Ellipsometry and Simulations

Abstract: Artificial lipid membranes are often investigated as a replica of the cell membrane in the form of supported lipid bilayers (SLBs). In SLBs, the phase state of a lipid bilayer strongly depends on the presence of molecules such as cholesterol, ceramide, and physical parameters such as temperature. Cholesterol is a key molecule of biological membranes and it exerts condensing effect on lipid bilayers. In this paper, we demonstrate the influence of excess cholesterol content on a supported lipid bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (fluid-phase) using spectroscopic ellipsometry (SE) and coarse-grained (CG) molecular dynamics (MD) simulations. The results show the condensation effect due to cholesterol addition up to 30% and interleaflet decoupling at excess cholesterol beyond 30%. SE results show the separation of individual leaflets of the bilayer and influence of cholesterol on the biophysical properties such as thickness and optical index. CG simulations were performed at different ratios of DOPC:cholesterol mixtures to explore cholesterol-driven bilayer properties and stability. The simulations displayed the accumulation of cholesterol molecules at the interface of the lower and upper leaflets of the bilayer, thus leading to undulations in the bilayer. This work reports the successful application of SE technique to study lipid–cholesterol interactions for the first time. Graphical abstract: [Figure not available: see fulltext.]

Lipid domains in the biological membrane surrounding milk fat globules: role of temperature and cholesterol on their morphology and nanomechanical properties

The biological membrane surrounding fat globules in milk, the MFGM, is poorly known despite its importance in the functional properties of many dairy products and in the mechanisms of milk lipids digestion occurring in the gastrointestinal tract of mammal newborns. Studies recently revealed the formation of lipid domains in the MFGM that have been interpreted as the lateral segregation of high phase transition temperature (Tm) lipids, mainly milk sphingomyelin (MSM; Tm 34°C) [1]. The role played by these MSM-rich domains in the MFGM is currently unknown and needs further investigation. In this context, the aim of this study was to investigate the role of temperature and cholesterol in the morphology and nanomechanical properties of the MFGM. Accessing the biophysical properties of biological membranes at nanoscale is possible using Atomic Force Microscopy (AFM). In the complex biomimetic membranes formed using a MFGM lipid extract, topographical AFM images revealed for T < 35°C the formation of µm-large domains that protruded above the continuous phase by ~1 nm [2]. The resistance of the MSM-rich gel phase domains to rupture was significantly higher than that of the liquid disordered continuous phase, i.e. breakthrough force FB of 23 nN vs. 6 nN at 25°C, respectively. The key role played by cholesterol in the lateral segregation of high Tm lipids, by decreasing the size of the lipid domains, their height and by fluidizing the MFGM has been demonstrated [3]. Such work on the heterogeneous distribution of polar lipids in the MFGM may open perspectives in dairy technology and infant nutrition, e.g. optimisation of the interface of processed lipid droplets in infant milk formulas to mimick the biological MFGM in breast milk

Lipid domains in the biological membrane surrounding milk fat globules: role of cholesterol on their morphology and nanomechanical properties, probed by AFM and force spectroscopy.

Comité d'organisation:Prof Perla RELKIN, AgroParisTech-Centre de Massy (President)SPAB, Department of Engineering and Science of Food and Bioproducts1 Avenue des Olympiades, 91300 MASSY, FranceDr Monique AXELOS, INRA, France (Vice-President)Head of the division for Science and Process Engineering of Agricultural ProductsRue de la Géraudière BP 71627INRA_logo44316 NANTES, Francea) Introduction. The biological membrane surrounding fat globules in milk is poorly known despite its importance in the functional properties of many dairy products and in the mechanisms of milk lipids digestion occurring in the gastrointestinal tract of mammal newborns. The milk fat globule membrane (MFGM) is essentially composed of polar lipids (of which 30% is milk sphingomyelin; SMmilk), cholesterol and membrane proteins. Studies recently revealed the heterogeneous distribution of polar lipids in the outer bilayer of the MFGM that has been interpreted as the lateral segregation of high phase transition temperature (Tm) lipids, mainly SMmilk, in the form of “lipid rafts” at the surface of the milk fat globule (Lopez et al., 2010). In cells, “raft” structures composed by SM and cholesterol have been associated in signaling and trafficking biological mechanisms. The role played by these SMmilk-rich domains in the MFGM is currently unknown and needs further investigation. Accessing the biophysical properties of biological membranes at nanoscale is possible using Atomic Force Microscopy (AFM) and could provide a better insight into the heterogeneous distribution of lipids and in the nanomechanichal properties of the MFGM. In this context, the aim of this study was to investigate the role of cholesterol in the morphology and nanomechanical properties of the MFGM. b) Materials & methods. Lipid films containing i) SMmilk/DOPC (50/50%mol), ii) SMmilk/DOPC/chol (40/40/20%mol), or iii) a complex MFGM lipid extract with 0 or 50 % mol. of cholesterol to the SMmilk were prepared in chloroform/methanol and evaporated under N2. The lipid films were then hydrated at T=60°C > Tm of SMmilk, at 1mg/mL in PIPES buffer pH 6.7 containing 2 mM Ca2+. Small unilamellar vesicles were obtained by sonication then fused onto freshly cleaved mica in PIPES buffer and at 60°C to form hydrated lipid bilayers, which were further cooled to room temperature in a programmed incubator to control the formation of lipid domains. Imaging and force spectroscopy experiments were conducted with an MFP-3D Asylum Research AFM in PIPES buffer and at room temperature. c) Results & discussions. In model membranes composed by SMmilk and DOPC, the lateral segregation of SMmilk to form 0.9 nm-salient and µm-wide domains was observed for T < 35°C, which corresponds to the liquid crystalline to gel phase transition temperature of SMmilk (Tm 34°C). These domains were interpreted as segregated high Tm lipids in the gel phase surrounded by a continuous fluid phase. In the presence of cholesterol, the SMmilk domains were scattered into numerous shorter, smaller, tortuous individuals and were less resistant to rupture than in absence of cholesterol, indicating a tendency to mixing into the continuous fluid phase (Guyomarc’h et al., 2014). In the complex biomimetic membranes formed using a MFGM lipid extract, topographical AFM images showed that in the absence of added cholesterol (Figure 1, top left), µm-large domains were formed that protruded above the continuous phase by ~1 nm. In the presence of cholesterol (Fig. 1, bottom left), scattered nm-sized domains were characterised. On the same locations, AFM force spectroscopy experiments were performed to estimate the mechanical strength of the bilayers. Without cholesterol (Fig 1, top row), the fluid phase ruptured at ~5 nN while the domains could not be pierced by the AFM tip at the 12 nN force load set point (black pixels in the force maps). This indicated higher stiffness of these domains than in model SMmilk domains (Guyomarc’h et al., 2014), possibly due to traces of ceramides (Sullan et al., 2009). In the presence of cholesterol the breakthrough force was less (~3 nN) and more homogeneous across the milk lipid membranes, i.e., the domains were softer than in absence of cholesterol. The results are interpreted in terms of the fluidizing effect of the cholesterol onto SM (Simons and Vaz, 2004).In conclusion, cholesterol plays a key role in the lateral segregation of high Tm lipids in the biological membrane surrounding milk fat globules, by decreasing the size of the lipid domains, their height and by fluidizing the MFGM. Such work on the heterogeneous distribution of lipids in the MFGM may open perspectives in dairy technology and infant nutrition (e.g. optimisation of the interface of processed lipid droplets in infant formulas as compared to the biological MFGM in breast milk)

Cholesterol affects the height and area of the lipid domains formed in the milk fat globule membrane, as revealed on Langmuir-Blodgett film monolayers

Comité d'organisation:Prof Perla RELKIN, AgroParisTech-Centre de Massy (President)SPAB, Department of Engineering and Science of Food and Bioproducts1 Avenue des Olympiades, 91300 MASSY, FranceDr Monique AXELOS, INRA, France (Vice-President)Head of the division for Science and Process Engineering of Agricultural ProductsRue de la Géraudière BP 71627INRA_logo44316 NANTES, FranceNational audiencea) Introduction. The organisation and biophysical properties of the biological membrane surrounding fat globules in milk are poorly known and need further investigation to better understand their technological and biological functions. The milk fat globule membrane (MFGM) is the interface between the triacylglycerol core of milk fat globules and their aqueous environment. This membrane is important for the physical stability of fat globules and is involved in the mechanisms of milk fat globule digestion in the gastrointestinal tract of mammal newborns. The MFGM is essentially composed of polar lipids (of which 30% mol is milk sphingomyelin; MSM), cholesterol and membrane proteins. The lateral segregation of polar lipids characterised by high phase transition temperature (e.g. the MSM with Tm=34°C but also DPPC) has been revealed in the external bilayer of the MFGM and interpreted as the formation of “lipid rafts” at the surface of the milk fat globule (Lopez et al., 2010). The properties of rafts in the biological cells, and the interactions between cholesterol and sphingolipids such as sphingomyelin, have been the subject of many studies (Simons and Vaz, 2004). Increasing knowledge about the role of cholesterol in the physical characteristics of the high Tm lipid domains formed in the MFGM is of primary interest. Previous results indicated that the cholesterol strongly impacted on the lateral packing of the MSM in MSM/DOPC/cholesterol bilayers (Guyomarc’h et al., 2014). The objectives of this study were to investigate the role of cholesterol on the lipid domains formed in the MFGM, using Langmuir-Blodgett monolayers probed at the nanoscale using Atomic Force Microscopy (AFM) to characterise the structural details.b) Materials & methods. Investigations of lipid heterogeneities have been performed using a MFGM lipid extract (39% MSM, 32% PC, 24% PE, 3% PI, 3% PS). MFGM lipid extracts with 0 to 50 % mol. of cholesterol to the milk sphingomyelin (MSM) were prepared in methanol/chloroform then deposited at the air-water interface of a Langmuir trough containing PIPES buffer at 20°C. The lipids were compressed to 30 mN/m at 5 cm2/min, transferred at constant pressure onto freshly cleaved mica then dried in a dessicator for at least 3 days. Imaging was conducted with an MFP-3D Asylum Research AFM at room temperature. c) Results & discussion.Monolayers of MFGM lipids showed the formation of domains, which have been interpreted as the lateral segregation of high Tm lipids form the other lipids (polar lipids containing unsaturated fatty acids and forming the fluid phase surrounding the domains). The lipid domains formed in the absence of added cholesterol (Fig.1, left), had a µm-large size and protruded above the continuous phase by ~1.4 nm height step. As the proportion of cholesterol was increased (Fig. 1), the domains were clearly more numerous while decreasing both in area and in height step, the latter being only ~0.9 nm from 35% mol cholesterol onwards. This indicated that cholesterol scattered and fluidified the lipid domains into the continuous fluid phase (Milhiet et al., 2001). Langmuir compression isotherms were recorded and clearly evidenced the condensing effect of cholesterol onto the MFGM polar lipids, most probably the MSM (Simons and Vaz, 2004)

Cholesterol strongly affects the organization of lipid monolayers studied as models of the milk fat globule membrane: Condensing effect and change in the lipid domain morphology

International audienceThe biological membrane that surrounds the milk fat globules exhibits phase separation of polar lipids that is poorly known. The objective of this study was to investigate the role played by cholesterol in the organization of monolayers prepared as models of the milk fat globule membrane (MFGM). Differential scanning calorimetry and X-ray diffraction experiments allowed characterization of the gel to liquid crystalline phase transition temperature of lipids, Tm ~ 35 °C, in vesicles prepared with a MFGM lipid extract. For temperature below Tm, atomic force microscopy revealed phase separation of lipids at 30 mN.m− 1 in Langmuir-Blodgett monolayers of the MFGM lipid extract. The high Tm lipids form liquid condensed (LC) domains that protrude by about 1.5 nm from the fluid liquid expanded (LE) phase. Cholesterol was added to the MFGM extract up to 30% of polar lipids (cholesterol/milk sphingomyelin (MSM) molar ratio of 50/50). Compression isotherms evidenced the condensing effect of the cholesterol onto the MFGM lipid monolayers. Topography of the monolayers showed a decrease in the area of the LC domains and in the height difference H between the LC domains and the continuous LE phase, as the cholesterol content increased in the MFGM lipid monolayers. These results were interpreted in terms of nucleation effects of cholesterol and decrease in the line tension between LC domains and LE phase in the MFGM lipid monolayers. This study revealed the major structural role of cholesterol in the MFGM that could be involved in biological functions of this interface (e.g. mechanisms of milk fat globule digestion

Domaines de sphingomyéline dans la membrane entourant les globules gras du lait - Apport de la microscopie AFM

Plusieurs équipes de recherche rennaises utilisent des expériences aux interfaces fluides planes pour connaître et comprendre les propriétés de biomolécules variées. Lors d’une journée d’échanges, des scientifiques de l'UMR STLO et d’autres équipes contribueront à dresser le panorama des thématiques concernées et montrer le potentiel de ces expériences.Une journée d'échanges scientifiques est organisée par l'Institut de Physique de Rennes, le 22 janvier 2015. Elle vise à illustrer le potentiel et à construire une vision d'ensemble des expériences aux interfaces planes, dans des domaines scientifiques aussi variés que la physique des mousses et émulsions, les technologies de transformation des aliments, la digestion néonatale des lipides laitiers ou l'interaction de protéines avec les membranes biologiques. L'opportunité de montrer que les expériences aux interfaces planes livrent des informations abondantes et pertinentes.National audienc

The MFGM and milk polar lipids: Highly reactive assemblies involving structural and mechanical heterogeneities

IntroductionThe biological membrane surrounding fat globules in milk, i.e. the milk fat globule membrane (MFGM), is a trilayer assembly of polar lipids including 25-40% of sphingomyelin (SM) and glycerophospholipids. It is involved in the mechanical stability of fat globules, in the mechanisms of their digestion, and in physiological functions that need to be further understood. Increasing knowledge about the MFGM is therefore of primary importance both for dairy technology and nutrition. AimThe objectives of this study were to explore the dynamics of the MFGM polar lipids in situ in milk and to describe the biophysical properties of milk polar lipids assemblies as a function of temperature and composition, e.g. presence of cholesterol. MethodsThe biophysical properties and organization of polar lipids in the MFGM were investigated by the combination of techniques: DSC, synchrotron radiation XRD, CLSM, electron microscopy, AFM imaging and force spectroscopy. ResultsThe MFGM is a dynamic patchwork exhibiting phase separation of the high melting temperature (Tm) polar lipids, mainly SM, to form domains in ordered phase dispersed in a continuous fluid phase composed of the unsaturated polar lipids. In absence of cholesterol, the SM-rich domains are formed for T < 35°C and can melt upon heating. These domains protrude by 1 nm and have a higher resistance to rupture than the fluid phase composed of the unsaturated polar lipids. When present, cholesterol exhibits attractive interactions with SM with a condensing effect, and affects the topography of the membrane with a fluidizing effect. ConclusionWe propose an updated model for the heterogeneous organization of polar lipids in the MFGM, highlighting the dynamic changes in the topography and nanomechanical properties that are governed by the temperature and the presence of cholesterol. These recent findings on the assemblies of polar lipids in the MFGM and specific biophysical properties of SM in interaction with cholesterol will undoubtedly contribute in the development of smart biomimetic fat globules, e.g. in infant milk formulas