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.

Abstract

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)