Cholesterol Decreases the Size and the Mechanical Resistance to

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Cholesterol decreases the size and the mechanical resistance to rupture of sphingomyelin rich domains, in lipid bilayers studied as model of the milk fat globule membrane Appala Venkata Ramana Murthy, Fanny Guyomarc'h, and Christelle Lopez Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b01040 • Publication Date (Web): 14 Jun 2016 Downloaded from http://pubs.acs.org on June 14, 2016

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Langmuir Murthy et al _ Revised version

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Cholesterol decreases the size and the mechanical resistance to rupture of

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sphingomyelin rich domains, in lipid bilayers studied as model of the milk

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fat globule membrane

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Appala Venkata Ramana MURTHY‡, Fanny GUYOMARC’H‡, Christelle LOPEZ‡*

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‡

UMR STLO 1253 Science et Technologie du Lait et de l'Œuf, AGROCAMPUS OUEST,

INRA, 35042 Rennes, France

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*

Corresponding author: Christelle LOPEZ

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1 ACS Paragon Plus Environment


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ABSTRACT

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Sphingomyelin-rich microdomains have been observed in the biological membrane

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surrounding milk fat globules (MFGM). The role played by cholesterol in these domains and

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in the physical properties and functions of the MFGM remains poorly understood. The

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objective of this work was therefore to investigate the phase state, topography and mechanical

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properties of MFGM polar lipid bilayers as a function of cholesterol concentration, by

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combining X-ray diffraction, atomic force microscopy imaging and force spectroscopy. At

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room temperature, i.e. below the phase transition temperature of the MFGM polar lipids, the

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bilayers showed the formation of sphingomyelin-rich domains in the solid ordered (so) phase

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that protruded about 1 nm above the liquid disordered (ld) phase. These so phase domains have

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a higher mechanical resistance to rupture than the ld phase (30 nN versus 15 nN). Addition of

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cholesterol in the MFGM polar lipid bilayers i) induced the formation of liquid-ordered (lo)

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phase for up to 27% mol in the bilayers, ii) decreased the height difference between the

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thicker ordered domains and the surrounding ld phase, iii) promoted the formation of small

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sized domains and iv) decreased the mechanical resistance to rupture of the sphingomyelin-

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rich domains down to ∼ 5 nN. The biological and functional relevance of the lo phase

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cholesterol/sphingomyelin-rich domains in the membrane surrounding fat globules in milk

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remains to be elucidated. This study brought new insight about the functional role of

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cholesterol in milk polar lipid ingredients, which can be used in the preparation of food

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emulsions e.g. infant milk formulas.

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INTRODUCTION

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Membranes have received many attention due to their essential biological functions, in

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particular plasma cell membranes. In contrast, the biological membrane surrounding fat

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globules in milk has been scarcely investigated and is therefore poorly known, despite its


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implication in the mechanisms of lipid digestion and protection in the gastrointestinal tract of

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newborns. Biological membranes from animal origin are a multicomponent mixture of lipids

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(glycerophospholipids, sphingolipids, cholesterol) and proteins. Abundant studies have

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demonstrated the importance of cholesterol in various mammalian cell membrane structures

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and processes1-3. Cholesterol is reported to be involved in a wide range of significant cellular

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events, e.g. membrane fusion processes such as endocytosis and exocytosis, formation of

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sphingolipid/cholesterol liquid-ordered (lo) phase segregated domains called rafts4,5.

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The milk fat globule membrane (MFGM) has raised attention in the last 10 years since

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the understanding of the relationship between its chemical composition, its structure and its

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functions will open perspectives in food formulation, human nutrition and pediatrics (e.g.

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improvement of infant milk formulas by mimicking human milk fat globules). Upon secretion

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of milk fat globules, the triacylglycerol droplet that was formed at the rough endoplasmic

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reticulum becomes coated with the apical plasma membrane of the lactating mammary gland

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to yield the MFGM6. Hence, the MFGM is a biological membrane and shows similar polar

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lipid composition to that of the mother’s lactating mammary cell7. The major polar lipids

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found in the MFGM are phosphatidylethanolamine (PE), phosphatidylcholine (PC), milk

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sphingomyelin (MSM), phosphatidylserine (PS) and phosphatidylinositol (PI)8. MSM, the

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main sphingolipid representing 20 to 45% wt of the milk polar lipids9, contains long chain

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saturated fatty acids contributing greatly in the gel to liquid crystalline phase transition of

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MFGM polar lipids (Tm = 36.4°C for MFGM10). The zwitterionic MSM and PC are mainly

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found on the external leaflet of the MFGM11. Cholesterol, that represents 308 to 606 mg / 100

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g fat in milk12, is located mostly in the MFGM but its transverse distribution in the membrane

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is not known12. Recent confocal laser scanning microscopy experiments performed in situ in

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milk revealed the heterogeneous distribution of proteins and polar lipids in the MFGM and

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the presence of µm-size lipid domains13-19. These lipid microdomains have been interpreted as



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the lateral segregation of high phase transition temperature (Tm) polar lipids, mainly MSM, in

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the solid-ordered (so) or in the liquid-ordered (lo) phase in presence of cholesterol, surrounded

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by a continuous liquid-disordered (ld) phase matrix of unsaturated PE, PS, PI and PC9,15,16,20.

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A central question is whether cholesterol plays an active role for the self-assembly of the

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high-Tm polar lipids to form phase-segregated domains in the plane of the membrane, and the

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potential consequences on the mechanical properties and biological functions of the MFGM.

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Answers to this question are important first in perinatal biology and nutrition, to better

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understand how the packing of lipids in the MFGM can affect biological mechanisms at the

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surface of the milk fat globule e.g. the sorting of bioactive membrane proteins, enzymes

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insertion and activity (lipase, sphingomyelinase), lipid digestion, interactions with viruses and

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bacteria in the gastrointestinal tract. It is also important in the context of food technology as

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regards to the physical stability of milk fat globules upon processing (e.g. in the manufacture

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of butter) or for the use of MFGM polar lipids enriched ingredients e.g. preparation of

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emulsion, design of encapsulation liposomes with targeted mechanical properties to control

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release.

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Elucidation of the phase separation, topography and mechanical properties of model

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membranes can be performed at nanoscale using atomic force microscopy (AFM) due to its

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high lateral and vertical resolutions. Moreover, AFM provides force spectroscopy ability that

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allows association of mechanical information on the nanometer lateral scale with structural

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information corresponding to phases of the lipid bilayer21-25. Recent AFM studies on MFGM

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lipid extract monolayers showed that upon cholesterol addition ranging 0 to 27 mol %, the

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morphology of the liquid-condensed phase domains drastically changed from large µm-

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diameter units protruding ~1.5 nm above the fluid liquid-expanded phase matrix to scattered

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numerous units protruding ~1 nm above the fluid phase and revealed a condensing effect of

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cholesterol10. In bilayers of MSM and dioleylphosphatidylcholine (DOPC), cholesterol also


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affected the morphology of the MSM domains26. Moreover, AFM force spectroscopy

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experiments showed that MSM domains ruptured at lower breakthrough force in presence of

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20 mol % cholesterol than in cholesterol-free bilayers, revealing an effect of cholesterol on

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the mechanical properties of the hydrated MSM/DOPC bilayers26. However, detailed

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information on the structural and mechanical behavior of hydrated bilayers with complex

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composition as that of the MFGM, as a function of cholesterol concentration, remains

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unknown.

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The objective of the present study was therefore to investigate the physical properties

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of hydrated MFGM polar lipid bilayers, below Tm and in presence of various amounts of

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cholesterol. The phase state of polar lipids was identified using X-ray diffraction, the

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topography of bilayers was characterized using AFM imaging and heterogeneities in their

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mechanical properties were elucidated using AFM force spectroscopy.

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EXPERIMENTAL SECTION

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Materials. The MFGM polar lipid extract used in this study was the same as in

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Murthy et al.10. The relative weight percentages of the five main classes of polar lipids in the

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MFGM lipid extract were 38.7% MSM, 31.6% PC, 23.5% PE, 3.4% PI and 2.8% PS. Milk

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polar lipids were composed by 65% saturated fatty acids with chain length ranging from 10 to

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24 atoms of carbon. The main saturated fatty acids of the MFGM polar lipid extract were due

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to the high amount of MSM, which is rich in C16:0, C22:0, C23:0 and C24:026,27. The

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cholesterol content of the MFGM lipid extract was 1.34% wt of the total polar lipids and was

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neglected. Cholesterol (Chol; from ovine wool, >98%) was purchased from Avanti Polar

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Lipids (Alabaster, AL) and used as received. PIPES 10 mM (1,4-piperazinediethane sulfonic

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acid ; purity ≥ 99%; Sigma Aldrich, Milwaukee, WI, USA) buffer was prepared with NaCl 50

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mM, (purity ≥ 99%; Sigma) in Milli-Q water and adjusted to pH 6.7 using NaOH 5 M. PIPES 5 ACS Paragon Plus Environment


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buffer was also prepared with 2 mM CaCl2 for AFM experiments. For XRD and AFM

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experiments, MFGM polar lipids/cholesterol samples were prepared by dissolving appropriate

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stock solutions of MFGM lipid extract or cholesterol in chloroform/methanol (4/1 v/v) and

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mixing them to reach 0 – 26.8 % mol cholesterol on a total MFGM polar lipids basis,

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corresponding to molar proportions of MSM/cholesterol from 100/0 to 50/50. The organic

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solvent was then evaporated under a stream of dry nitrogen at 50°C. The samples were stored

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at -20°C before use.

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X-ray diffraction (XRD). MFGM polar lipids and MFGM polar lipids /

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cholesterol samples were hydrated at 65°C with PIPES buffer (NaCl 50 mM, pH 6.7), to reach

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a final concentration of 40% wt lipids. The dispersions were heated at 75°C and thoroughly

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mixed in a vortex stirrer to form multilamellar vesicles. Small volumes (~20 µL) of samples

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containing fully hydrated MFGM polar lipids and MFGM polar lipids/cholesterol

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multilamellar vesicles were loaded in thin quartz capillaries of 1.5 mm diameter (GLAS W.

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Muller, Berlin, Germany). X-ray scattering experiments were performed on the SAXS/WAXS

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beamline at Australian synchrotron (Melbourne, Australia). Two 2-dimensional detectors

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allowed the recording of XRD patterns in the small- (0.02-0.2 Å-1) and wide- (0.8-3.5Å-1)

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angles regions of interest to characterize the long range organization of the phase and to

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identify the molecular packing of the fatty acid chains respectively. Diffraction patterns

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displayed a series of concentric rings as a function of the radial scattering vector q = 4 π sinθ /

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λ, where 2θ is the scattering angle and λ the wavelength of the incident beam. The channel to

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scattering vector q calibration of the detector was carried out with silver behenate28. A

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temperature controlled stage (Linkam Scientific Instruments, Tadworth, UK) inserted in the

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beamline permitted experiments at 20°C, i.e. below the phase transition temperature of the

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MFGM lipid extract (Tm=36.4°C10).


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Atomic force microscopy (AFM). The MFGM polar lipids and MFGM polar

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lipids / cholesterol dried mixtures were hydrated with PIPES-NaCl buffer (without calcium)

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and at 65°C to reach a final concentration of 0.1 % wt lipids then thoroughly vortexed. Small

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unilamellar vesicles (SUV) were produced at ~65°C by sonication, to yield monodisperse

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suspensions of vesicles below 100 nm in mean diameter as measured by dynamic light

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scattering (not shown). Homogeneous size distribution of the SUV ensures that the bilayers

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composition is representative of the sample’s complexity. The SUV were diluted 10-fold (v/v)

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with hot PIPES-NaCl-2 mM CaCl2 buffer, deposited onto freshly cleaved mica in an Asylum

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Research AFM liquid cell, then incubated at ~65°C for 60 min. Slow cooling of the samples

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was performed using a programmed incubator at rates sequentially decreasing from ~1°C.min-

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1

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for biological membranes, even though their contact with the flat mica substrate might affect

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the ld/so phase transition by a few °C or slightly alter lipid diffusion and packing29. Operating

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fusion at temperatures well above Tm, as was done, favors leaflet registry and limits substrate

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effects. Once equilibrated at room temperature (23 ± 0.7°C), the bilayers were extensively

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rinsed with PIPES-NaCl-CaCl2 buffer. AFM imaging of the bilayers was performed in contact

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mode using an MFP-3D Bio AFM (Asylum Research, Santa Barbara, CA, USA), silicon

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MSNL probes (nominal spring constant k ~0.03 N.m-1 - Bruker Nano Surfaces, Santa Barbara,

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CA, USA) and loading forces typically below ~1 nN. The typical scan rate was 0.5 Hz for 256

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× 256 pixels images. Up to 10 images of 10 × 10 µm2 scan size were recorded at various

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locations on each sample and were found similar for a given lipid composition. The images

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were typically planefitted at order 0, flattened at order 1 then planefitted again after masking

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of the protruding domains. Sections were drawn across images to measure the height

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difference H between features of the images. Force mapping was performed over 10 × 10 µm2

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(32 × 32 pixels) bilayer area using the same AFM instrument and the same MSNL probes as

to ~0.1°C.min-1 to yield supported lipid bilayers (SLB). SLB have proven valuable models



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in imaging. The probes were calibrated extemporaneously using the thermal noise method.

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Temperature inside the liquid cell was 24.5 ± 0.7°C, due to heating by e.g. the AFM laser.

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Typically, an applied load of up to 35 nN and a piezo speed of 2 µm.s-1 were used. The

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collected force curves were batch-analyzed using a self-developed algorithm adapted from Li

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et al.25. The mean breakthrough force values were obtained from manual Gaussian fits of the

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force-distribution histograms. Statistical analysis (t-test) was performed using Excel

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(Microsoft, Redmond, WA, USA).

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RESULTS AND DISCUSSION

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Phase state as a function of cholesterol concentration determined

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below Tm of MFGM polar lipids. XRD experiments allowed identification of the

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phase state of MFGM polar lipids and MFGM polar lipids /cholesterol bilayers at 20°C (i.e.

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below Tm of the MFGM polar lipids10), as a function of cholesterol concentration. For

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MFGM polar lipid bilayers, the small-angle XRD patterns were characterized by broad first

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(01)- and second (02)-order rings of scattering corresponding to lamellar structures (Figure 1).

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The lamellar repeat spacing deduced from the (01) reflection was d = 80.6 Å. At wide angles,

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the single peak observed at the d-spacing of 4.18 Å (q = 1.5 Å-1) was typical of hexagonaly

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packed, extended hydrocarbon chains in the gel or so phase (Figure 1). This is in agreement

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with previous results reported below the phase transition temperature of MFGM lipid

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extract10, MSM bilayers10 and egg-SM bilayers30.

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Cholesterol induced structural reorganization of MFGM polar lipid bilayers below the

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phase transition temperature, as revealed by the evolution of the XRD patterns. Increasing

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cholesterol concentration induced the formation of sharp rings of scattering with a progressive

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decrease of the lamellar repeat spacing of the lipid phase down to 74 Å for 27% mol.

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cholesterol. The wide-angle XRD peak associated to the so phase progressively broadened and 8 ACS Paragon Plus Environment

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decreased in intensity as a function of cholesterol addition, in particular for cholesterol

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concentration above 3% mol. in the MFGM polar lipid bilayer. The wide-angle XRD peak

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was not observed for 27% mol. cholesterol in the MFGM polar lipid bilayer. As previously

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reported, differential scanning calorimetry (DSC) thermograms recorded on heating of

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multilamellar vesicles of MFGM polar lipids exhibited a broad endothermal peak with a

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maximum at Tm = 36.4°C, corresponding to the gel (also noted so) - ld phase transition of

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high Tm saturated polar lipids10. The addition of cholesterol to MFGM polar lipid bilayers

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decreased and then eliminated this endothermal event for 27% mol cholesterol, which

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demonstrated the solubilization of cholesterol in MFGM polar lipid bilayers (results not

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shown). Both XRD and DSC data revealed the formation of the lo phase in MFGM polar

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lipids / cholesterol bilayers for 27% mol cholesterol, as already reported for egg-

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SM/cholesterol bilayers30. It is known that cholesterol in large concentrations (above 25%

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mol) eliminates the so to ld phase transition of saturated lipids (e.g. SM and DPPC) and

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produces a new type of fluid phase, i.e. lo phase31,32.

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Figure 1: X-ray diffraction (XRD) patterns of MFGM polar lipids and MFGM polar lipids/cholesterol

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bilayers fully hydrated with Pipes buffer at 20°C. (A) Small-angle XRD patterns, (B) wide-angle XRD

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patterns (so: solid-ordered phase, lo: liquid ordered phase). The MFGM polar lipids/cholesterol ratios

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are indicated in the figure.

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Topography and mechanical properties of the MFGM polar lipid

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bilayers: role of cholesterol. AFM imaging was employed to visualize how different

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cholesterol amounts influence the lateral organization of hydrated supported lipid bilayers

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prepared from MFGM polar lipids. AFM height images of the MFGM polar lipid bilayer

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showed the coexistence of µm-wide domains (lighter regions) dispersed in a continuous phase

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(darker matrix; Figure 2, top image in the left column). Cross-section analysis, as sketched in


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Figure 3, showed that the domains protruded above the continuous phase by a height

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difference H of 0.96 ± 0.16 nm.

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Upon addition of 3.9 or 8.4% mol. cholesterol to the total polar lipids, area of the wide

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domains in the MFGM polar lipid bilayers decreased while a number of smaller domains were

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formed (order of magnitude 0.01 µm2 – Figure 2, left column).

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Figure 2. Typical 10x10 µm2 AFM height images of supported lipid bilayers of mixtures of milk fat

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globule membrane polar lipids (MFGM) and cholesterol (chol) as indicated (left column), the

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corresponding breakthough force (FB) mapping experiments (center column; mind the changes in the

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nN scale) and the resulting FB histograms obtained after batch analysis of the individual force curves

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(right column). The colors of the square symbols refer to those of the symbols in Figure 5 (same color,

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same phase). The bilayers were imaged in PIPES/NaCl/CaCl2 buffer, pH 6.7 at room temperature. 12 ACS Paragon Plus Environment

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The height difference H between the domains and the continuous phase significantly

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decreased from ∼ 0.96 nm in absence of cholesterol down to an overall average of 0.82 ± 0.16

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nm in presence of 3.9 or 8.4% mol. cholesterol in the MFGM polar lipid bilayers (Figure

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3&4). The large and small domains visible on the AFM height images for 3.9 or 8.4%

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cholesterol (Figures 2&3) did not show a difference in H, in agreement with previous

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monolayer observations10 and indicating similar compositions and phases of the domains.

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Upon addition of 16.5 or 26.8% mol. cholesterol to the total polar lipids, the AFM height

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images did not show any visible separation between phases in the MFGM polar lipid bilayers

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(Figure 2). As a consequence, the height difference H dropped down to virtually zero (Figure

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4). In essence, the results observed with MFGM polar lipids/cholesterol bilayers agreed well

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with those found with MFGM polar lipids/cholesterol monolayers, except that the numerous

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0.01 µm2 domains visible in monolayers at the higher cholesterol levels were absent or not

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visible in the present hydrated bilayer conditions10.

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Figure 3. Topography of the milk fat globule membrane (MFGM) polar lipid bilayers, as a function of

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cholesterol concentration. 3D-representation of the MFGM polar lipid bilayers with 0% (A1) and 8%

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cholesterol (A2) showed in Figure 2 and sketch of the sections taken along a scan line (in blue), (B1,

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B2) respective height profiles showing the height difference H between the ordered domains (solid

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ordered so; liquid ordered lo) and the liquid disordered ld continous phase.

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Figure 4. Topography of the milk fat globule membrane (MFGM) polar lipid bilayers, as a function of

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cholesterol concentration. Histogram of the height differences H between the ordered domains and the

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liquid disordered continous phase recorded for the different concentrations of added cholesterol (N=25

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taken on at least 3 different images). Different star symbols indicate significantly different values

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(p

Cholesterol Decreases the Size and the Mechanical Resistance to

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