# The impact of sphingomyelin and cholesterol on ordered lipid domain formation in the bovine milk fat globule membrane using artificial giant unilamellar vesicles as a model

## TL;DR

The study uses artificial vesicles to show that cholesterol, more than sphingomyelin, is key in forming ordered lipid domains in milk fat globule membranes.

## Contribution

Demonstrates that cholesterol, not sphingomyelin, is primarily responsible for ordered lipid domain formation in model milk fat globule membranes.

## Key findings

- Cholesterol is more effective than sphingomyelin in forming ordered lipid domains in phospholipid bilayers.
- Dark regions in vesicles indicate ordered lipid domains, even in the absence of cholesterol.
- Phospholipids with high melting temperatures also contribute to lipid phase separation.

## Abstract

Summary: Giant unilamellar vesicles (GUV) were constructed by an electroformation method. Solutions of lipid mixture containing a blend of phospholipids and different contents of cholesterol or milk sphingomyelin (SM) were deposited on the surface of indium tin oxide (ITO)-coated glass slides to form a dried thin film of lipid mixture. The dried layer of lipid mixture was then hydrated by sucrose solution. An alternating current (AC) field was applied to the small sample reservoir within an O-ring space. The applied electrical field stimulated a swelling effect of the deposited lipid layers. Consequently, lipid layers constructed by the mixed lipidic components were formed; these systems include GUV, multilamellar vesicles (MLV), and multivesicular vesicles (MVV). By analyzing the (nonfluorescent) dark regions in GUV constructed by different lipid mixtures containing different amounts of SM and cholesterol, it was suggested that cholesterol might be an effective component in determining ordered lipid domain (OLD) formation. CLSM = confocal laser scanning microscopy; DIC = differential interfacial contrast.

Summary: Giant unilamellar vesicles (GUV) were constructed by an electroformation method. Solutions of lipid mixture containing a blend of phospholipids and different contents of cholesterol or milk sphingomyelin (SM) were deposited on the surface of indium tin oxide (ITO)-coated glass slides to form a dried thin film of lipid mixture. The dried layer of lipid mixture was then hydrated by sucrose solution. An alternating current (AC) field was applied to the small sample reservoir within an O-ring space. The applied electrical field stimulated a swelling effect of the deposited lipid layers. Consequently, lipid layers constructed by the mixed lipidic components were formed; these systems include GUV, multilamellar vesicles (MLV), and multivesicular vesicles (MVV). By analyzing the (nonfluorescent) dark regions in GUV constructed by different lipid mixtures containing different amounts of SM and cholesterol, it was suggested that cholesterol might be an effective component in determining ordered lipid domain (OLD) formation. CLSM = confocal laser scanning microscopy; DIC = differential interfacial contrast.

•We used GUV to study the structural nature of milk fat globule membrane.•The GUV were made by polar lipids and cholesterol relevant to milk fat globule membrane.•Compared with SM, cholesterol appeared more responsible for OLD formation.

We used GUV to study the structural nature of milk fat globule membrane.

The GUV were made by polar lipids and cholesterol relevant to milk fat globule membrane.

Compared with SM, cholesterol appeared more responsible for OLD formation.

Giant unilamellar vesicle (GUV) bilayers were constructed from polar lipids and cholesterol by electroformation as a model system to investigate the formation of ordered lipid domains (OLD) within the milk fat globule membrane (MFGM). Dark regions without fluorescent staining on the surfaces of GUV, observed by confocal laser scanning microscopy, were characterized as OLD. Lipid formulations were designed by mixing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; milk sphingomyelin; and cholesterol with designated molar ratios to reveal the key components responsible for segregated OLD formation. Cholesterol, rather than milk sphingomyelin, was more responsible for OLD formation. Dark regions were observed in GUV, which contained sphingomyelin but no cholesterol. This observation revealed that at room temperature (below the melting transition temperature [Tm]), not only do cholesterol-mediated ordered domains contribute to lipid phase separation, but phospholipids with high Tm in MFGM are also segregated from the bright fluorescent liquid-disordered domains. This work provides visible evidence demonstrating the comparative roles of sphingomyelin and cholesterol in forming OLD in phospholipid bilayers.