Cholesterol does not induce segregation of liquid-ordered domains in bilayers modeling the inner leaflet of the plasma membrane

A fluorescence-quenching method has been used to assess the potential formation of segregated liquid-ordered domains in lipid bilayers combining cholesterol with mixtures of amino and choline phospholipids like those found in the cytoplasmic leaflet of the mammalian cell plasma membrane. When present in proportions >20-30 mol %, different saturated phospholipids show a strong proclivity to form segregated domains when combined with unsaturated phospholipids and cholesterol, in a manner that is only weakly affected by the nature of the phospholipid headgroups. By contrast, mixtures containing purely unsaturated phospholipids and cholesterol do not exhibit detectable segregation of domains, even in systems whose components differ in headgroup structure, mono- versus polyunsaturation and/or acyl chain heterogeneity. These results indicate that mixtures of phospholipids resembling those found in the inner leaflet of the plasma membrane do not spontaneously form segregated liquid-ordered domains. Instead, our findings suggest that factors extrinsic to the inner-monolayer lipids themselves (e.g., transbilayer penetration of long sphingolipid acyl chains) would be essential to confer a distinctive, more highly ordered organization to the cytoplasmic leaflet of "lipid raft" structures in animal cell membranes.