Cholesterol Transbilayer Distribution in Mammalian cells: Mechanisms and Functions

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Eukaryotic plasma membrane (PM) is known for its assymetric distribution of phospholipids between the leaflets. Cholesterol is another major component of the PM Yet, its bilayer distribution remains uncertain. Currently, cholesterol is thought to preferentially bind to SM in the outer leaflet to form lipid raft. Contrarily, recent studies have provided solid evidence that a fluorescent sterol is mainly in the inner leaflet of the PM (70-80%). With 3H-cholesterol, we also have found about 70% cholesterol in the inner leaflet. This is quite unexpected. Cholesterol spontaneously flip-flops within bilayers (t1/2 << 1 s) and prefers SM over other phospholipids in model membranes. Thus, cholesterol should prefer the outer leaflet where SM resides. However, other factors may also play a role. First, studies where cholesterol prefers SM were all carried out in symmetric membranes. Recent evidence reveals a strong inter-leaflet coupling in an asymmetric membrane. Such coupling creates an integrated state that greatly differs from the symmetric membrane with the same lipid composition. Secondly, the PM is not only asymmetric, its inner leaflet is also negatively charged and rich in PE, a cone-shaped lipid. This combination in an asymmetric membrane was recently proposed to provide strong force to retain cholesterol. If indeed cholesterol is more in the inner leaflet of all mammalian cells, how does it contribute to lipid raft? The nature of these nano-domains has being controversial, particular when defined by biochemical techniques. However, with the advancement of optical technology, lipid rafts can now be detected in live cells. By combining these technologies with cholesterol bilayer distribution, it is now possible to gain a broad understanding how cholesterol interacts with phospholipids in asymmetric membranes to form lipid rafts. We will focus on the hypothesis that there is a cholesterol asymmetry in all mammalian PM (aim 1). This asymmetry defines the PM lipid rafts (aim 2). We also hypothesize that the phospholipid asymmetry in the PM is one of the major driving forces for cholesterol enrichment in the inner leaflet of the PM (aim 3).