Cholesterol Transbilayer Distribution in Mammalian cells: Mechanisms and Functions

真核生物质膜（PM）以其磷脂在小叶之间的不对称分布而闻名。胆固醇是PM的另一个主要组成部分，但其双分子层分布仍不确定。目前，胆固醇被认为优先与外小叶中的SM结合形成脂质筏。相反，最近的研究提供了确凿的证据，表明荧光甾醇主要存在于PM的内部小叶中（70-80%）。对于3h -胆固醇，我们也发现大约70%的胆固醇在内小叶。这真是出乎意料。胆固醇自发地在双分子层内进行翻转（t1/2 << 1s），并且在模型膜中更倾向于SM而不是其他磷脂。因此，胆固醇更倾向于SM所在的外小叶。然而，其他因素也可能起作用。首先，胆固醇倾向于SM的研究都是在对称膜中进行的。最近的证据表明，在不对称膜中有很强的叶间偶联。这种耦合产生了一种与具有相同脂质组成的对称膜大不相同的集成状态。其次，PM不仅不对称，其内部小叶也带负电荷，富含PE（一种锥形脂质）。不对称膜中的这种组合最近被提出提供强大的力量来保留胆固醇。如果胆固醇确实存在于所有哺乳动物细胞的内叶中，那么它是如何促成脂质堆积的呢？这些纳米结构域的性质一直是有争议的，特别是当由生化技术定义时。然而，随着光学技术的进步，现在可以在活细胞中检测到脂筏。通过将这些技术与胆固醇双分子层分布相结合，现在可以广泛地了解胆固醇如何与不对称膜中的磷脂相互作用以形成脂筏。我们将重点关注所有哺乳动物PM中胆固醇不对称的假设（目的1）。这种不对称性定义了PM脂筏（目的2）。我们还假设，PM中的磷脂不对称是PM内小叶中胆固醇富集的主要驱动力之一（目的3）。

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).