Cholesterol Transbilayer Distribution in Mammalian cells: Mechanisms and Functions

哺乳动物细胞中胆固醇跨双层分布：机制和功能

• 批准号: 402010-2013
• 负责人: Zha, Xiaohui
• 金额: $ 1.97万
• 依托单位: University of Ottawa
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=633605
• 关键词: Cholesterol; Transbilayer; Distribution; Mammalian; cells

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).

真核细胞质膜（PM）是已知的，其不对称分布的磷脂之间的小叶。胆固醇是PM的另一个主要成分，但其双层分布仍不确定。目前，胆固醇被认为优先与外叶中的SM结合以形成脂筏。此外，最近的研究提供了坚实的证据表明，荧光甾醇主要存在于PM的内小叶中（70-80%）。对于3 H-胆固醇，我们还发现内瓣叶中约有70%的胆固醇。这是完全出乎意料的。胆固醇在双层内自发地翻转（t1/2 << 1 s），并且在模型膜中比其他磷脂更喜欢SM。因此，胆固醇应该更喜欢SM所在的外叶。然而，其他因素也可能发挥作用。首先，胆固醇偏好SM的研究都是在对称膜中进行的。最近的证据表明，在不对称膜中存在强烈的瓣叶间耦合。这种耦合产生了一种整合状态，与具有相同脂质组成的对称膜有很大不同。其次，PM不仅是不对称的，它的内小叶也带负电荷，富含PE，一种锥形脂质。最近有人提出，这种不对称膜的结合可以提供强大的力来保留胆固醇。如果所有哺乳动物细胞的内叶中确实有更多的胆固醇，那么它是如何形成脂筏的呢？这些纳米结构域的性质一直存在争议，特别是当通过生物化学技术定义时。然而，随着光学技术的进步，脂筏现在可以在活细胞中检测到。通过将这些技术与胆固醇双层分布相结合，现在可以广泛了解胆固醇如何与磷脂在不对称膜中相互作用形成脂筏。我们将重点放在假设，有一个胆固醇不对称的所有哺乳动物PM（目的1）。这种不对称性定义了PM脂筏（目的2）。我们还假设PM中的磷脂不对称性是PM内小叶中胆固醇富集的主要驱动力之一（目的3）。