Components And Kinetics In Exocytosis

这个项目是集中在胞吐作用的机制，普遍存在的真核细胞的过程中，囊泡融合到质膜和释放其内容物。我们报告了今年的两个子项目，这两个项目都与主要的胞吐蛋白质聚集的事实有关。 1.胆固醇促进流感血凝素诱导的膜融合中的半融合和孔增宽。 流感病毒的成功感染需要包膜刺突蛋白血凝素（HA）催化病毒包膜和靶细胞的胞内体膜之间的融合，并产生足够大的孔以释放病毒基因组。 越来越多的人认识到，膜脂质在这一关键事件中发挥作用，主要来自脂质组成与膜单层曲率应力的关系的实验和理论。 最近，已经考虑到膜相行为和膜微结构域对脂质与膜蛋白的横向分布、分选和相互作用的作用，特别是病毒包膜糖蛋白。胆固醇是真核细胞膜的主要和重要组成部分。 其独特的结构，一个小的亲水性头基和刚性，疏水性，融合环，有利于优先协会与饱和的酰基链脂质和鞘脂形成液体有序的微域（称为脂筏）在磷脂双层膜的正确组成。脂筏被假设存在于细胞质膜中的特定位点，在该位点处，与有序的、富含胆固醇的环境具有有利关联的蛋白质被浓缩。 由流感病毒蛋白血凝素催化的膜融合的特定中间体受胆固醇调节。 表达HA的Sf9细胞（HAS细胞）中早期脂质转移的程度与先前在哺乳动物细胞系统中观察到的相似，但初始融合孔较小，孔扩张受阻。 细胞胆固醇的三倍增加（参见方法）导致1）更快的脂质染料转移动力学，2）增加的水性染料转移量，3）增加的水性染料转移程度，和4）孔电导率的增加。 融合效率的胆固醇依赖性增加需要完整的HA TMD和最佳pH。总的来说，这些结果支持宿主细胞胆固醇在膜融合中的两个阶段起作用的假设：1）融合孔开放之前的早期阶段，和2）融合孔扩张期间的后期阶段。 胆固醇的物理性质如何影响融合？ 我们已经表明，胆固醇促进流感HA介导的细胞-细胞膜融合中的脂质转移（半融合）和融合孔扩张。 胆固醇效应需要完整的TMD和最佳pH值。我们假设胆固醇促进融合孔扩张1）凭借其负的固有曲率和2）相同的特异性胆固醇/脂质/HA相互作用，介导HA在膜平面中的1 - 10 nm尺度聚集，HA在成纤维细胞中的流动性和流感包膜的相行为。 这些结构力在病毒入侵的融合阶段起作用，以促进融合孔变宽。 2.蛋白质脂质润湿引起的膜结构域形成 经典的流体镶嵌模型认为质膜的脂质环境基本上是均匀的。但在过去的十年里，人们越来越认识到，膜中的非均匀性是依赖于膜内蛋白质-蛋白质相互作用的生物功能的核心。膜的非均匀性必然存在，因为由疏水、货车范德华力、静电和化学力介导的脂质和蛋白质相互作用将导致一些脂质和蛋白质聚集成结构域，而另一些脂质和蛋白质排斥。30多年前，电子自旋共振证实了微小的空间不均匀性的存在。结果发现，与不与蛋白质相关的脂质相比，蛋白质周围的边界脂质表现出约10倍的跳跃时间减少。在我们的项目中，筏和其他结构域在细胞膜中的形成被认为是蛋白质被脂质润湿。膜被建模为一个连续的弹性介质。这种方法产生了形成宏观润湿膜所需的条件;也可以确定其厚度。 我们计算了润湿脂质膜的热力学函数，通过使用液晶的平均场理论，适用于生物膜。我们表明，无论是分子或宏观膜可以形成，这取决于参数的值，如膜厚度，疏水高度失配，自发曲率的脂质，和蛋白质半径。 我们发现，一个单一的蛋白质的r1 nm是不够大，以诱导局部相变，形成蛋白质-脂质筏。 但是，在直径超过几十纳米的脂质/蛋白质聚集体周围可以形成宏观润湿膜。 我们已经假设，在这样的聚集体中的脂质是在液体有序状态，类似于固体表面的润湿。这一假设的有效性是支持的计算和模拟的上下文中的毛细波模型的Reichitsen。覆盖聚集体的润湿膜可能是相当重要的，因为它们促进域的合并。此外，润湿膜防止蛋白质离开聚集体，从而促进蛋白质的积累和聚集。

This project is centered on the mechanisms of exocytosis, the ubiquitous eukaryotic process by which vesicles fuse to the plasma membrane and release their contents. We report two subprojects this year, both related to the fact that the major exocytotic proteins are clustered. 1. Cholesterol promotes hemifusion and pore widening in membrane fusion induced by influenza hemagglutinin. Successful infection by influenza virus requires that the envelope spike protein, hemagglutinin (HA), catalyzes fusion between the viral envelope and the intracellular endosomal membrane of the target cell and creates a pore large enough to release the viral genome. There is a growing appreciation that membrane lipids play a role in this critical event, coming mostly from experiments and theory on lipid composition in relationship to membrane monolayer curvature stress. Recently there has been consideration given to the role of membrane phase behavior and membrane micro-domains on the lateral distribution, sorting and interactions of lipids with membrane proteins in general, and viral envelope glycoproteins in particular. Cholesterol is a major and vital constituent of eukaryotic cell membranes. Its unique structure, a small hydrophilic head group and rigid, hydrophobic, fused-rings, favors preferential association with saturated acyl-chain lipids and sphingolipids to form liquid-ordered micro-domains (termed lipid rafts) in phospholipid bilayer membranes of the right composition. Lipid rafts are hypothesized to exist in the cell plasma membrane at specialized sites where proteins, having favorable associations with the ordered, cholesterol-rich, environment, are concentrated. Specific intermediates of membrane fusion, catalyzed by the influenza virus protein hemagglutinin, are regulated by cholesterol. The extent of early lipid transfer in Sf9 cells expressing HA (HAS cells) is similar to that previously observed in mammalian cell systems, but the initial fusion pore is small and pore expansion is stunted. A three-fold increase in cellular cholesterol (see Methods) leads to 1) faster lipid dye transfer kinetics, 2) increased amount of aqueous dye transferred, 3) increased extent of aqueous dye transferred, and 4) an increase in the rate of pore conductance. The cholesterol dependent increase in fusion efficiency required an intact HA TMD and optimal pH. Overall, these results support the hypothesis that host cell cholesterol acts at two stages in membrane fusion: 1) an early, lipidic stage prior to fusion pore opening and, 2) a later stage during fusion pore expansion. How can the physical properties of cholesterol influence fusion? We have shown that cholesterol promotes both lipid transfer (hemi-fusion) and fusion pore expansion in the cell-cell membrane fusion mediated by influenza HA. The cholesterol effect requires a complete TMD and optimal pH. We hypothesize that cholesterol promotes fusion pore expansion 1) by virtue of its negative intrinsic curvature and 2) the same specific cholesterol/lipid/HA interactions that mediate the 1-10 nm scale clustering of HA in the plane of the membrane, the mobility of HA in fibroblasts, and the phase behavior of the influenza envelope. These structural forces act at the fusion stage of viral invasion to facilitate fusion pore widening. 2. Domain formation in membranes caused by lipid wetting of protein The classic fluid mosaic model views the lipid environment of a plasma membrane as essentially homogenous. But over the past decade it has been increasingly realized that non-homogeneities in membranes are central to biological functions that depend on protein-protein interactions within membranes. Membrane non-uniformities must exist because lipid and protein interactions mediated by hydrophobic, Van der Waals, electrostatic and chemical forces will cause some lipids and proteins to cluster into domains and others to repel. Over thirty years ago it was demonstrated by electron spin resonance that small spatial inhomogeneities existed. It was found that boundary lipids surrounding a protein exhibit about a 10-fold reduction in hop time compared to lipids that are not associated with proteins. In our project, formation of rafts and other domains in cell membranes is considered as wetting of proteins by lipids. The membrane is modeled as a continuous elastic medium. This approach yields the conditions necessary for a macroscopic wetting film to form; its thickness could also be determined. We calculated thermodynamic functions of wetting lipid films by using a mean-field theory of liquid crystals as adapted to biomembranes. We show that either molecular or macroscopic films can form, depending on the values of parameters such as membrane thickness, hydrophobic height mismatches, spontaneous curvature of lipids, and protein radius. We show that a single protein of r 1 nm is not large enough to induce a local phase transition to form a protein-lipid raft. But a macroscopic wetting film can form around a lipid/protein aggregate of more than tens of nanometers in diameter. We have assumed that the lipids in such aggregates are in a liquid-ordered state, analogous to wetting of solid surface. The validity of this assumption is supported by calculations and simulations made in the context of the capillary wave model of Mouritsen. Wetting films that coat an aggregate could be quite important because they facilitate merger of domains. Also, a wetting film prevents a protein from leaving an aggregate and thereby promotes accumulation and clustering of proteins.