Components And Kinetics In Exocytosis

这个项目的中心是胞吐作用的机制，这是一种普遍存在的真核过程，通过这种过程，囊泡融合到质膜上并释放其内容物。我们今年报告了两个子项目，都与主要胞吐蛋白聚集的事实有关。 1.在流感血凝素诱导的膜融合中，胆固醇促进膜的半融合和扩孔。 流感病毒的成功感染需要包膜尖峰蛋白血凝素(HA)催化病毒包膜和靶细胞内膜之间的融合，并创造一个足够大的孔来释放病毒基因组。越来越多的人认识到膜脂在这一关键事件中发挥了作用，主要来自与膜单分子层曲率应力相关的脂类组成的实验和理论。最近，人们考虑了膜相行为和膜微区对脂类与膜蛋白，特别是与病毒包膜糖蛋白的横向分布、分类和相互作用的作用。胆固醇是真核细胞膜的主要和重要成分。其独特的结构，一个小的亲水头基和刚性的，疏水的，稠环，有利于与饱和的酰链脂和鞘脂优先结合，在组成合适的磷脂双层膜中形成液体有序的微域(称为脂筏)。假设脂筏存在于细胞质膜中的特定位置，在那里蛋白质与有序的、富含胆固醇的环境有良好的联系。 膜融合的特定中间产物由流感病毒蛋白血凝素催化，受胆固醇调节。在表达HA(HAS细胞)的Sf9细胞中，早期脂质转移的程度与以前在哺乳动物细胞系统中观察到的相似，但最初的融合孔很小，孔扩张受阻。细胞胆固醇增加三倍(见方法)会导致1)更快的脂类染料转移动力学，2)水性染料转移的量增加，3)水性染料转移的程度增加，以及4)孔电导的增加。胆固醇依赖的融合效率的提高需要完整的HA、TMD和最适pH。总体而言，这些结果支持宿主细胞胆固醇在膜融合中作用于两个阶段的假设：1)融合孔开放之前的早期脂类阶段和2)融合孔扩张的后期阶段。胆固醇的物理性质如何影响融合？ 我们已经证明，在流感HA介导的细胞-细胞膜融合中，胆固醇既促进脂质转移(半融合)，又促进融合孔扩张。胆固醇效应需要完整的TMD和最适pH。我们假设，胆固醇促进融合孔扩张1)通过其负的固有曲率和2)相同的特定的胆固醇/脂/HA相互作用，调节HA在膜平面上1-10 nm尺度的聚集，HA在成纤维细胞中的流动性，以及流感病毒被膜的相行为。这些结构性作用力在病毒入侵的融合阶段起作用，以促进融合孔的扩大。 2.蛋白质脂质润湿引起的膜结构域形成 经典的流体马赛克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，人们越来越意识到膜上的非均质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。由于疏水性、范德华力、静电和化学力等因素介导的脂类和蛋白质相互作用会导致某些脂类和蛋白质聚集在结构域中，而另一些则相互排斥，因此必然存在膜的不均匀。三十多年前，电子自旋共振证实了微小的空间不均匀性的存在。研究发现，与与蛋白质无关的脂类相比，蛋白质周围的边界脂类化合物的跳跃时间减少了约10倍。在我们的项目中，细胞膜中木筏和其他结构域的形成被认为是蛋白质被脂类润湿。膜被模拟为连续的弹性介质。这种方法产生了形成宏观润湿膜的必要条件；它的厚度也可以确定。 我们用适用于生物膜的液晶平均场理论计算了润湿类脂膜的热力学函数。我们发现，根据膜厚度、疏水高度失配、脂类自发曲率和蛋白质半径等参数的取值，可以形成分子膜或宏观膜。我们发现，单个r1 nm的蛋白质不足以诱导局部相变形成蛋白质-脂筏。但宏观润湿膜可以在直径超过几十纳米的脂质/蛋白质聚集体周围形成。我们假设这种聚集体中的脂类处于液体有序状态，类似于固体表面的润湿。在Mouritsen的毛细管波模型的背景下进行的计算和模拟支持了这一假设的有效性。包裹聚集体的润湿膜可能非常重要，因为它们促进了结构域的合并。此外，润湿膜防止蛋白质离开聚集体，从而促进蛋白质的聚集和聚集。

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.