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。去年，在第一个项目中，我们描述了脂质膜中宏观筏结构域的创建。 我们定量描述了多组分脂双层膜中相分离结构域的产生和演变。早期阶段，称为成核阶段，和独立生长阶段，是非常迅速的（特征时间分别为亚毫秒和毫秒），该系统由平均半径约5 - 50 nm的纳米畴。其次，域的流动性变得重要;域合并和裂变成为物质交换的主要机制，并且线张力是任何时间点的域尺寸分布的主要决定因素。对于足够小的线张力，熵项的减少，从域合并的结果是大于边界能量的减少，只有纳米畴存在。 对于大的线张力，在边界能量的减少占主导地位的不利熵的合并，合并导致纳米畴迅速扩大到微米尺度的半径。在中间线张力和有限的时间内，纳米畴可以保持分散，并与新的全球相共存。快速形成大筏所需的线张力的理论临界值与根据巨单层囊泡中出芽域曲率的实验估计值雅阁。今年，我们将继续详细研究这一机制。研究了外加横向张力对两个不同结构域之间的线张力的影响 计算脂双层膜中的厚度。厚域被视为液体有序相，以模拟生物膜中的筏;薄域被认为是液体无序相，以模拟周围区域。在我们的模型中，单层弹性扭曲的边界，以创建一个平滑的，而不是阶梯状的边界，以避免暴露的厚筏的疏水内部的水。这种变形的能量由展曲和倾斜的基本变形来描述。每单位边界长度的能量产生筏板的线张力。施加横向张力改变基本变形，使得线张力增加。当筏板的自发曲率大于围壁的自发曲率时，线张力的增加较大;如果筏板的自发曲率小于围壁的自发曲率，则由于施加横向张力而引起的线张力的增加较为适度。 第二个项目是实验性的，并使用一个模型的胞吐蛋白融合蛋白在成纤维细胞中表达。生物膜的组织在长度尺度上跨越了许多数量级，但远场光学显微镜的有限分辨率阻碍了许多生物膜模型之间的区分。不均匀分布的膜蛋白的一个典型例子是来自流感病毒的血凝素（HA），其与有争议的富含胆固醇的脂筏相关。使用荧光光活化定位显微镜（FPAL M），我们能够成像的分布成千上万的HA分子与亚衍射分辨率（30-40 nm）在活的和固定的成纤维细胞。HA分子在30 nm至许多微米的长度尺度上形成不规则簇，与电子显微镜的结果一致。在活细胞中，观察和量化簇内HA分子的动力学以确定有效扩散系数。结果被解释在几个已建立的模型的生物膜。

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. Last year, in the first project we described the creation of macroscopic raft domains in lipid membranes. We describe quantitatively the creation and evolution of phase-separated domains in a multicomponent lipid bilayer membrane. The early stages, termed the nucleation stage, and the independent growth stage, are extremely rapid (characteristic times are submillisecond and millisecond, respectively) and the system consists of nanodomains of average radius about 5 -50 nm. Next, mobility of domains becomes consequential; domain merger and fission become the dominant mechanisms of matter exchange, and line tension is the main determinant of the domain size distribution at any point in time. For sufficiently small line tension, the decrease in the entropy term that results from domain merger is larger than the decrease in boundary energy, and only nanodomains are present. For large line tension, the decrease in boundary energy dominates the unfavorable entropy of merger, and merger leads to rapid enlargement of nanodomains to radii of micrometer scale. At intermediate line tensions and within finite times, nanodomains can remain dispersed and coexist with a new global phase. The theoretical critical value of line tension needed to rapidly form large rafts is in accord with the experimental estimate from the curvatures of budding domains in giant unilamellar vesicles. This year we continue to study this mechanism in detail. The effect of an external applied lateral tension on the line tension between two domains of different thickness in a lipid bilayer membrane is calculated. The thick domain is treated as a liquid-ordered phase in order to model a raft in a biological membrane; the thin domain is considered a liquid-disordered phase to model the surrounding region. In our model, the monolayers elastically distort at the boundary to create a smooth rather than steplike boundary to avoid exposure of the hydrophobic interior of the thick raft to water. The energy of this distortion is described by the fundamental deformations of splay and tilt. This energy per unit length of boundary yields the line tension of the raft. Applying lateral tension alters the fundamental deformations such that line tension increases. This increase in line tension is larger when the spontaneous curvature of a raft is greater than that of the surround; if the spontaneous curvature of the raft is less than that of the surround, the increase of the line tension due to application of the lateral tension is more modest. The second project is experimental in nature, and uses a model for the exocytotic proteins a fusion protein expressed in fibroblasts. Organization in biological membranes spans many orders of magnitude in length scale, but limited resolution in far-field light microscopy has impeded distinction between numerous biomembrane models. One canonical example of a heterogeneously distributed membrane protein is hemagglutinin (HA) from influenza virus, which is associated with controversial cholesterol-rich lipid rafts. Using fluorescence photoactivation localization microscopy (FPALM), we are able to image distributions of tens of thousands of HA molecules with sub-diffraction resolution (30-40 nm) in live and fixed fibroblasts. HA molecules form irregular clusters on length scales from 30 nm up to many micrometers, consistent with results from electron microscopy. In live cells, the dynamics of HA molecules within clusters is observed and quantified to determine an effective diffusion coefficient. The results are interpreted in terms of several established models of biological membranes.