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。去年，在第一个项目中，我们描述了在脂膜中创建宏观的RAFT结构域。我们定量地描述了多组分脂质双层膜中相分离结构域的产生和演化。早期阶段称为成核阶段和独立生长阶段，速度非常快(特征时间分别为亚毫秒和毫秒)，体系由平均半径约为5-50 nm的纳米微区组成。其次，磁区的迁移性变得重要；磁区合并和分裂成为物质交换的主要机制，而线张力是任何时间点磁区尺寸分布的主要决定因素。对于足够小的线张力，由磁区合并引起的熵项的下降大于边界能的下降，并且只存在纳米磁区。对于较大的线张力，边界能的降低主导了合并的不利熵，合并导致纳米微区迅速扩大到微米级的半径。在中线张力下，在有限的时间内，纳米结构域可以保持分散，并与新的全局相共存。快速形成大筏子所需的线张力的理论临界值与由巨大单层囊泡萌发区域的曲率估算的实验结果相一致。今年，我们将继续详细研究这一机制。外加横向拉力对不同区域间线张力的影响 计算了脂质双层膜的厚度。为了模拟生物膜中的浮筏，厚区域被视为液体有序相，而薄区域被视为液体无序相来模拟周围区域。在我们的模型中，单层在边界处弹性扭曲，以创建平滑的而不是阶梯状的边界，以避免厚筏的疏水内部暴露在水中。这种变形的能量由张开和倾斜的基本变形来描述。每单位长度边界的能量产生了木筏的线张力。施加横向张力会改变基本变形，从而增加线张力。当筏子的自发曲率大于周围的曲率时，绳索张力的增加较大；如果筏子的自发曲率小于环绕物体的自发曲率，则由于施加横向张力而引起的绳索张力增加较小。 第二个项目在本质上是实验性的，并使用了一个胞吐蛋白的模型，这是一种在成纤维细胞中表达的融合蛋白。生物膜中的组织在长度尺度上跨越许多数量级，但远场光学显微镜的有限分辨率阻碍了对众多生物膜模型的区分。异质分布的膜蛋白的一个典型例子是流感病毒的血凝素(HA)，它与有争议的富含胆固醇的脂筏有关。使用荧光光活化定位显微镜(FPALM)，我们能够成像数以万计的亚衍射分辨率(30-40 nm)的HA分子在活的和固定的成纤维细胞中的分布。HA分子在30纳米到许多微米的长度尺度上形成不规则的团簇，这与电子显微镜的结果一致。在活细胞中，观察和量化簇内HA分子的动态，以确定有效扩散系数。结果用几种已建立的生物膜模型进行了解释。

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.