Components And Kinetics In Exocytosis

这个项目集中在胞吐作用的机制上，胞吐作用是真核生物普遍存在的一个过程，通过这个过程，囊泡融合到质膜并释放它们的内容物。我们今年报告了两个子项目，都与主要的胞外蛋白聚集有关。去年，在第一个项目中，我们描述了在脂质膜中创建宏观筏结构域。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进化。早期阶段，称为成核阶段和独立生长阶段，非常快（特征时间分别为亚毫秒和毫秒），系统由平均半径约为5 -50 nm的纳米畴组成。接下来，领域的移动性变得重要；畴合并和裂变成为物质交换的主要机制，而线张力是决定任何时间点畴尺寸分布的主要因素。当线张力足够小时，由畴合并引起的熵项的减少大于边界能的减少，并且只存在纳米畴。当线张力较大时，边界能的降低主导了不利的合并熵，合并导致纳米畴迅速扩大到微米尺度半径。在中间线张力和有限时间内，纳米畴可以保持分散并与新的全局相共存。快速形成大木筏所需的线张力的理论临界值与由巨大单层囊泡的出芽区曲率所得到的实验值是一致的。今年我们将继续深入研究这一机制。外部施加的侧向张力对两个不同域之间的线张力的影响

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.