Components And Kinetics In Exocytosis

本项目主要研究钙触发胞吐作用的机制，这是一种普遍存在的真核生物过程，通过这种过程，囊泡融合到质膜上并释放其内容物。尽管胞吐和钙之间的关系对突触和非神经元分泌功能都是至关重要的，但由于钙的时间和空间特性，以及囊泡运输、启动、回收和再循环是耦合的，因此分析是有问题的。通过对海胆卵分泌囊泡体外胞吐动力学的分析，确定了胞吐的最后步骤。这些步骤被建模为一个三态系统：激活、提交和融合，其中州际转换由激活的融合复合体提交的概率（alpha）和提交的融合复合体导致融合的概率(p)给出。每个囊泡对接点的提交复合体的数量是泊松分布，平均为n-bar。实验结果表明，p和n-bar随钙的增加而增加，而α和p/n-bar的比值保持不变，从而将动力学描述简化为只有一个钙依赖的控制变量n-bar。平均而言，最大速率（Rmax）的钙依赖性和达到Rmax （Tpeak）的时间由n-bar的钙依赖性来描述。因此，游离钙浓度与胞吐率之间的非线性关系可以完全用囊泡对接部位融合复合物分布对钙的依赖性来解释。

This project is centered on the mechanisms of calcium-triggered exocytosis, the ubiquitous eukaryotic process by which vesicles fuse to the plasma membrane and release their contents. Although the relationship between exocytosis and calcium is fundamental both to synaptic and nonneuronal secretory function, analysis is problematic because of the temporal and spatial properties of calcium, and the fact that vesicle transport, priming, retrieval, and recycling are coupled. By analyzing the kinetics of sea urchin egg secretory vesicle exocytosis in vitro, the final steps of exocytosis are resolved. These steps are modeled as a three-state system: activated, committed, and fused, where interstate transitions are given by the probabilities that an active fusion complex commits (alpha) and that a committed fusion complex results in fusion, p. The number of committed complexes per vesicle docking site is Poisson distributed with mean n-bar. Experimentally, p and n-bar increase with increasing calcium, whereas alpha and the p/n-bar ratio remain constant, reducing the kinetic description to only one calcium-dependent, controlling variable, n-bar. On average, the calcium dependence of the maximum rate (Rmax) and the time to reach Rmax (Tpeak) are described by the calcium dependence of n-bar. Thus, the nonlinear relationship between the free calcium concentration and the rate of exocytosis can be explained solely by the calcium dependence of the distribution of fusion complexes at vesicle docking sites. Although immunoblotting (Western blotting) is widely used for the detection of specific proteins, it is often thought to be an inadequate technique for accurate and precise measurements of protein concentration. However, an accurate and precise technique is essential for quantitative testing of hypotheses, and thus for the analysis and understanding of proposed molecular mechanisms. The analysis of Calcium-triggered exocytosis, the ubiquitous eukaryotic process by which vesicles fuse to the plasma membrane (PM) and release their contents, requires such an unambiguous identification and a quantitative assessment of the membrane surface density of specific molecules. Newly refined immunoblotting and analysis approaches permit one to obtain a quantitative analysis of the SNARE protein complement (VAMP, SNAP-25, and syntaxin) of functional secretory vesicles. The method illustrates the feasibility of the routine quantification of femtomole to attomole amounts of known proteins by immunoblotting. The results indicate that sea urchin egg secretory vesicles and synaptic vesicles have a marked similarity in SNARE densities. We have also carried out theoretical work on the processes of membrane fission, essential to endocytosis. The biochemical and biophysical mechanisms of membrane remodeling are critically dependent upon the composition of the local piece of membrane called upon by the cell to roll up into a new biological entity. The time is ripe for detailed study of the ways that physical forces and cell membrane inhomogeneities team up to allow for controlled and organized vesiculation in the general vacuolar system of cells, release of infectious viral particles, and internalization of membrane bound material. Consideration of lipid microdomains introduces new variables for membrane structure that have ramifications for proposed mechanisms for membrane budding and fission. Many investigators believe that microdomains of ordered lipids exist in both leaflets of a lipid bilayer, to explain the effects of lipid composition on cytoplasmic leaflet signaling. Moreover, the coordination of these two leaflets into a ?bilayer raft? is an attactive, albeit unproven idea. If one allows for the inner and outer leaflets of a microdomain to independently assemble or dissasemble, it is known that more ordered structures are higher in density. Thus lipid microdomains should be higher in density than non-raft areas. Thus the area per phospholipid head group will be smaller. If one leaflet can be raft and the other not, then this difference in one leaflet?s area compared to the other would curve the membrane (if there is not flip-flop of membrane compartments to relieve the asymmetry of area, and if the lipids are restricted from diffusing by molecular fences). Thus, if biology could turn on and off monolayer order, then it could bend membranes at will. That leads to a provocative suggestion: that transmembrane signalling may proceed through monolayer ordering. Consider a non-ordered membrane. Ordering one leaflet may lead to the ordering of the trans leaflet through the same forces that would stabilize bilayer rafts. This order is information, and can cause protein aggregation onto the newly ordered trans leaflet. For example, polymerization of PH domains can lead to PIP2 aggregation, which can order the internal leaflet, which can in turn order the outer leaflet, which can aggregate extracellular (or lumenal) domains. It is thus possible to build a mechanism for trans-bilayer signalling that need not involve protein transmembrane domains. If microdomains are sitting on either side of a neck, they are effectively in a ring topology. That is, if a vesicle buds out of a membrane from a large domain (a 150 nm diameter raft has more than twice the area of a 50 nm vesicle), then the donor membrane one is left with a hitherto unconsidered geometry of a domain: a ring. In other words, dynamin, outside the bilayer, would have a counterpart in the bilayer, a ?ring raft?. This ring could act to facilitate fission, as discussed above. In addition, since rafts must adapt their three-dimensional geometry to the needs of the dynamic situation, e.g. tubes to cups to spheres to elipsoids to planes to villi to etc, then it is of fundamental importance to consider the differential effects of curvature and geometry on ordered and disordered membrane domains. Ring rafts can also play an important role as a barrier to lipid diffusion, which can facilitate fission by reducing the number of lipid molecules involved in the fission reaction. Ultimately, these physical consideration of lipid microdomain topology must be regulated and organized by the underlying cellular architecture and organizing principals.