Components And Kinetics In Exocytosis

胞吐作用的组成和动力学

• 批准号: 6671872
• 负责人: JOSHUA ZIMMERBERG
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6671872
• 关键词: calcium ion; cell component structure /function; cell components; cell membrane; egg /ovum; exocytosis; granule; membrane fusion; sea urchins; western blottings; calcium ion; cell component structure /function; cell components; cell membrane; egg /ovum; exocytosis; granule; membrane fusion; sea urchins; western blottings

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.

该项目集中在钙触发的胞吐作用的机理上，这是无处不在的真核过程，囊泡通过该过程融合到质膜并释放其含量。尽管胞吐和钙之间的关系对于突触和非神经分泌功能都是基本的，但由于钙的时间和空间特性，分析是有问题的，并且囊泡传输，启动，检索和回收是偶联的。通过在体外分析海胆卵分泌囊泡胞吐作用的动力学，可以解决胞吞作用的最后一步。这些步骤被建模为一个三州系统：激活，投入和融合，在该系统中，州际过渡是由主动融合复合物（Alpha）的概率给出的，并且构成的融合复合物导致融合，p。每个囊泡对接位点所致加的复合物的数量是带有平均N杆的泊松。在实验上，p和n杆随着钙的增加而增加，而α和p/n-bar比率保持恒定，将动力学描述降低到仅钙依赖性的，控制变量，n杆。平均而言，最大速率（RMAX）和达到RMAX（TPEAK）的时间的钙依赖性由N杆的钙依赖性描述。因此，可以仅通过囊泡对接位点融合络合物的分布的钙依赖性来解释游离钙浓度与胞吞作用速率之间的非线性关系。 尽管免疫印迹（蛋白质印迹）被广泛用于检测特定蛋白质，但通常认为它是一种不足的技术，无法准确，精确地测量蛋白质浓度。但是，准确而精确的技术对于假设的定量测试至关重要，因此对于对所提出的分子机制的分析和理解。钙触发的胞吐作用的分析，即普遍存在的真核过程，囊泡通过囊泡融合到质膜（PM）并释放其含量，需要对特定分子的膜表面密度进行这种明确的鉴定和定量评估。新精制的免疫印迹和分析方法允许人们对功能性分泌囊泡的SNARE蛋白质补体（VAMP，SNAP-25和语法）进行定量分析。该方法说明了通过免疫印迹将tmole量常规定量定量为attomole量的已知蛋白质的可行性。结果表明，海胆卵分泌囊泡和突触囊泡在圈套密度方面具有明显的相似性。 我们还对膜裂变过程进行了理论工作，这对于内吞作用至关重要。膜重塑的生化和生物物理机制严重取决于细胞所要求的局部膜的组成，以卷成新的生物学实体。详细研究的时间已经成熟，以研究物理力和细胞膜不均匀性的方式，以允许在一般细胞系统中进行控制和有组织的囊泡，传染性病毒颗粒的释放以及膜结合材料的内在化。脂质微区域的考虑引入了膜结构的新变量，这些变量对膜出芽和裂变的拟议机制产生了影响。 许多研究者认为，脂质双层的两个小叶中都存在有序脂质的微区，以解释脂质组成对细胞质小叶信号传导的影响。此外，这两个传单的协调为双层筏吗？是一个诚实的，虽然未经证实的想法。如果一个人允许微域的内部和外部小叶独立组装或分解，则众所周知，有序的结构的密度更高。因此，脂质微域在密度上应高于非收割区域。因此，每个磷脂头组的面积将较小。如果一个传单可以是木筏，而另一个则不能，那么与另一个小叶区域相比，与另一个小叶区域相比会弯曲膜（如果没有膜隔室的触发器以减轻面积的不对称性，并且如果脂质限制了通过分子栅极扩散的限制））。因此，如果生物学可以打开和关闭单层秩序，则可以随意弯曲膜。这导致了一个挑衅的建议：跨膜信号可以通过单层订购进行。考虑一个非订购的膜。订购一个传单可能会导致跨性别的叶子订购，从而稳定双层筏。该顺序是信息，可能会导致蛋白质聚集到新有序的反式传单上。例如，pH结构域的聚合可以导致PIP2聚集，这可以订购内部小叶，从而依次可以排序外部小叶，该小叶可以聚集细胞外（或腔内）结构域。因此，可以构建一种不需要涉及蛋白质跨膜结构域的反式双层信号传导的机制。 如果微域坐在脖子的两侧，则有效地处于环拓扑中。也就是说，如果一个囊泡从大域中从膜上芽出来（直径为150 nm的筏的面积是50 nm囊泡的面积的两倍以上），则将供体膜一个带有一个迄今未经考虑的域的几何形状：一个环：一个环。换句话说，在双层外，dynamin在双层中会有一个木筏吗？如上所述，该戒指可以起作用来促进裂变。此外，由于筏必须使其三维几何形状适应动态情况的需求，例如将杯子的管子带到埃利普斯到villi的平面，然后考虑曲率和几何形状对有序和无序膜域的差异效应至关重要。环筏还可以作为脂质扩散的障碍发挥重要作用，这可以通过减少参与裂变反应涉及的脂质分子的数量来促进裂变。最终，这些对脂质微域拓扑的物理考虑必须受到潜在的细胞结构和组织原理的调节和组织。