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

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.

该项目的重点是钙触发的胞吐作用的机制，这是一种普遍存在的真核过程，囊泡通过该过程与质膜融合并释放其内容物。尽管胞吐作用和钙之间的关系对于突触和非神经元分泌功能都是基础，但由于钙的时间和空间特性以及囊泡运输、启动、回收和再循环相互耦合的事实，分析存在问题。通过对海胆卵分泌囊泡体外胞吐动力学的分析，解决了胞吐的最后步骤。这些步骤被建模为三状态系统：激活、提交和融合，其中状态间转换由主动融合复合体提交 (alpha) 和提交融合复合体导致融合的概率 p 给出。每个囊泡对接位点的定型复合物的数量服从平均n-bar的泊松分布。实验上，p 和 n-bar 随着钙的增加而增加，而 alpha 和 p/n-bar 比率保持不变，将动力学描述减少为只有一个钙依赖性控制变量 n-bar。平均而言，最大速率 (Rmax) 的钙依赖性和达到 Rmax 的时间 (Tpeak) 由 n-bar 的钙依赖性描述。因此，游离钙浓度和胞吐速率之间的非线性关系可以仅通过囊泡对接位点融合复合物分布的钙依赖性来解释。 尽管免疫印迹（蛋白质印迹）广泛用于检测特定蛋白质，但通常认为它不足以准确测量蛋白质浓度。然而，准确而精确的技术对于假设的定量检验以及对所提出的分子机制的分析和理解至关重要。钙触发的胞吐作用是囊泡与质膜 (PM) 融合并释放其内容物的普遍存在的真核过程，分析需要对特定分子的膜表面密度进行明确的识别和定量评估。新改进的免疫印迹和分析方法允许人们对功能性分泌囊泡的 SNARE 蛋白补体（VAMP、SNAP-25 和 Syntaxin）进行定量分析。该方法说明了通过免疫印迹对已知蛋白质的飞摩尔至阿托摩尔量进行常规定量的可行性。结果表明，海胆卵分泌小泡和突触小泡的 SNARE 密度具有明显的相似性。 我们还对内吞作用所必需的膜裂变过程进行了理论工作。膜重塑的生化和生物物理机制关键取决于细胞要求卷成新生物实体的局部膜片的组成。详细研究物理力和细胞膜不均匀性如何共同作用的时机已经成熟，以允许细胞的一般液泡系统中受控和有组织的囊泡形成、感染性病毒颗粒的释放以及膜结合材料的内化。对脂质微区的考虑引入了膜结构的新变量，这些变量对提出的膜出芽和裂变机制产生影响。 许多研究人员认为，脂质双层的两个小叶中都存在有序脂质的微结构域，以解释脂质成分对细胞质小叶信号传导的影响。此外，这两个小叶协调成“双层筏”？这是一个有吸引力的想法，尽管尚未得到证实。如果允许微域的内部和外部小叶独立地组装或分解，则已知结构越有序，密度越高。因此，脂质微区的密度应高于非筏区域。因此每个磷脂头基的面积将会更小。如果一个小叶可以是筏，而另一个小叶则不是，那么一个小叶与另一小叶相比的面积差异将使膜弯曲（如果没有膜室的翻转来缓解面积的不对称性，并且如果脂质被分子栅栏限制扩散）。因此，如果生物学可以打开和关闭单层秩序，那么它就可以随意弯曲膜。这导致了一个令人兴奋的建议：跨膜信号传导可能通过单层排序进行。考虑无序膜。排序一个小叶可能会通过稳定双层筏的相同力导致反式小叶的排序。该顺序是信息，并且可以导致蛋白质聚集到新排序的反式小叶上。例如，PH结构域的聚合可以导致PIP2聚集，其可以对内部小叶进行排序，而内部小叶又可以对外部小叶进行排序，而外部小叶可以聚合细胞外（或腔内）结构域。因此，有可能建立一种不需要涉及蛋白质跨膜结构域的跨双层信号传导机制。 如果微域位于颈部的两侧，则它们实际上是环形拓扑。也就是说，如果囊泡从大域的膜中芽出（直径为 150 nm 的筏的面积是 50 nm 囊泡面积的两倍以上），那么供体膜就会留下迄今为止未考虑的域几何形状：一个环。换句话说，双层外的动力在双层中会有一个对应物，即“火筏”。如上所述，该环可以起到促进裂变的作用。此外，由于筏必须调整其三维几何形状以适应动态情况的需要，例如，管到杯到球体到椭球体到平面到绒毛等等，那么考虑曲率和几何形状对有序和无序膜域的不同影响至关重要。环筏还可以作为脂质扩散的屏障发挥重要作用，它可以通过减少参与裂变反应的脂质分子的数量来促进裂变。最终，脂质微域拓扑的这些物理考虑必须由底层细胞结构和组织原理来调节和组织。