Structure and dynamics of exocytotic fusion pores

项目总结/摘要 在胞吐过程中，融合孔形成第一个水连接，允许神经递质逃逸 和分泌囊泡中的激素。尽管SNARE复合物催化融合是公认的， 熔合孔的结构和组成仍然未知。这是该领域的核心问题， 膜融合，作为融合的机制不能解决，直到第一个关键中间体的结构 在这条途径中，融合孔，已经被阐明。该提案的主要目的是获得新的 深入了解融合孔的组成，结构和动力学，使用重建和基于细胞的 接近。细胞融合孔的生物化学研究的一个主要限制是其丰度低 短暂的自然例如，在神经内分泌细胞中，融合的初始开放状态的持续时间 孔的数量级为毫秒;然后孔或者关闭（吻-跑胞吐作用），或者扩张以产生完全融合。 为了克服这一局限性，我们已经开始研究融合孔结构，在体外，通过利用刚性 纳米盘的框架。带有SNARE的蛋白脂质体与含有同源物的纳米盘对接和融合 陷阱。由于纳米盘被膜支架蛋白所束缚，因此孔不能扩张， 可以进行生物化学研究。使用这个系统，我们已经开始询问重组蛋白的性质， 融合孔我们的初步数据表明，与融合孔纯粹是 尽管如此，它们实际上是混合结构，由脂质和蛋白质组成。我们将使用模拟 方法推导出融合孔结构的新模型，并进行低温电子显微镜研究， 想象一下这个结构。我们还将使用各种不同直径的货物， 传感器报告它们在融合过程中的释放，以确定孔的大小，并确定是否 孔径是“可塑的”，并且随着SNARE蛋白的数量而变化。我们还将探索 货物和SNARE跨膜结构域之间的静电相互作用。 纳米圆盘系统将适用于单分子研究，以监测细胞的孔隙打开和关闭。 实时测量单个孔隙，并直接评估调节因素对孔隙稳定性的影响。这些在 体外实验将通过我们正在进行的嗜铬细胞中融合孔的直接测量来补充， 使用碳纤维安培法，通过比较SNARE突变在这两个系统中的影响。我们将 在这些系统之间进行比较，以便我们能够得出统一的，生理相关的模型， 毛孔最后，我们还将设计新型的光学探针，基于pH敏感染料与量子 点，以研究培养的神经元中的融合孔。这些后面的研究将解决高度争议的话题 和完全融合的区别总之，这里描述的工作将提供无与伦比的 体外和基于细胞的观察之间的比较，并将揭示新的见解，在第一个关键 胞吐途径的中间体：神秘的融合孔。

PROJECT SUMMARY/ABSTRACT During exocytosis, fusion pores form the first aqueous connection that allows escape of neurotransmitters and hormones from secretory vesicles. Although it is well established that SNARE complexes catalyze fusion, the structure and composition of fusion pores remain unknown. This is the central question in the field of membrane fusion, as the mechanism of fusion cannot be solved until the structure of the first key intermediate in this pathway, the fusion pore, has been elucidated. The main objective of this proposal is to gain new insights into fusion pore composition, structure, and dynamics, using both reconstitution and cell-based approaches. A major limitation in the biochemical study of fusion pores in cells concerns their low abundance and ephemeral nature. For example, in neuroendocrine cells, the duration of the initial open state of the fusion pore is of the order of msec; the pore then either closes (kiss-and-run exocytosis), or dilates to yield full fusion. To overcome this limitation, we have begun to study fusion pore structure, in vitro, by exploiting the rigid framework of nanodiscs. SNARE-bearing proteoliposomes dock and fuse with nanodiscs that harbor cognate SNAREs. Since nanodiscs are bounded by membrane scaffolding proteins, the pores cannot dilate, and hence can be studied biochemically. Using this system, we have begun to interrogate the properties of reconstituted fusion pores. Our preliminary data indicate that, contrary to the common view that fusion pores are purely lipidic, they are in fact hybrid structures, composed of both lipids and proteins. We will use a simulation approach to derive a new model for fusion pore structure, and conduct cryo-electron microscopy studies to visualize this structure. We will also use a variety of cargos of varying diameter, in conjunction with optical sensors that report their release during fusion, to determine the size of the pore, and to determine whether pore diameter is `plastic' and varies with the number of SNARE proteins. We will also probe for interactions between cargo and SNARE transmembrane domains by exploiting electrostatic interactions between them. The nanodisc system will be adapted to single molecule studies, to monitor pore opening and closing of individual pores in real-time, and to directly assess the impact of regulatory factors on pore stability. These in vitro experiments will be complimented by our ongoing direct measurements of fusion pores in chromaffin cells, using carbon fiber amperometry, by comparing the effects of SNARE mutations in these two systems. We will draw parallels between these systems so that we can arrive at unified, physiologically relevant models for pores. Finally, we will also design novel optical probes, based on pH sensitive dyes conjugated to quantum dots, to study fusion pores in cultured neurons. These latter studies will address the highly controversial topic of kiss-and-run exocytosis versus full fusion. Together, the work described here will provide unparalleled comparisons between in vitro and cell based observations, and will reveal new insights in the first crucial intermediate in the exocytotic pathway: the enigmatic fusion pore.