Structure and dynamics of exocytotic fusion pores

项目摘要/摘要 在胞吐过程中，融合毛孔形成第一个水溶液连接，使神经递质得以逃逸。 以及来自分泌囊的荷尔蒙。尽管众所周知，SNARE复合体催化融合， 融合孔的结构和成分尚不清楚。这是……领域的中心问题 膜融合，作为融合的机理要等到第一个关键中间体的结构才能解决 在这个途径中，融合孔已经被阐明。这项提议的主要目的是获得新的 对融合孔组成、结构和动力学的洞察，使用重建和基于细胞 接近了。细胞中融合孔的生物化学研究的一个主要限制是它们的丰度低。 和转瞬即逝的自然。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 毛孔的大小约为毫秒；然后毛孔要么关闭(接吻并跑出胞吐)，要么扩张以产生完全融合。 为了克服这一局限，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。携带SNARE的蛋白脂质体与含有同源基因的纳米盘对接并融合 陷阱。由于纳米盘被膜支架蛋白结合，所以孔不能扩张，因此 可以用生物化学方法来研究。利用这个系统，我们已经开始询问重组的属性 融合毛孔。我们的初步数据表明，与通常的观点相反，融合毛孔纯粹是 脂类，它们实际上是混合结构，由脂类和蛋白质组成。我们将使用模拟 方法推导出一个新的融合孔结构模型，并进行冷冻电子显微镜研究 想象一下这个结构。我们还将使用各种不同直径的货物，与光学 传感器在融合过程中报告它们的释放，以确定毛孔的大小，并确定 孔径是“塑料的”，并随SNARE蛋白的数量而变化。我们还将探索互动 通过利用它们之间的静电相互作用，在Cargo和SNARE跨膜结构域之间进行相互作用。 纳米盘系统将适用于单分子研究，以监测孔的打开和关闭 实时检测单个孔隙，并直接评估调节因素对孔隙稳定性的影响。这些输入 我们正在进行的对嗜铬细胞融合孔的直接测量将对体外实验起到补充作用， 使用碳纤维安培法，通过比较这两个系统中SNARE突变的影响。我们会 画出这些系统之间的相似之处，这样我们就可以得到统一的、生理上相关的模型 毛孔。最后，我们还将设计基于pH敏感染料与量子共轭的新型光学探针 DOTS，以研究培养神经元中的融合毛孔。这些后一项研究将解决这个极具争议性的话题 接吻即跑的胞吐与完全融合的对比。总之，这里描述的工作将提供无与伦比的 在体外和基于细胞的观察之间的比较，并将揭示第一个关键的新见解 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.