Regulation of Membrane Fusion in Exocytosis

项目总结/摘要 几乎相同的SNARE机器如何根据上下文以截然不同的速度运行， 往往比单个SNAREpin快得多这是当今推动该领域发展的核心问题之一， 它所体现的问题在神经元突触上表现得最为突出，所以我们在这里集中讨论 结构，生物物理学，和关键蛋白质机器的生理特性。我们的总体假设是 同步释放的多个SNAREpin，每一个都已经接近触发融合点， 实现核聚变的速度比任何单独的核聚变都要快得多。在目前的支持期间，我们发现， 钙传感器突触结合蛋白（通常锚定在突触囊泡中）可以在体外自组装成Ca2 +- 敏感的环状低聚物，直径约为30 nm，并建议在聚合物之间形成这种环。 突触囊泡（或胰岛素分泌囊泡）和质膜将阻止释放，直到它们被释放。 被Ca2+破坏。我们的具体假设是，突触结合蛋白（Syt）的这种环状寡聚体是一种中心的 胞吐作用的组织原理，使多个细胞能够夹紧和快速同步释放， SNAREpin。这一假设得到了最近实验的有力支持，在这些实验中，靶向突变（F349A） 使Syt1环不稳定的蛋白质显著增加自发和诱发释放， 神经元，并显着降低同步性的释放与动作电位。我们建议：（1） 检验突触结合蛋白的环状寡聚体调节胞吐作用的假设; 2）检验突触结合蛋白的环状寡聚体调节胞吐作用的假设。 Syt1和Syt7在同步和异步释放中扮演着不同的结构和功能角色。 3）阐明Munc13及其提出的寡聚体的动力学和拓扑结构， 假定作为囊泡系链和外环分子伴侣模板SNAREpin的双重作用;和4）通过单克隆获得 体外和体内功能性释放位点的粒子冷冻EM和冷冻EM断层扫描高分辨率结构 在定义的功能状态中原位捕获。类似的机制介导神经内分泌生理学， 包括胰腺胰岛素分泌，所以我们希望答案将是高度相关的使命， NIDDK。此外，在后瘦素时代，神经系统在代谢中的关键作用几乎没有疑问。 平衡与疾病

Project Summary/Abstract How can virtually the same SNARE machine operate at dramatically different speeds depending on context, often far faster than a single SNAREpin? This is one of the central questions driving the field today, and the problem it embodies stands in boldest relief at the neuronal synapse, so it is here that we focus on the structures, biophysics, and physiological properties of the key protein machinery. Our overall hypothesis is that multiple SNAREpins released synchronously, each already close to the point of triggering fusion, co-operate to achieve fusion dramatically faster than any one alone. During the current period of support we discovered that the calcium sensor Synaptotagmin (normally anchored in synaptic vesicle) can self-assemble in vitro into Ca2+- sensitive, ring-like oligomers ~30 nm in diameter and have suggested that such rings forming between the synaptic vesicle (or insulin secretory vesicle) and the plasma membrane would prevent release until they are disrupted by Ca2+. Our specific hypothesis is that such ring oligomers of Synaptotagmin (Syt) are a central organizing principle for exocytosis, enabling the clamping and rapid synchronous release of multiple SNAREpins. This hypothesis is strongly supported by recent experiments in which a targeted mutation (F349A) that de-stabilizes Syt1 rings dramatically increases spontaneous and evoked release and in hippocampal neurons, and dramatically reduces the synchronicity of release with the action potential. We propose to 1) Test the hypothesis that ring-like oligomers of Synaptotagmins regulate exocytosis; 2) Test the hypothesis that Syt1 and Syt7 play distinct structural and functional roles in synchronous and asynchronous release from the same docked vesicles; 3) Elucidate the dynamics and topology of Munc13 and its proposed oligomers and the posited dual roles as vesicle tether and outer ring chaperone templating SNAREpins; and 4) Obtain by single particle cryo-EM and cryo EM tomography high resolution structures of functional release sites in vitro and in situ trapped in defined functional states. Similar machinery mediates neuroendocrine secretory physiology, including pancreatic insulin secretion, so we expect the answers will be highly relevant to the mission of NIDDK. Further, there is little doubt in the post-leptin era of the key role of the nervous system in metabolic balance and diseases.