Regulation of Membrane Fusion in Exocytosis

项目摘要/摘要 几乎相同的诱捕机器如何根据环境以显著不同的速度运行， 通常比单个SNAREpin快得多？这是当今推动该领域的核心问题之一，而 它所体现的问题在神经元突触上表现得最大胆，所以我们就在这里关注 关键蛋白质机械的结构、生物物理和生理特性。我们的总体假设是 同时发布的多个SNAREPin，每个都已经接近触发融合的点，合作 实现核聚变比单独实现任何一个都要快得多。在当前的支持期间，我们发现 钙感受器突触素(通常固定在突触小泡中)在体外可以自我组装成钙离子。 敏感的环状低聚物直径约30 nm，并表明这种环在 突触小泡(或胰岛素分泌小泡)和质膜将阻止释放，直到它们 被钙离子干扰。我们的具体假设是，这种突触素的环状寡聚体(SYT)是一个中心 胞吐组织原理，可夹紧并快速同步释放 SNAREPINS。最近的实验有力地支持了这一假设，在这些实验中，靶向突变(F349A) 破坏Syt1环的稳定显著增加了自发和诱发的释放，并在海马区 并显著降低释放与动作电位的同步性。我们建议1) 检验突触素环状寡聚体调节胞吐作用的假说；2)检验以下假说 Syt1和Syt7在从 3)阐明了Munc13及其寡聚体的动力学和拓扑结构以及 定位为囊泡锚链和外环伴侣的双重作用模板SNAREpins；4)通过单一 颗粒低温电子显微镜和低温电子显微镜断层扫描功能释放部位的高分辨结构 原位被困在定义的功能状态中。类似的机制调节神经内分泌分泌生理学， 包括胰腺胰岛素分泌，所以我们预计答案将与 NIDDK。此外，在后瘦素时代，神经系统在新陈代谢中的关键作用几乎是毫无疑问的。 平衡和疾病。

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.