Regulation of Vesicle Traffic

囊泡交通的调节

• 批准号: 9070963
• 负责人: JAMES ROTHMAN
• 金额: $ 49.37万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9070963
• 关键词: Adhesives; Animals; Biochemical; Brain; Cells; Cellular biology; Complex; Coupled; Engineering; Eukaryota; Exocytosis; Fatty Acids; Funding; GRASP gene; Genetic study; Goals; Golgi Apparatus; Image; Individual; Life; Membrane; Membrane Fusion; Modeling; Nanoscopy; Organelles; Palmitates; Physical Chemistry; Physiology; Proteins; Reaction; Regulation; Research; SNAP receptor; Signal Transduction; Synaptic Vesicles; Testing; Time; Vesicle; Yeasts; base; enzyme substrate; information processing; millisecond; molecular assembly/self assembly; nano; neurotransmitter release; operation; protein transport; public health relevance; sensor; synaptotagmin; synthetic biology; temporal measurement; trafficking

 DESCRIPTION (provided by applicant): This MIRA proposal consolidates funded research on three central problems in cell biology concerning membrane dynamics: synaptic vesicle exocytosis, dynamic membrane contacts, and synthetic biology-enabled tests of Golgi models. Synaptic vesicles fuse to release neurotransmitters in less than 1 msec after Ca++ enters, enabling effective information processing in the brain. In spite of all of the progress in our understanding of membrane fusion generally, it is remarkable that we have no clear explanation of this. Our goal for the next 5-10 years is to develop a detailed structural biochemical and biophysical understanding of how neurotransmitter release is coupled to Ca++ and how it can occur so very rapidly. Two related problems must be solved to provide an answer: how is the half-zippered SNARE complex stabilized ("clamped") from completing fusion spontaneously? How can fusion be completed 100-1000 times faster than permitted by the physical chemistry of individual SNAREs? Our general hypothesis is that sub-millisecond exocytosis is achieved in some way by a supra-molecular assembly involving the Ca++ sensor synaptotagmin and the clamp/activator complexin that harnesses and synchronizes the force of many SNAREs co-operatively to enable explosive release. Membrane contacts generally are dynamic entities, forming and breaking as the result of signals and shifts in physiology. The Golgi stack is based on such membrane contacts, and despite the fact that it looks like a stable entity, our recent and surprising results reveal that the principal proteins responsible for registered stacking (GRASP proteins) cycle on and off the membrane every minute! Nanoscopy-based imaging of suggests this is due to rapid cycles of fatty acid (palmitate) addition and cleavage in specialized transien adhesive nano-domains. Over the next 5 years we hope to elucidate the mechanisms involved using real-time nanoscopy of the enzymes, substrates and their products in living cells combined with detailed biochemical, biophysical, and genetic studies. The Golgi apparatus is found in all eukaryotes, and yet has the dubious and remarkable distinction of being the last remaining membrane organelle whose basic principle of operation is still unknown! In spite of decades of intensive research we still debate how proteins traffic across the Golgi stack in animal cells, simply because we lack the ability to directly observe protein transfer reactions in living cells at sufficient spatial/temporal resolution. The new idea we are developing is to engineer the conversion of the animal Golgi stack into a more yeast-like unstacked topology in living cells, so that we can now much more easily track individual cisternae, vesicles and tubules in relation to transport cargo and machinery, especially with the help of dynamic nanoscopy now possible in our department.

 描述(由申请人提供)：这项MIRA提案整合了细胞生物学中与膜动力学有关的三个中心问题的资助研究：突触小泡胞吐作用、动态膜接触以及高尔基模型的合成生物学启用测试。突触小泡在钙离子进入后不到1毫秒内融合释放神经递质，使大脑能够进行有效的信息处理。尽管我们对膜融合的理解总体上有了很大的进步，但值得注意的是，我们对此没有明确的解释。我们未来5-10年的目标是对神经递质释放是如何与钙离子耦合的，以及它如何如此迅速地发生的详细的结构、生化和生物物理的理解。必须解决两个相关的问题才能提供答案：如何使半拉链的圈套复合体稳定(“夹住”)，使其不会自发完成融合？如何以比单个陷阱的物理化学所允许的速度快100-1000倍的速度完成融合？我们的一般假设是，亚毫秒级的胞吐在某种程度上是通过涉及钙离子感受器突触凝集素和钳制/激活复合体的超分子组装实现的，该复合体协同利用并同步许多陷阱的力量，以实现爆炸性释放。膜接触通常是动态的实体，由于信号和生理变化而形成和断开。高尔基体堆叠是基于这样的膜接触，尽管它看起来像一个稳定的实体，但我们最近令人惊讶的结果显示，负责注册堆叠的主要蛋白质(GRAP蛋白质)每分钟都在膜上和膜下循环！基于纳米显微镜的成像表明，这是由于脂肪酸(棕榈酸酯)在专门的过渡胶粘接纳米域中快速循环添加和切割所致。在接下来的5年里，我们希望利用活细胞中酶、底物及其产物的实时纳米显微镜结合详细的生化、生物物理和遗传学研究来阐明所涉及的机制。高尔基体在所有真核生物中都有发现，但有一个令人怀疑和显著的区别：它是最后剩下的膜细胞器，其基本工作原理尚不清楚！尽管经过了几十年的密集研究，我们仍然在争论蛋白质如何在动物细胞中通过高尔基体堆叠，仅仅是因为我们缺乏以足够的空间/时间分辨率直接观察活细胞中蛋白质转移反应的能力。我们正在开发的新想法是将动物高尔基体堆叠转化为活细胞中更像酵母的非堆叠拓扑结构，这样我们现在就可以更容易地跟踪与运输货物和机械有关的单个池子、小泡和小管，特别是在我们部门现在可以使用动态纳米显微镜的帮助下。