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（由适用提供）：该MIRA建议巩固了有关膜动力学细胞生物学三个中心问题的资助研究：合成蔬菜胞吐作用，动态膜接触和高尔基模型的合成生物学测试。 CA ++后，突触蔬菜融合以在小于1毫秒内释放神经递质，从而在大脑中有效地处理。尽管我们对膜融合的理解中的所有进步都大致，但值得注意的是，我们对此没有明确的解释。我们接下来5 - 10年的目标是对神经递质释放如何与Ca ++结合以及如何如此迅速地发生详细的结构生化和生物物理理解。必须解决两个相关的问题以提供答案：半拉链的军鼓复合物如何稳定（“夹紧”），从而获得了融合的融合？融合如何比单个贪食的物理化学允许的速度快100-1000倍？我们的总体假设是，涉及Ca ++传感器突触剂的上分子组件和夹具/激活蛋白以某种方式实现了亚毫秒的胞吐作用，该组合可以刺激并同步许多SNARES的力，从而同步许多SNARES的力量，以启用爆炸性释放。膜接触通常是动态实体，由于生理学的信号和变化而形成和破裂。 Golgi堆栈基于这种膜接触，并承担这样一个事实，即它看起来像一个稳定的实体，我们最近的令人惊讶的结果表明，主蛋白负责注册堆叠（GRASP蛋白）每分钟和膜下的循环！基于纳米镜检查的成像表明，这是由于未来5年的脂肪酸（棕榈酸酯）添加和裂解的快速循环，我们希望在活细胞中使用酶，底物及其产物的实时纳米纳米镜阐明涉及的机制，并结合了详细的生物化学，生物生物物质研究和遗传研究。在所有真核生物中都发现了高尔基体设备，但具有令人怀疑和显着的区别，即是最后剩下的膜细胞器，其基本操作的基本原理仍然未知！尽管进行了数十年的深入研究，但我们仍在争论动物细胞中高尔基体堆栈的蛋白质如何流动，这仅仅是因为我们缺乏直接观察活细胞中蛋白质转移反应以足够的空间/时间分辨率的能力。我们正在开发的新想法是要设计动物高尔基堆积的转化为活细胞中更类似酵母的未堆积拓扑，以便我们现在可以更轻松地跟踪单个cisternae，蔬菜和块茎，尤其是在我们部门的动态纳米镜头的帮助下，尤其是在我们部门的帮助下。