Molecular Mechanisms for Co‐Assembly of Endocytic and Exocytic Machineries at a Synapse

突触内吞和胞吐机器共同组装的分子机制

• 批准号: 10570512
• 负责人: Javier Emperador Melero
• 金额: $ 11.31万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10570512
• 关键词: Ablation; Affect; Amino Acid Sequence; Binding; Biological Assay; Brain; Brain Diseases; Cell Line; Cell membrane; Cells; Clathrin Adaptors; Complement; Data; Dynamin; Dynamin I; Electrons; Electrophysiology (science); Endocytosis; Engineering; Ensure; Enzymes; Excitatory Synapse; Genes; Goals; Grant; Inhibitory Synapse; Invertebrates; Knock-out; Knockout Mice; Knowledge; Learning; Liquid substance; Literature; Measures; Mediating; Mental disorders; Methodology; Modeling; Molecular; Mutation; Neurons; Neurotransmitters; PHluorin; Pathway interactions; Phase; Phenotype; Physical condensation; Positioning Attribute; Property; Proteins; Recruitment Activity; Role; Signal Transduction; Site; Solid; Structure; Synapses; Synaptic Potentials; Synaptic Receptors; Synaptic Transmission; Synaptic Vesicles; Testing; Training; Vertebrates; Vesicle; Visualization; Work; amphiphysin; experience; experimental study; in vivo; information processing; insight; mutant; nano; nanoscale; postsynaptic; presynaptic; presynaptic neurons; reconstitution; recruit; scaffold; skills; superresolution microscopy; tool; transmission process; vesicular release

Project summary At presynaptic nerve terminals, the precise nano-scale positioning of two protein machineries, the active zone and the endocytic apparatus, is critical for high fidelity synaptic transmission. The active zone, which generates release sites for synaptic vesicles, is aligned to postsynaptic receptors to ensure efficient signal transmission. The endocytic apparatus, which restores vesicles after release, is assembled adjacent to active zones. Most components of these machineries are associated with brain disorders, underlining their importance for brain function. This grant generates fundamental insight into the molecular mechanisms that position the endocytic apparatus next to the active zone at vertebrate synapses. The presynaptic scaffold Liprin-α is an ideal candidate to spatially organize these two machineries for three reasons. First, the broad synaptic interactome of Liprin-α and the generalized phenotypes caused by ablating the single invertebrate gene predict that it organizes multiple presynaptic compartments. Second, my previous work and preliminary data show that deletion of a subset of the four vertebrate Liprin-α proteins already causes mislocalization of active zone and endocytic components, which suggests co-organizing roles for vertebrate Liprin-α. Finally, Liprin-α undergoes phase separation, which is a potential synaptic organizing mechanism, and my ongoing work establishes that both active zone and endocytic proteins are recruited to Liprin-α phase condensates. Therefore, I hypothesize that Liprin-α positions the endocytic apparatus next to active zones at vertebrate synapses. I will use newly-generated Liprin-α quadruple mutants, in which all four Liprin-α genes can be deleted, to test this hypothesis. Aim 1 (training) will dissect roles for Liprin-α in active zone organization by comparing the nano- scale positioning of active zone proteins and active zone function after Liprin-α quadruple ablation in excitatory and inhibitory synapses in cultured neurons and at the Calyx of Held. Aim 2 (training) will determine roles for Liprin-α in organizing the endocytic apparatus by using the same approaches. Both aims will be complemented with experiments in cell lines to assess the ability of Liprin-α to recruit endocytic and active zone proteins. I will also do structure-function experiments to dissect the relevance of the protein sequences that mediate Liprin-α phase separation in presynaptic assembly. These aims will establish the roles of Liprin-α in the organization of active zones and endocytic assemblies, and will provide an essential training experience in the studies of the endocytic machinery and in the assessment of their in vivo organization using super-resolution microscopy. Aim 3 (independent) will test the complementary model that the endocytic apparatus mediates the spatial organization of these machineries. In particular, I will dissect structural roles for Dynamin, which is fundamental for synaptic endocytosis, and also undergoes phase separation. I will use Dynamin triple mutants and build upon the methodology implemented during (training) aims 1 & 2 to determine these roles.

项目摘要 在突触前神经末梢，两种蛋白质机器的精确纳米级定位，即活性区， 以及内吞器，对于高保真突触传递至关重要。活动区，它产生 突触囊泡的释放位点与突触后受体对齐，以确保有效的信号传递。 内吞装置在释放后恢复囊泡，并组装在活性区附近。最 这些机制的组成部分与大脑疾病有关，强调了它们对大脑的重要性。 功能这项资助产生了对定位细胞的分子机制的基本见解。 脊椎动物突触活动区附近的内吞装置。 突触前支架Liprin-α是空间组织这两种机制的理想候选者， 原因首先，Liprin-α的广泛突触相互作用组和消融引起的广泛表型 单无脊椎动物基因预测，它组织多个突触前区室。第二，我以前 工作和初步数据表明，四种脊椎动物Liprin-α蛋白的一个亚组的缺失已经导致 活性区和内吞成分的错误定位，这表明脊椎动物的协同组织作用 Liprin-α。最后，Liprin-α经历相分离，这是一种潜在的突触组织机制， 我正在进行的工作表明，活性区和内吞蛋白都被募集到Liprin-α相 冷凝物因此，我假设Liprin-α将内吞装置定位在活动区附近 脊椎动物的突触 我将使用新产生的Liprin-α四重突变体，其中所有四个Liprin-α基因都可以删除，以测试 这个假设。目标1（培训）将通过比较纳米脂质体， Liprin-α四重消融后激活区蛋白的尺度定位和激活区功能 和抑制性突触在培养的神经元和在杯的赫尔德。目标2（培训）将确定 Liprin-α通过使用相同的方法组织内吞装置。这两个目标将相辅相成 通过细胞系实验评估Liprin-α招募内吞和活性区蛋白的能力。我会 还进行结构-功能实验以剖析介导Liprin-α的蛋白质序列的相关性 突触前组装的相位分离。这些目标将确立Liprin-α在组织中的作用 活性区和内吞组件，并将提供一个重要的培训经验，在研究的 内吞机制和使用超分辨率显微镜评估其体内组织。目的 3（独立）将测试内吞装置介导空间的互补模型， 组织这些机构。特别是，我将剖析动力蛋白的结构作用，这是最基本的， 对于突触内吞作用，也经历相分离。我会用发动蛋白三重突变体 在（培训）期间采用的方法旨在确定这些作用。