Dissecting the assembly of neurotransmitter release sites

剖析神经递质释放位点的组装

• 批准号: 10682464
• 负责人: Pascal Simon Kaeser
• 金额: $ 66.66万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-13 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10682464
• 关键词: Ablation; Acute; Affect; Affinity; Binding; Binding Proteins; Brain; Brain Diseases; Cell Line; Cell membrane; Cells; Collection; Communication; Complex; Docking; Electron Microscopy; Electrophysiology (science); Fluorescence Recovery After Photobleaching; Freezing; Gene Family; Genes; Goals; Hippocampus; Image; Individual; Knock-out; Light; Link; Liquid substance; Mediating; Membrane; Methodology; Microscopy; Modeling; Molecular; Mutant Strains Mice; Nerve; Neurons; Neurotransmitters; Phase; Phosphatidylinositol 4,5-Diphosphate; Physical condensation; Process; Property; Protein Dynamics; Proteins; Role; Scaffolding Protein; Site; Slice; Structure; Surface; Synapses; Synaptic Receptors; Synaptic Vesicles; Tertiary Protein Structure; Testing; Transfection; Vesicle; Work; Zinc Fingers; experimental study; flexibility; insight; knockout gene; mouse genetics; mutant; neurotransmitter release; novel strategies; operation; postsynaptic; predictive modeling; pressure; presynaptic; presynaptic neurons; protein complex; reconstitution; recruit; resilience; scaffold; stoichiometry

Project Summary Neurotransmitter release at synapses critically depends on the precise assembly of the secretory machine. Within a presynaptic nerve terminal, synaptic vesicles fuse at the active zone, a protein scaffold that forms release sites apposed to postsynaptic receptors. This protein complex contains RIM, ELKS, Munc13, RIM-BP, Liprin-α and Bassoon/Piccolo as central components. Recent work provides ground for new models of how these proteins assemble into functional release sites. First, the active zone is remarkably resilient and ablation of individual genes has at most modest effects on its assembly. Instead, combined deletions of RIM, ELKS, or RIM-BP strongly disrupt active zone assembly, establishing scaffolding redundancy. Second, current studies have led to a working model of assembly through liquid-liquid phase separation, with robust contributions of multivalent low-affinity interactions to assembly. Regardless of exact mechanisms, an overarching model that arises from these and other studies is that the active zone is a dynamic protein network that is held together by redundant, low-affinity protein binding. This is different from conventional models in which master organizers mediate assembly through rigid complexes with well-defined stoichiometries. Here, we build on our and other’s recent progress with the goal to identify what mechanisms mediate assembly of the initial active zone scaffold, and how opposing surfaces of these active zone protein networks interact with the target plasma membrane and with the synaptic vesicle cluster, respectively. We will use a three-pronged approach to answer these questions. Aim 1 defines roles and mechanisms of RIM in active zone assembly. We build on our finding that RIM drives recruitment of interacting proteins after removing scaffolding redundancy through RIM+ELKS knockout. We test the model that RIM organizes active zones through a two-step process that mechanistically separates RIM-targeting to active zones from RIM’s activity in recruiting other active zone proteins. Aim 2 dissects how synaptic vesicle clusters and active zones, two presynaptic sub- compartments, interact with one another. We rely on a new, “in-synapse” reconstitution approach and test parallel models to define which binding activities are sufficient to mediate vesicle docking. Aim 3 determines active zone anchoring mechanisms at the target plasma membrane. This aim makes use of our unique collection of conditional and compound mutants to solve the long-standing question of how the active zone scaffolds are physically attached to the right place at the target membrane. We use state-of-the-art methodology including conditional gene knockout, stimulated emission depletion (STED) microscopy, fluorescence recovery after photobleaching (FRAP), high pressure freezing- and correlative light-electron microscopy (CLEM), and electrophysiology to answer these questions. Our work will establish mechanistic models on how the target membrane, the active zone, and the vesicle cluster interact with one another to support both stability and dynamics in the synaptic vesicle cycle.

项目摘要 神经递质在突触的释放在很大程度上取决于分泌机的精确组装。 在突触前神经末梢内，突触小泡在活动区融合，形成一种蛋白质支架 释放部位与突触后受体相对。该蛋白质复合体含有RIM、ELKS、Munc13、RIM-BP、 利普林-α和巴松管/短笛作为核心组件。最近的工作为新的模型提供了基础 这些蛋白质如何组装成功能释放部位。首先，活动区具有显著的弹性和 单个基因的去除对其组装的影响至多是轻微的。相反，综合删除RIM， ELK或RIM-BP强烈破坏活动区组装，建立脚手架冗余。第二，当前 研究已经产生了一种通过液-液相分离进行组装的工作模型，具有很强的贡献 多价低亲和力相互作用到组装。不管确切的机制如何，一个重要的模型 从这些和其他研究中得出的结论是，活动区是一个动态的蛋白质网络，它被保持在一起 通过多余的、低亲和力的蛋白质结合。这不同于传统的主组织者模式 通过具有定义明确的化学计量比的刚性络合物来调节组装。 在这里，我们建立在我们和其他人最近的进展基础上，目标是确定哪些机制调解组装 以及这些活动区蛋白质网络的相对表面是如何相互作用的 靶细胞膜和突触囊泡团。我们将采取三管齐下的办法 回答这些问题的方法。目标1定义了RIM在活动区组装中的作用和机制。 我们基于我们的发现，RIM在去除支架冗余后推动相互作用蛋白的招募 通过RIM+ELKS淘汰赛。我们测试了RIM通过两步过程组织活动区域的模型 这在机制上将RIM针对活动区域的活动与RIM招募其他活动区域的活动分开 蛋白质。目的2解剖突触小泡簇和活动区、两个突触前亚单位 隔间，相互作用。我们依靠一种新的“突触内”重建方法和测试 定义哪些结合活性足以调节囊泡对接的并行模型。目标3决定 目标质膜上的活性区域锚定机制。这一目标利用了我们独特的 收集条件和复合突变体来解决长期存在的问题，即活动区如何 支架被物理地附着在靶膜上的正确位置。我们使用最先进的方法 包括条件性基因敲除、受激发射耗尽(STED)显微镜、荧光恢复 经过光漂白(FRAP)、高压冷冻和相关的光电子显微镜(Clem)，以及 电生理学来回答这些问题。 我们的工作将建立关于靶膜、活动区和囊泡如何 簇之间相互作用，以支持突触囊泡周期的稳定性和动态性。

项目成果

Spatial and temporal scales of dopamine transmission.

• DOI: 10.1038/s41583-021-00455-7
• 发表时间: 2021-06
• 期刊: Nature reviews. Neuroscience
• 作者: Liu C;Goel P;Kaeser PS
• 通讯作者: Kaeser PS

Rebuilding essential active zone functions within a synapse.

• DOI: 10.1016/j.neuron.2022.01.026
• 发表时间: 2022-05-04
• 期刊: NEURON
• 影响因子: 16.2
• 作者: Tan, Chao;Wang, Shan Shan H.;de Nola, Giovanni;Kaeser, Pascal S.
• 通讯作者: Kaeser, Pascal S.

Firing Rate Homeostasis Can Occur in the Absence of Neuronal Activity-Regulated Transcription.

在缺乏神经元活动调节转录的情况下，可能会发生放电率稳态。

• DOI: 10.1523/jneurosci.1108-19.2019
• 发表时间: 2019
• 期刊: The Journal of neuroscience : the official journal of the Society for Neuroscience
• 作者: Tyssowski,KelseyM;Letai,KatherineC;Rendall,SamuelD;Tan,Chao;Nizhnik,Anastasia;Kaeser,PascalS;Gray,JesseM
• 通讯作者: Gray,JesseM

PKC-phosphorylation of Liprin-α3 triggers phase separation and controls presynaptic active zone structure.

• DOI: 10.1038/s41467-021-23116-w
• 发表时间: 2021-05-24
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Emperador-Melero J;Wong MY;Wang SSH;de Nola G;Nyitrai H;Kirchhausen T;Kaeser PS
• 通讯作者: Kaeser PS

A Presynaptic Liquid Phase Unlocks the Vesicle Cluster.

突触前液相解锁囊泡簇。

• DOI: 10.1016/j.tins.2018.07.009
• 发表时间: 2018
• 期刊: Trends in neurosciences
• 影响因子: 15.9
• 作者: Wang,ShanShanH;Kaeser,PascalS
• 通讯作者: Kaeser,PascalS