Dissecting the assembly of neurotransmitter release sites

剖析神经递质释放位点的组装

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要突触处的神经递质释放在很大程度上取决于秘密机器的精确组装。在突触前神经末端，在活性区域的突触蔬菜融合，这是形成的蛋白质支架释放位点应用于突触后受体。该蛋白质复合物包含边缘，麋鹿，munc13，rim-bp， liprin-α和巴松/短笛作为中心成分。最近的工作为新模型提供了基础这些蛋白如何组装到功能释放位点中。首先，活动区非常有弹性和单个基因的消融对其组装最多具有适度的影响。相反，将轮辋的删除结合在一起，麋鹿或RIM-BP强烈破坏活跃的区域组件，建立脚手架的冗余。第二，当前研究导致了通过液体液相分离的组装工作模型，并具有强大的贡献与组装的多价低亲和力相互作用。不管确切的机制，总体模型这些研究和其他研究产生的是，活性区是一个动态蛋白网络，共同存在通过冗余，低亲和力的蛋白结合。这与总体组织者的传统模型不同通过具有明确定义的石学计量法的刚性复合物进行介导组件。在这里，我们以我们的最新进展为基础，目的是确定媒体集会的机制初始活跃区支架的，以及这些活性区蛋白网络的相反表面如何与靶质膜和突触囊泡簇分别。我们将使用三管回答这些问题的方法。 AIM 1定义了RIM在活动区组装中的作用和机制。我们以我们的发现为基础，即删除脚手架的冗余后，轮辋驱动了相互作用的蛋白质通过RIM+麋鹿淘汰赛。我们测试了RIM通过两步过程组织活动区域的模型从机械上将边缘靶向与活动区域分开与RIM在招募其他活动区域的活动中蛋白质。 AIM 2剖析合成囊泡簇和活性区域如何，两个突触前亚 - 隔间，彼此互动。我们依靠一种新的“鼻内”重建方法和测试平行模型来定义哪些结合活动足以介导蔬菜对接。 AIM 3决定在靶质膜上的主动区域锚定机制。这个目标利用了我们的独特有条件和复合突变体的收集，以解决活跃区的长期问题脚手架物理连接到目标膜的正确位置。我们使用最先进的方法包括条件基因敲除，刺激的发射耗竭（STED）显微镜，荧光恢复光漂白（FRAP）后，高压冻结和相关光电子显微镜（CLEM）和电生理来回答这些问题。我们的工作将建立有关目标膜，活性区和囊泡的机械模型簇相互相互作用，以支持突触囊泡周期中的稳定性和动力学。

项目成果

期刊论文数量（13）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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
影响因子：
0
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.

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

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
影响因子：
0
作者：
Tyssowski,KelseyM;Letai,KatherineC;Rendall,SamuelD;Tan,Chao;Nizhnik,Anastasia;Kaeser,PascalS;Gray,JesseM
通讯作者：
Gray,JesseM

Munc13 supports fusogenicity of non-docked vesicles at synapses with disrupted active zones.