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.

项目摘要突触中的神经递质释放至关重要，取决于分泌机器的精确组装。在突触前神经末端，在活性区域的突触囊泡融合，蛋白质支架形式释放位点，该蛋白质复合物包含边缘，Munc13，Rim-BP， Liprin-α和低音/短笛作为中央组件。这些蛋白质如何组装到功能释放位点。单个基因的消融最多具有适度的效果，占轮辋的结合缺失，麋鹿或RIM-BP强大的活跃区组件，建立脚手架的冗余研究导致了通过液体液相分离的组装模型，并具有强大的贡献与组装的多价低亲和力相互作用。由OTER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，低亲和力的蛋白质结合。通过具有明确定义的石学仪的刚性复合物进行介导组件。在这里，我们基于我们的最新进展，目的是确定哪些机制介导了组装初始活跃区支架的，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，我们将使用树木供应的树木。回答这些问题的方法。我们以我们的发现为基础，即删除冗余后，轮辋驱动互动蛋白的募集通过RIM+麋鹿淘汰赛。该机械机械地将边缘靶向与活动区域的活动分开，从招募其他活动区域的活动中蛋白质。隔间，彼此互动。定义哪些结合活性的平行模型足以介导囊泡对接在目标质膜上的主动区域锚定机制可让我们独特收集条件和化合物muts以解决活跃区的长期问题脚手架在乌龟膜上物理上固定在正确的位置。包含条件基因敲除，刺激的发射耗竭（STED）显微镜，荧光恢复光裂（FRAP）后，高压冻结和相关光电子显微镜（CLEM）和D 电生理来回答这些问题。 Ourk的工作将建立有关目标孟买，活动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.