Spatial and temporal regulation of synapse formation through phase separation

通过相分离调节突触形成的空间和时间

基本信息

批准号：
10445090
负责人：
Nathan McDonald
金额：
$ 12.29万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-15 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10445090
关键词：
Actins Action Potentials Aging Animals Automobile Driving Award Biological Assay Caenorhabditis elegans Calcium Channel Cell Adhesion Molecules Common Core Communication Complement Complex Cues Data Defect Degenerative Disorder Depressed mood Development Educational process of instructing Elements Enhancers Event F-Actin Future Genetic Genetic Screening Goals Image In Vitro Individual Ion Channel Link Liquid substance Location Mass Spectrum Analysis Membrane Mentorship Methods Mission Molecular Mutate Mutation National Institute of Neurological Disorders and Stroke Natural regeneration Nervous system structure Neurons Neurophysiology - biologic function Neurosciences Pathway interactions Phase Phosphorylation Phosphorylation Site Phosphotransferases Play Positioning Attribute Post-Translational Protein Processing Process Property Regulation Research Research Methodology Resolution Scaffolding Protein Side Signal Pathway Signaling Molecule Site Specific qualifier value Structure Synapses Synaptic Vesicles System T-Lymphocyte Testing Thinking Training Universities Work Writing active control autism spectrum disorder career development cognitive function experimental study genome-wide immunological synapse in vitro Assay in vivo in vivo imaging insight mutant nervous system disorder neural circuit neurodevelopment novel optogenetics phosphoproteomics presynaptic reconstitution scaffold screening spatiotemporal synaptogenesis vesicular release

项目摘要

Synapses are the basic unit of neuronal communication, and consequently their location, number, and properties govern the function of neural circuits and nervous systems. How synapses form remains a fundamental question in contemporary neuroscience. A wide variety of synaptic cell adhesion molecules (syCAMs) are capable of initiating synapse formation. Downstream of these diverse connections, however, common pre- and post-synaptic specializations are formed. On the presynaptic side, an “active zone” structure is formed comprised of large multi-valent scaffolding proteins. The active zone coordinates the central functions of the presynapse by tethering and priming synaptic vesicles, clustering calcium channels, and aligning with the postsynapse through transmembrane connections. Recently the candidate showed conserved C. elegans active zone scaffolds SYD-2/Liprin-α and ELKS form liquid-liquid phase separated condensates at developing synapses, and this activity was required for active zone assembly. This work positioned active zone phase separation as a central assembly hub in the presynapse. The overall goal of the proposed studies is to determine molecular mechanisms and novel components that drive active zone phase separation, and thus presynapse formation. In Aim 1, phosphorylation will be investigated as a mechanism that controls active zone phase separation. Preliminary data suggest SAD-1 kinase phosphorylation of SYD-2 controls its activity. This phosphoregulation will be investigated with live animal in vivo imaging at single-synapse resolution in combination with in vitro phase separation assays. In Aim 2, the connection between synaptogenic syCAMs and active zone phase separation will be investigated. Multiple syCAMs have been identified to build local F- actin networks, which may link these upstream molecules to active zone phase separation. The requirement and sufficiency of F-actin networks in active zone formation and phase separation will be determined with optogenetic methods in vivo, and reconstitution of syCAM signaling and active zone phase separation in vitro. Finally, automated forward genetic screens will be performed to identify novel regulators of active zone formation. As all synapses across the diversity of neurons build a conserved core active zone, these studies have the potential to uncover common synapse assembly pathways. A fundamental understanding of synapse assembly will yield insight into synapse regeneration and future treatment of synaptopathies and is of primary relevance to the NINDS mission. The candidate will perform the K99 phase at Stanford University under the mentorship of Dr. Kang Shen, an expert in molecular and developmental neuroscience. This award will support the candidate’s career development with personalized training in research methods (genetic screening, phosphoregulation, and in vitro reconstitution) and career development (scientific writing, teaching, and management). This training will enable the candidate to successfully complete the proposed research and transition to an independent position investigating synapse formation.

突触是神经元通信的基本单位，因此它们的位置，数量和特性控制着神经元电路和神经系统的功能。突触形式如何仍然是当代神经科学中的一个基本问题。多种突触细胞粘附分子（Sycams）能够引发突触形成。然而，在这些不同的联系的下游，形成了常见的前后突触后专业。在突触前的一侧，“活动区”结构由大型多价脚手架蛋白形成。活性区通过束缚和启动突触蔬菜，聚类钙通道以及通过跨膜连接与后散布对齐，从而协调了降临前的中心功能。最近，候选人显示了秀丽隐杆线虫活跃区支架Syd-2/liprin-α，麋鹿形成液态液相的液相分离的冷凝物，在发育的突触中，这种活性是活性区组装所必需的。这项工作将活动区分离定位为散发前的中央组件集线器。拟议研究的总体目标是确定驱动活性区相分离的分子机制和新成分，从而驱动了非丧失前的形成。在AIM 1中，将研究磷酸化作为控制活跃区相分离的机制。初步数据表明SAD-1激酶磷酸化的SYD-2控制其活性。该磷化调节将在单共聚的分辨率下与体外相分离测定法相结合，以活体内成像进行研究。在AIM 2中，将研究突触造型结胶与活动区分离之间的联系。已经确定了多个Sycams来构建局部F-肌动蛋白网络，这些网络可能会将这些上游分子与活动区相分离联系起来。 F-肌动蛋白网络在活性区形成和相分离中的需求和充分性将由体内的光遗传学方法确定，并在体外重新建立了Sycam信号传导和活跃区相分离的体外。最后，将执行自动远期遗传筛选，以识别活跃区形成的新调节剂。随着神经元多样性的所有突触建立一个保守的核心活性区，这些研究可能会发现共同的突触组装途径。对突触大会的基本理解将使人们能够深入了解突触疾病突触的再生和未来治疗，并且与NINDS任务至关重要。候选人将根据分子和发育神经科学专家Kang Shen博士的心态在斯坦福大学进行K99阶段。该奖项将通过在研究方法（基因筛查，磷酸盐调节和体外重构）和职业发展（科学写作，教学和管理）方面的个性化培训（基因筛查，磷酸盐调节和体外重构）来支持候选人的职业发展。该培训将使候选人能够成功完成提议的研究并过渡到独立的职位，以调查突触形成。