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.

突触是神经通讯的基本单位，因此它们的位置、数量和特性控制着神经回路和神经系统的功能，突触的形成仍然是当代神经科学中的一个基本问题。然而，在这些不同连接的下游，形成了共同的突触前和突触后专门化，形成了“活性区”结构。活性区由大的多价支架蛋白组成，通过束缚和启动突触小泡、聚集钙通道以及通过跨膜连接与突触后对齐来协调突触前的中心功能。 SYD-2/Liprin-α 和 ELKS 在发育突触时形成液-液相分离的冷凝物，并且这种活性这项工作将活性区相分离定位为突触前的中央组装中心。拟议研究的总体目标是确定驱动活性区相分离并从而形成突触前的分子机制和新颖成分。在目标 1 中，将研究磷酸化作为控制活性区相分离的机制。初步数据表明 SYD-2 的 SAD-1 激酶磷酸化控制其活性，并将在活体动物体内研究这种磷酸化。单突触分辨率成像与体外相分离测定相结合在目标 2 中，将研究突触生成 syCAM 和活性区相分离之间的联系，以构建局部 F-肌动蛋白网络。 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通过体内光遗传学方法以及syCAM信号和活性的重建来确定。最后，将进行自动正向遗传筛选，以确定活性区形成的新调节因子，因为跨神经元多样性的所有突触都构建了保守的核心活性区，这些研究有可能揭示常见的突触组装途径。对突触组装的基本了解将有助于深入了解突触再生和突触病的未来治疗，并且与 NINDS 任务密切相关。候选人将在康博士的指导下在斯坦福大学进行 K99 阶段。沉博士是分子和发育神经科学领域的专家。该奖项将通过研究方法（基因筛查、磷酸调节和体外重建）和职业发展（科学写作、教学和管理）方面的个性化培训来支持候选人的职业发展。候选人成功完成拟议的研究并过渡到研究突触形成的独立职位。