Spatial and temporal regulation of synapse formation through phase separation

突触是神经元交流的基本单位，因此它们的位置、数量和性质决定着神经回路和神经系统的功能。突触如何形成仍然是当代神经科学的一个基本问题。各种各样的突触细胞粘附分子（syCAMs）能够启动突触的形成。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特化。在突触前侧，形成了一个由大的多价支架蛋白组成的“活性区”结构。活跃区通过系结和启动突触囊泡、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枢功能。最近的研究表明，保守的秀丽隐杆线虫活性区支架SYD-2/Liprin-α和ELKS在发育突触形成液-液相分离凝聚物，这种活性是活性区组装所必需的。这项工作将活动区相分离定位为突触前的中央组装枢纽。提出的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，从而形成突触前。在Aim 1中，磷酸化将作为控制活性区相分离的机制进行研究。初步数据表明，SYD-2的SAD-1激酶磷酸化控制其活性。这种磷酸化调节将通过活体动物体内成像在单突触分辨率结合体外相分离分析进行研究。在Aim 2中，将研究突触源性syCAMs与活动区相分离之间的联系。多个sycam已被确定用于构建局部F-肌动蛋白网络，该网络可能将这些上游分子与活性区相分离联系起来。F-actin网络在活性区形成和相分离中的需求和充分性将在体内通过光遗传学方法确定，并在体外通过syCAM信号重构和活性区相分离来确定。最后，将进行自动前向遗传筛选，以确定活性区形成的新调节因子。由于不同种类神经元的所有突触都建立了一个保守的核心活跃区，这些研究有可能揭示共同的突触组装途径。对突触组装的基本理解将有助于深入了解突触再生和突触病变的未来治疗，并且与NINDS任务具有主要相关性。该候选人将在斯坦福大学的分子和发育神经科学专家沈康博士的指导下进行K99阶段。该奖项将为候选人的职业发展提供个性化培训，包括研究方法（基因筛选、磷酸化调节和体外重建）和职业发展（科学写作、教学和管理）。这项培训将使候选人能够成功完成拟议的研究，并过渡到一个独立的位置调查突触的形成。

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.