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、数量和性质支配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中的一个基本问题。多种突触细胞黏附分子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。在突触前一侧，一个由大的多价支架蛋白组成的“活动区”结构形成。活动区通过捆绑和启动突触小泡，聚集钙通道，并通过跨膜连接与突触后结合来协调突触前的中枢功能。最近的候选研究表明，线虫的活动区支架SYD-2/Liprin-α和ELKs在发育中的突触形成液-液分离的凝聚体，这种活性是活动区组装所必需的。这项工作将活动区相分离定位为突触前的中心组装中心。拟议研究的总体目标是确定驱动活动区相分离的分子机制和新的成分，从而形成突触前。在目标1中，将研究磷酸化作为控制活动区相分离的机制。初步数据表明，SYD-2的SAD-1激酶磷酸化控制其活性。这种磷调节将用活体动物体内成像在单一突触分辨率下结合体外相分离分析来研究。在目标2中，将研究突触源性SyCAMs与活动区相分离之间的联系。已经确定了多个syCAM来构建局部F-肌动蛋白网络，该网络可能将这些上游分子连接到活动区相分离。F-肌动蛋白网络在活动区形成和相分离中的需求和充分性将在体内用光遗传学方法确定，并在体外用Sycam信号重建和活动区相分离来确定。最后，将进行自动正向遗传筛选，以确定活跃区形成的新调节因子。由于不同神经元之间的所有突触都建立了一个保守的核心活动区，这些研究有可能揭示共同的突触组装路径。对突触组装的基本了解将有助于洞察突触再生和未来突触病变的治疗，并对NINDS任务具有主要意义。候选人将在分子和发育神经科学专家沈康博士的指导下，在斯坦福大学进行K99阶段的测试。该奖项将通过在研究方法(基因筛选、磷调节和体外重组)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。这项培训将使候选人能够成功地完成拟议的研究，并过渡到一个独立的职位，研究突触的形成。

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.