Dissecting the assembly of neurotransmitter release sites

项目摘要 神经递质在突触处的释放关键取决于分泌机器的精确组装。 在突触前神经末梢内，突触囊泡在活性区融合，活性区是一种蛋白质支架， 与突触后受体并列的释放位点。这种蛋白质复合物含有RIM、ELKS、Munc 13、RIM-BP、 Liprin-α和巴松管/短笛作为中心成分。最近的工作为新的模型提供了基础， 这些蛋白质是如何组装成功能性释放位点的。首先，活跃区具有显著的弹性， 单个基因的切除对其组装的影响最多是适度的。相反，RIM的组合缺失， ELKS或RIM-BP强烈破坏活性区组装，建立支架冗余。第二，当前 研究已经产生了通过液-液相分离组装的工作模型，具有强大的贡献 多价低亲和力相互作用的集合体。不管确切的机制如何，一个总体模型 从这些和其他研究中得出的结论是，活性区是一个动态的蛋白质网络， 通过冗余的低亲和力蛋白质结合。这是不同于传统的模式，其中主组织者 通过具有明确化学计量的刚性复合物介导组装。 在这里，我们建立在我们和其他人的最新进展的目标，以确定什么机制调解组装 以及这些活性区蛋白质网络的相对表面如何与 靶质膜和突触囊泡簇。我们将使用三管齐下的 方法来回答这些问题。目的1定义了RIM在活动区组装中的作用和机制。 我们建立在我们的发现，RIM驱动招聘相互作用的蛋白质后，删除支架冗余 通过RIM+ELKS击倒。我们测试了RIM通过两步流程组织活动区域的模型 从机制上将RIM针对活跃区域的活动与RIM招募其他活跃区域的活动分开 proteins.目的2研究突触囊泡如何聚集和活动区，两个突触前亚区， 彼此之间，互相影响。我们依靠一种新的“突触内”重建方法和测试， 平行模型来定义哪些结合活性足以介导囊泡对接。目标3确定 靶质膜上的活性区锚定机制。这一目标利用我们独特的 收集条件和复合突变体，以解决长期存在的问题，即活性区如何 支架被物理地附着到靶膜的正确位置。我们使用最先进的方法 包括条件基因敲除、受激发射损耗（STED）显微术、荧光恢复 光漂白后（FRAP），高压冷冻和相关光电子显微镜（CLEM），和 电生理学来回答这些问题。 我们的工作将建立关于靶膜、活性区和囊泡如何 簇相互作用以支持突触囊泡周期中的稳定性和动力学。

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.