Molecular and Cellular Mechanisms Mediating Structural and Functional Active Zone Maturation

目前的提案描述了确定神经元如何调节的实验方法 活动区(AZs)的结构和功能成熟，AZs是突触 交流就会发生。尽管膜转运机制高度保守 在整个细胞中，额外的突触特异性调节已经进化为调节快速的钙离子- 精确排列的特定突触前AZ处的依赖突触小泡(SV)融合 到突触后感受器。在AZs中发现了多种进化上保守的蛋白质，包括 RIM，RIM结合蛋白，SyD-1，Liprin-α， Elks/Cast/BruchPilot(BRP)，大管/短笛/长笛 和UNC13。我们实验室以前的研究表明，数百个AZ由一个 果蝇单个谷氨酸能运动神经元的突触异质性分布 实力，邻近的AZS经常显示出释放概率的50倍之差 (PR)。我们发现，AZ的成熟驱动突触强度的增加发生在多个- 日间发育期，新形成的AZ在成熟前作为弱Pr部位发育 通过一组核心蛋白质的协调积累转化为高PR的AZs。在当前 应用，我们将确定神经元如何调节AZ的结构和功能成熟， 以及这些过程中的变化如何推动突触多样性。AZ的调控机制 成熟分为两大类：控制关键建筑在整个细胞范围内的可用性的成熟 阻碍种植AZs(目标1)以及影响捕获和保留新材料的AZ 个别区域(目标2)。我们将确定是否产生特定的AZ蛋白和 运输的数量超过了它们被纳入不断增长的AZ的范围，或者它们在 突触终末对AZ的成熟具有限速作用。此外，我们将对效率进行表征 在整个AZ成熟周期中，每个AZs的物质捕获情况。最后，我们会 检查材料的可获得性和捕获在主音和相运动神经元中的差异 支配相同的突触后肌肉，但在AZ组织中显示出显著的差异 和SV释放特性(目标3)。这些研究将为突触如何 力量在神经元的AZ队列中发展，以及突触多样性如何 对神经元亚类进行更广泛的控制。

The current proposal describes experimental approaches to determine how neurons regulate structural and functional maturation of active zones (AZs), a key signaling hub where synaptic communication occurs. Although membrane trafficking mechanisms are highly conserved across cells, additional synapse-specific regulation has evolved to mediate rapid Ca2+- dependent synaptic vesicle (SV) fusion at specialized presynaptic AZs that are precisely aligned to postsynaptic receptors. Multiple evolutionarily conserved proteins are found at AZs, including RIM, RIM binding protein, Syd-1, Liprin-α, ELKS/CAST/Bruchpilot (BRP), Bassoon/Piccolo/Fife and Unc13. Previous studies in our lab demonstrated that the hundreds of AZs formed by a single glutamatergic motoneuron in Drosophila have a heterogeneous distribution of synaptic strength, with neighboring AZs often showing >50-fold differences in the probability of release (Pr) of SVs. We found that AZ maturation drives increased synaptic strength occur over a multi- day developmental period, with newly formed AZs developing as weak Pr sites before maturing into high Pr AZs through the coordinated accumulation of a core set of proteins. In the current application, we will determine how neurons regulate structural and functional maturation of AZs, and how variations in these processes drive synaptic diversity. The mechanisms regulating AZ maturation fall into two broad categories: those that control cell-wide availability of key building blocks to growing AZs (Aim 1) and those that affect capture and retention of new material at individual AZs (Aim 2). We will determine whether specific AZ proteins are produced and transported in excess of their incorporation into growing AZs, or whether their availability at the synaptic terminal is rate-limiting for AZ maturation. In addition, we will characterize the efficiency of material capture at individual AZs throughout the AZ maturation cycle. Finally, we will examine how material availability and capture differ in tonic and phasic motoneurons that innervate the same postsynaptic muscle, but display striking differences in their AZ organization and SV release properties (Aim 3). These studies will provide new insights into how synaptic strength develops across the cohort of AZs of a neuron, as well as how synaptic diversity can be more broadly controlled across neuronal subclasses.