Molecular and Cellular Mechanisms Mediating Structural and Functional Active Zone Maturation

目前的提议描述了确定神经元如何调节的实验方法 活动区（AZ）的结构和功能成熟，这是突触传递的关键信号中枢， 通信发生。虽然膜运输机制是高度保守的 在细胞间，额外的突触特异性调节已经进化为介导快速的Ca 2 +- 依赖性突触囊泡（SV）在精确对齐的特化突触前AZ处融合 突触后受体在AZ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），巴松管/短笛/法伊夫 Unc13我们实验室以前的研究表明，由一种 果蝇单个突触能运动神经元突触分布不均匀 相邻AZ的释放概率通常相差>50倍 (Pr)的SV。我们发现AZ成熟驱动突触强度增加，发生在多细胞中。 日发育期，新形成的AZ在成熟前发育为弱Pr位点 通过一组核心蛋白质的协同积累转化为高Pr AZ。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驱动突触多样性。调节AZ的机制 成熟分为两大类：控制关键建筑物细胞范围可用性的成熟 阻止生长的AZ（目标1）和那些影响捕获和保留新物质的物质， 个体AZ（目标2）。我们将确定是否产生特定的AZ蛋白， 运输超过其纳入日益增长的AZ，或是否在他们的可用性 突触终末是AZ成熟的限速因子。此外，我们将描述效率 在整个AZ成熟周期中，在各个AZ处的材料捕获。最后我们将 研究材料的可用性和捕获如何在紧张性和相位运动神经元中不同， 支配相同的突触后肌肉，但在AZ组织中显示出显著差异 和SV释放性质（目标3）。这些研究将提供新的见解如何突触 强度在神经元的AZ群中发展，以及突触多样性如何 在神经元亚类中得到更广泛的控制。

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.