Organization and function of the periactive zone

项目摘要 这个提议的目的是了解突触前周期性突触的空间和功能组织， 区（PAZ），其被发现邻近突触囊泡释放的位点。PAZ是一个微米级的结构， 由几十种蛋白质占据，这些蛋白质在多价组装中共同作用，以偶联膜重塑 到产生力的肌动蛋白聚合在许多系统中对PAZ蛋白的研究表明， 蛋白质在突触囊泡周期的多个步骤以及在其他突触膜功能中起作用 (e.g.突触形态发生和受体运输）。目前尚不清楚微米级组织和 PAZ蛋白的调节将它们的膜和细胞骨架重塑活性引导到这些不同的 神经元特异性功能我们将使用果蝇幼虫神经肌肉接头（NMJ），这是一个强大的模型 突触，以破译PAZ蛋白组装，活动和细胞功能是如何联系在一起的。使用高- 分辨率成像，我们最近发现PAZ蛋白占据重叠和不同的结构域内， PAZ和PAZ蛋白在这些结构域之间适当分离取决于它们的多价性 相互影响。我们最近还描述了PAZ依赖的动态肌动蛋白丝 结构，其代表了PAZ蛋白在这些不同结构域中的活性的直接读数。使用这些 工具，我们将问突触如何调节PAZ蛋白的活动和相互作用的空间和时间，以及如何 PAZ组织是其多种神经元特异性功能的基础，响应于突触活动， 传输在目标1中，我们将使用以下方法确定PAZ蛋白在静息和活动突触中的组织方式 互补的固定，实时和超分辨率成像方法，并开发新的定量方法， 描述它们在PAZ域中的几何关系。在目标2中，我们将检验突触肌动蛋白 斑块代表网格蛋白依赖性突触囊泡回收事件，并确定 突触肌动蛋白片组装和动力学。在目标3中，我们将探讨PAZ的组织和突触活动是如何 控制多种细胞生物学PAZ功能，包括释放位点清除和组织细胞粘附 配合物总的来说，我们的实验将解释PAZ如何组织成不同的子域， 是突触的多种功能和结构特性的基础。PAZ蛋白参与多种 因此，破译它们的体内功能对于理解神经系统疾病的病因至关重要。 这些人类疾病。

PROJECT SUMMARY The goal of this proposal is to understand the spatial and functional organization of the presynaptic periactive zone (PAZ), which is found adjacent to sites of synaptic vesicle release. The PAZ is a micron-scale structure, occupied by dozens of proteins that work together in multivalent assemblies to couple membrane remodeling to force-generating actin polymerization. Studies of PAZ proteins in many systems have suggested that these proteins act at multiple steps of the synaptic vesicle cycle as well as in other synaptic membrane functions (e.g. synaptic morphogenesis and receptor traffic). It remains unknown how the micron-scale organization and regulation of PAZ proteins direct their membrane and cytoskeleton remodeling activities to these different neuron-specific functions. We will use the Drosophila larval neuromuscular junction (NMJ), a powerful model synapse, to decipher how PAZ protein assemblies, activities, and cellular functions are linked. Using high- resolution imaging, we recently found that PAZ proteins occupy both overlapping and distinct domains within the PAZ, and that proper segregation of PAZ proteins between these domains depends on their multivalent interactions with each other. We have also recently described PAZ-dependent dynamic actin filament structures, which represent a direct readout of PAZ protein activities in these different domains. Using these tools, we will ask how synapses regulate PAZ protein activities and interactions in space and time, and how PAZ organization underlies its diverse neuron-specific functions, in response to synaptic activity and transmission. In Aim 1, we will determine how PAZ proteins are organized at resting and active synapses using complementary fixed, live, and super-resolution imaging methods, and develop new quantitative methods to describe their geometric relationships in PAZ domains. In Aim 2, we will test the hypothesis that synaptic actin patches represent clathrin-dependent synaptic vesicle recycling events, and identify the determinants of synaptic actin patch assembly and dynamics. In Aim 3, we will ask how PAZ organization and synaptic activity control diverse cell biological PAZ functions, including release site clearance and organization of cell adhesion complexes. Overall, our experiments will explain how organization of the PAZ into distinct subdomains underlies multiple functional and structural properties of synapses. PAZ proteins are implicated in multiple neurological disorders, so deciphering their in vivo functions will be critical for understanding the etiology of these human diseases.