Organization and Function of the Periactive Zone

项目总结 这项建议的目的是了解突触前兴奋性神经元的空间和功能组织 带(PAZ)，与突触小泡释放部位相邻。PAZ是一个微米级的结构， 被数十种蛋白质占据，这些蛋白质在多价组装中共同作用，偶联膜重塑 以推动肌动蛋白聚合。对PAZ蛋白在许多系统中的研究表明，这些 蛋白质在突触小泡周期的多个步骤以及其他突触膜功能中起作用 (例如，突触形态发生和受体交通)。目前尚不清楚微米级的组织和 PAZ蛋白的调节将其膜和细胞骨架的重塑活性引导到这些不同的 神经元特有的功能。我们将使用果蝇幼虫神经肌肉接头(NMJ)，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蛋白同时占据重叠的和不同的结构域 PAZ，以及PAZ蛋白在这些结构域之间适当分离取决于它们的多价 相互之间的互动。我们最近还描述了依赖于PAZ的动态肌动蛋白细丝 结构，这代表了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，我们将询问突触如何调节Paz蛋白的活动和在空间和时间上的相互作用，以及如何 PAZ组织是其不同的神经元特异性功能的基础，以响应突触活动和 变速箱。在目标1中，我们将确定PAZ蛋白是如何在静止和活跃的突触中组织起来的 补充固定、实时和超分辨率成像方法，并开发新的定量方法来 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 斑块代表依赖于笼蛋白的突触小泡回收事件，并识别决定因素 突触肌动蛋白贴片组装和动力学。在目标3中，我们将询问PAZ如何组织和突触活动 控制不同的细胞生物学功能，包括释放部位的清除和细胞黏附的组织 复合体。总体而言，我们的实验将解释PAZ如何组织成不同的子域 突触的多种功能和结构特性。PAZ蛋白与多个 神经功能障碍，因此破译它们在体内的功能对于理解糖尿病的病因至关重要。 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.