The spatial distribution of proteins and their arrangement within the cellular ultrastructure regulates the opening of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in response to glutamate release at the synapse. Fluorescence microscopy imaging revealed that the postsynaptic density (PSD) and scaffolding proteins in the presynaptic active zone (AZ) align across the synapse to form a trans-synaptic “nanocolumn,” but the relation to synaptic vesicle release sites is uncertain. Here, we employ focused-ion beam (FIB) milling and cryoelectron tomography to image synapses under near-native conditions. Improved image contrast, enabled by FIB milling, allows simultaneous visualization of supramolecular nanoclusters within the AZ and PSD and synaptic vesicles. Surprisingly, membrane-proximal synaptic vesicles, which fuse to release glutamate, are not preferentially aligned with AZ or PSD nanoclusters. These synaptic vesicles are linked to the membrane by peripheral protein densities, often consistent in size and shape with Munc13, as well as globular densities bridging the synaptic vesicle and plasma membrane, consistent with prefusion complexes of SNAREs, synaptotagmins, and complexin. Monte Carlo simulations of synaptic transmission events using biorealistic models guided by our tomograms predict that clustering AMPARs within PSD nanoclusters increases the variability of the postsynaptic response but not its average amplitude. Together, our data support a model in which synaptic strength is tuned at the level of single vesicles by the spatial relationship between scaffolding nanoclusters and single synaptic vesicle fusion sites.

中文翻译：

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冷冻电子断层扫描揭示突触小泡和支架复合物的纳米级结构


蛋白质的空间分布及其在细胞超微结构内的排列调节 α-氨基-3-羟基-5-甲基-4-异恶唑丙酸 (AMPA) 受体的开放，以响应突触处谷氨酸的释放。荧光显微镜成像显示突触前活性区（AZ）中的突触后密度（PSD）和支架蛋白在突触上排列形成跨突触“纳米柱”，但与突触小泡释放位点的关系尚不确定。在这里，我们采用聚焦离子束（FIB）铣削和冷冻电子断层扫描在接近自然的条件下对突触进行成像。 FIB 铣削提高了图像对比度，可以同时可视化 AZ 和 PSD 内的超分子纳米簇以及突触囊泡。令人惊讶的是，融合释放谷氨酸的近膜突触小泡并不优先与 AZ 或 PSD 纳米簇对齐。这些突触小泡通过外周蛋白密度与膜相连，其大小和形状通常与 Munc13 一致，以及桥接突触小泡和质膜的球状密度，与 SNARE、突触结合蛋白和复合蛋白的预融合复合物一致。使用我们的断层图引导的生物现实模型对突触传递事件进行蒙特卡罗模拟，预测 PSD 纳米团簇内的 AMPAR 聚类会增加突触后反应的变异性，但不会增加其平均幅度。总之，我们的数据支持一个模型，其中突触强度通过支架纳米团簇和单个突触囊泡融合位点之间的空间关系在单个囊泡水平上进行调整。