STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis
STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis
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DOI：
10.1038/nature04592
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发表时间：
2006-04-13
期刊：
影响因子：
64.8
通讯作者：
Hell, SW
中科院分区：
文献类型：
--
作者：
Willig, KI;Rizzoli, SO;Hell, SW
突触传递由储存在突触囊泡中并在激活时通过胞吐作用释放的神经递质介导。囊泡膜随后通过内吞作用恢复，突触囊泡再生并重新充满神经递质（1）。虽然了解了囊泡回收的许多方面，但融合后囊泡的命运仍不清楚。它们的成分是在质膜上扩散，还是保持在一起？这个问题一直很难回答，因为突触囊泡太小（直径近似40 nm），而且过于密集，无法通过现有的荧光显微镜进行解析。在这里，我们使用受激发射损耗（STED）（2）将焦点面积减少到衍射极限以下一个数量级，从而分辨突触中的单个囊泡。我们发现，突触结合蛋白I，一种蛋白质驻留在囊泡膜，仍然聚集在孤立的补丁突触前膜上，无论神经末梢是轻度活跃或强烈刺激。这表明至少一些囊泡成分在回收过程中保持在一起。我们的研究还表明，涉及几十纳米尺度的细胞结构的问题可以用传统的远场光学和可见光来解决。
Synaptic transmission is mediated by neurotransmitters that are stored in synaptic vesicles and released by exocytosis upon activation. The vesicle membrane is then retrieved by endocytosis, and synaptic vesicles are regenerated and re-filled with neurotransmitter(1). Although many aspects of vesicle recycling are understood, the fate of the vesicles after fusion is still unclear. Do their components diffuse on the plasma membrane, or do they remain together? This question has been difficult to answer because synaptic vesicles are too small (similar to 40 nm in diameter) and too densely packed to be resolved by available fluorescence microscopes. Here we use stimulated emission depletion (STED)(2) to reduce the focal spot area by about an order of magnitude below the diffraction limit, thereby resolving individual vesicles in the synapse. We show that synaptotagmin I, a protein resident in the vesicle membrane, remains clustered in isolated patches on the presynaptic membrane regardless of whether the nerve terminals are mildly active or intensely stimulated. This suggests that at least some vesicle constituents remain together during recycling. Our study also demonstrates that questions involving cellular structures with dimensions of a few tens of nanometres can be resolved with conventional far-field optics and visible light.