Clathrin-Mediated Endocytosis near Active Zones in Snake Motor Boutons
Clathrin-Mediated Endocytosis near Active Zones in Snake Motor Boutons
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DOI：
10.1523/jneurosci.20-21-07986.2000
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发表时间：
2000-11
期刊：
影响因子：
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通讯作者：
H. Teng;R. S. Wilkinson
中科院分区：
文献类型：
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作者：
H. Teng;R. S. Wilkinson
我们使用活性依赖性探针 FM1-43 和电子显微镜 (EM) 来检查脊椎动物神经肌肉突触的内吞作用。在低温下进行非常短暂的神经刺激后固定制剂，并将内化的 FM1-43 光转化为电子密集的反应产物。为了定位反应产物，我们从串行 EM 部分重建了各个终端按钮的计算机渲染图。大多数反应产物出现在 40-60 nm 的囊泡中。所有标记的囊泡均包有网格蛋白，其中 92% 位于质膜 300 nm 范围内，表明它们从内吞位点回收后几乎没有经过任何处理。囊泡（以及由此推断的位点）并不是随机分散在膜平面附近，而是明显聚集在活性区域附近。在假定的巨脂质体中发现了额外的反应产物；这些似乎是由活动区附近的深层膜内陷形成的。因此，在短暂刺激后，两种内吞作用的机制是明显的。活性区附近的内吞作用与局部外切/内吞循环池的存在一致。当囊泡膜和质膜互换时，这种机制还可能有助于在活动期间保持活性区与突触后折叠的对齐。
We have used the activity-dependent probe FM1-43 with electron microscopy (EM) to examine endocytosis at the vertebrate nerve–muscle synapse. Preparations were fixed after very brief neural stimulation at reduced temperature, and internalized FM1-43 was photoconverted into an electron-dense reaction product. To locate the reaction product, we reconstructed computer renderings of individual terminal boutons from serial EM sections. Most of the reaction product was seen in 40–60 nm vesicles. All of the labeled vesicles were clathrin-coated, and 92% of them were located within 300 nm of the plasma membrane, suggesting that they had undergone little processing after retrieval from their endocytic sites. The vesicles (and by inference the sites) were not dispersed randomly near the plane of the membrane but instead were clustered significantly near active zones. Additional reaction product was found within putative macropinosomes; these appeared to form from deep membrane invaginations near active zones. Thus two mechanisms of endocytosis were evident after brief stimulation. Endocytosis near active zones is consistent with the existence of local exo/endocytic cycling pools. This mechanism also might serve to maintain alignment of active zones with postsynaptic folds during periods of activity when vesicular and plasma membranes are interchanged.