Dominant negative SNARE peptides stabilize the fusion pore in a narrow, release-unproductive state
Dominant negative SNARE peptides stabilize the fusion pore in a narrow, release-unproductive state
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DOI：
10.1007/s00018-016-2213-2
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发表时间：
2016-10-01
影响因子：
8
通讯作者：
Zorec, Robert
中科院分区：
文献类型：
--
作者：
Gucek, Alenka;Jorgacevski, Jernej;Zorec, Robert
基于囊泡的胶质递质释放的关键支持来自对具有星形胶质细胞特异性表达突触传导蛋白2 （dnSNARE）的显性阴性结构域的转基因小鼠的研究。为了确定这种肽如何影响胞外分泌，我们使用超分辨率刺激发射耗尽显微镜和结构照明显微镜研究星形胶质细胞中单个囊泡的解剖结构。较小的囊泡含有氨基酸和肽能递质，较大的囊泡含有ATP。膜电容的离散增加表明单囊泡融合，表明星形胶质细胞刺激增加了小囊泡中主要短暂融合事件的频率，而大囊泡则过渡到完全融合。为了确定这是否反映了较大囊泡中SNARE蛋白密度较低，我们用肉毒杆菌神经毒素D和E处理星形胶质细胞，这两种囊泡类型的胞外事件都减少了。dnSNARE肽将两种囊泡类型的融合孔直径稳定到狭窄，释放不产生的直径，无论囊泡直径如何。
Key support for vesicle-based release of gliotransmitters comes from studies of transgenic mice with astrocyte-specific expression of a dominant-negative domain of synaptobrevin 2 protein (dnSNARE). To determine how this peptide affects exocytosis, we used super-resolution stimulated emission depletion microscopy and structured illumination microscopy to study the anatomy of single vesicles in astrocytes. Smaller vesicles contained amino acid and peptidergic transmitters and larger vesicles contained ATP. Discrete increases in membrane capacitance, indicating single-vesicle fusion, revealed that astrocyte stimulation increases the frequency of predominantly transient fusion events in smaller vesicles, whereas larger vesicles transitioned to full fusion. To determine whether this reflects a lower density of SNARE proteins in larger vesicles, we treated astrocytes with botulinum neurotoxins D and E, which reduced exocytotic events of both vesicle types. dnSNARE peptide stabilized the fusion-pore diameter to narrow, release-unproductive diameters in both vesicle types, regardless of vesicle diameter.