In vivo single-molecule imaging of syntaxin1A reveals polyphosphoinositide- and activity-dependent trapping in presynaptic nanoclusters.
In vivo single-molecule imaging of syntaxin1A reveals polyphosphoinositide- and activity-dependent trapping in presynaptic nanoclusters.
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DOI：
10.1038/ncomms13660
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发表时间：
2017-01-03
影响因子：
16.6
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Synaxin1A以纳米簇的形式组织，这对于神经分泌细胞中的分泌囊泡的对接和启动至关重要。这些纳米簇是否以及如何受到生物神经末梢释放的神经递质的影响尚不清楚。在这里，我们用单粒子跟踪光活化定位显微镜对果蝇幼虫运动神经末梢中的光可转换合成素1A-mEos2进行了成像。光和热遗传神经元刺激增加了突触蛋白1A-mEos2的迁移率，并降低了纳米团簇的大小和分子密度，表明突触蛋白1A的活性依赖于从纳米团簇的限制中释放。通过突变其多聚磷脂酰肌醇结合部位或通过共表达破伤风毒素轻链来阻止SNARE复合体的组装，增加了Synaxin1A的流动性。相反，通过阻止SNARE复合体的拆解，Synaxin1A的迁移率降低。我们的数据表明，多磷肌醇有利于句法蛋白1A的捕获，并表明SNARE络合物的解离导致了句法蛋白1A从纳米簇中解离。因此，突触素1A在纳米团簇中的横向扩散和捕获动态地调节神经递质的释放。Synaxin1A(Sx1A)是在神经分泌细胞中以纳米簇的形式组织起来的，但这些纳米簇如何受到生物体内神经递质释放的影响尚不清楚。在这里，作者在活的苍蝇幼虫中进行了单分子成像分析，并表明Sx1A在纳米簇中的横向扩散和捕获受到突触活性的改变。
Syntaxin1A is organized in nanoclusters that are critical for the docking and priming of secretory vesicles from neurosecretory cells. Whether and how these nanoclusters are affected by neurotransmitter release in nerve terminals from a living organism is unknown. Here we imaged photoconvertible syntaxin1A-mEos2 in the motor nerve terminal of Drosophila larvae by single-particle tracking photoactivation localization microscopy. Opto- and thermo-genetic neuronal stimulation increased syntaxin1A-mEos2 mobility, and reduced the size and molecular density of nanoclusters, suggesting an activity-dependent release of syntaxin1A from the confinement of nanoclusters. Syntaxin1A mobility was increased by mutating its polyphosphoinositide-binding site or preventing SNARE complex assembly via co-expression of tetanus toxin light chain. In contrast, syntaxin1A mobility was reduced by preventing SNARE complex disassembly. Our data demonstrate that polyphosphoinositide favours syntaxin1A trapping, and show that SNARE complex disassembly leads to syntaxin1A dissociation from nanoclusters. Lateral diffusion and trapping of syntaxin1A in nanoclusters therefore dynamically regulate neurotransmitter release. Syntaxin1A (Sx1A) is organized in nanoclusters in neurosecretory cells but how these nanoclusters are affected by neurotransmitter release in a living organism is unknown. Here the authors perform single molecule imaging analysis in live fly larvae and show that the lateral diffusion and trapping of Sx1A in nanoclusters are altered by synaptic activity.