Activity-Dependent Dynamics of the Transcription Factor of cAMP-Response Element Binding Protein in Cortical Neurons Revealed by Single-Molecule Imaging
Activity-Dependent Dynamics of the Transcription Factor of cAMP-Response Element Binding Protein in Cortical Neurons Revealed by Single-Molecule Imaging
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DOI：
10.1523/jneurosci.0943-16.2016
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发表时间：
2017-01-04
影响因子：
5.3
通讯作者：
Yamamoto, Nobuhiko
中科院分区：
文献类型：
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作者：
Kitagawa, Hironobu;Sugo, Noriyuki;Yamamoto, Nobuhiko
转录调节对于控制神经元回路形成和突触可塑性的神经元活动依赖性过程至关重要。一个有趣的问题是神经元活动如何影响转录因子与其靶位点之间的时空相互作用。在这里，我们使用单分子成像技术，研究了小鼠皮层神经元中 cAMP 反应元件结合蛋白（CREB）的 DNA 结合和解离事件的活性依赖性，CREB 是活性依赖性转录的主要因素。为了在单分子水平上可视化 CREB，通过高度倾斜和层压光学片显微镜观察活体分离皮质神经元中荧光标记的 CREB。我们发现大部分 CREB 斑点在细胞核的限制位置停留了几秒钟（解离速率常数：0.42 s(-1)）。相比之下，两个不能与 cAMP 响应元件结合的突变 CREB 几乎没有表现出长期驻留。为了测试 CREB 动力学依赖于神经元活动的可能性，将药物治疗和涉及视紫红质通道蛋白 2 的光遗传学方法应用于培养的皮层神经元。神经元活动的增加似乎不会影响 CREB 点的停留时间，但显着增加了限制位置（热点）的数量，其中 CREB 点经常驻留且停留时间较长（> 1 秒）。这些结果表明，神经元活动通过增加 CREB 与高度本地化的基因组位置结合的频率来促进 CREB 依赖性转录。
Transcriptional regulation is crucial for neuronal activity-dependent processes that govern neuronal circuit formation and synaptic plasticity. An intriguing question is how neuronal activity influences the spatiotemporal interactions between transcription factors and their target sites. Here, using a single-molecule imaging technique, we investigated the activity dependence of DNA binding and dissociation events of cAMP-response element binding protein (CREB), a principal factor in activity-dependent transcription, in mouse cortical neurons. To visualize CREB at the single-molecule level, fluorescent-tagged CREB in living dissociated cortical neurons was observed by highly inclined and laminated optical sheet microscopy. We found that a significant fraction of CREB spots resided in the restricted locations in the nucleus for several seconds (dissociation rate constant: 0.42 s(-1)). In contrast, two mutant CREBs, which cannot bind to the cAMP-response element, scarcely exhibited long-term residence. To test the possibility that CREB dynamics depends on neuronal activity, pharmacological treatments and an optogenetic method involving channelrhodopsin-2 were applied to cultured cortical neurons. Increased neuronal activity did not appear to influence the residence time of CREB spots, but markedly increased the number of restricted locations (hot spots) where CREB spots frequently resided with long residence times (>1s). These results suggest that neuronal activity promotes CREB-dependent transcription by increasing the frequency of CREB binding to highly localized genome locations.