Reproducible and sustained regulation of Gαs signalling using a metazoan opsin as an optogenetic tool.
Reproducible and sustained regulation of Gαs signalling using a metazoan opsin as an optogenetic tool.
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DOI：
10.1371/journal.pone.0030774
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Lucas RJ
中科院分区：
文献类型：
--
作者：
Bailes HJ;Zhuang LY;Lucas RJ
光遗传学最初是为了调节神经元的兴奋性而开发的，现在也越来越多地用于以前所未有的时空分辨率控制其他细胞过程。所有主要G蛋白信号传导途径（Gq、Gi和Gs）的光遗传调节已经使用哺乳动物视杆视蛋白的变体实现。我们在这里表明，由这种视杆视蛋白为基础的工具驱动的光响应在重复曝光下消散，与这种色素的已知漂白特性一致。我们继续表明，用来自Carybdea rastonii，箱形水母（JellyOp）的耐漂白视蛋白代替视杆视蛋白克服了这一限制。可见光诱导表达JellyOp的哺乳动物细胞中cAMP的高幅度、可逆和可再现的增加。虽然单次闪光产生短暂的cAMP峰值，但重复刺激可维持升高的水平达10分钟。JellyOp比目前可用的光遗传学工具更光敏，响应于辐照度≥1 µW/cm 2的白色光。我们的结论是，JellyOp是一个很有前途的新工具，用于模拟具有良好的时空分辨率的GS-偶联G蛋白偶联受体的活性。
Originally developed to regulate neuronal excitability, optogenetics is increasingly also used to control other cellular processes with unprecedented spatiotemporal resolution. Optogenetic modulation of all major G-protein signalling pathways (Gq, Gi and Gs) has been achieved using variants of mammalian rod opsin. We show here that the light response driven by such rod opsin-based tools dissipates under repeated exposure, consistent with the known bleaching characteristics of this photopigment. We continue to show that replacing rod opsin with a bleach resistant opsin from Carybdea rastonii, the box jellyfish, (JellyOp) overcomes this limitation. Visible light induced high amplitude, reversible, and reproducible increases in cAMP in mammalian cells expressing JellyOp. While single flashes produced a brief cAMP spike, repeated stimulation could sustain elevated levels for 10s of minutes. JellyOp was more photosensitive than currently available optogenetic tools, responding to white light at irradiances ≥1 µW/cm2. We conclude that JellyOp is a promising new tool for mimicking the activity of Gs-coupled G protein coupled receptors with fine spatiotemporal resolution.
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