Visualization of a blue light transmission area in living animals using light-induced nuclear translocation of fluorescent proteins
Visualization of a blue light transmission area in living animals using light-induced nuclear translocation of fluorescent proteins
复制标题
使用荧光蛋白的光诱导核易位对活体动物中的蓝光透射区域进行可视化
DOI：
10.1016/j.bbrc.2019.11.023
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发表时间：
2020
影响因子：
3.1
通讯作者：
Onaka Tatsushi
中科院分区：
文献类型：
--
作者：
Inutsuka Ayumu;Kimizuka Norihiro;Takanohashi Natsuki;Yakabu Hisashi;Onaka Tatsushi
光学操纵被广泛用于分析活体神经元的功能。蓝光经常用于激活通道视紫红质或LOV结构域，尽管其吸收和散射的程度高于较长波长的光。高空间分辨率的光学操纵在体外实验中很容易实现，但由于多种因素的影响，光在组织中的散射和吸收是不均匀的。在活体内空间测量蓝光透射面积是困难的。在这里，我们提出了一种遗传方法来可视化蓝光传输在大脑和其他器官使用光诱导的核转位的荧光蛋白与LOV域。光诱导核定位信号（LINUS）由与核定位信号（NLS）融合的LOV 2结构域组成。我们证实，蓝光照射在HEK 293细胞中诱导NES-tdTomato-LINUS在30分钟内从细胞质到细胞核的可逆易位。通过使用可穿透血脑屏障的PHP.eb衣壳，眶后窦注射腺相关病毒（AAV）载体诱导核输出信号（内斯）-tdTomato-LINUS在脑中的分散表达。我们证实，30分钟的经颅蓝光照射诱导NES-tdTomato-LINUS在皮层，海马，甚至丘脑室旁核的核转位。我们还发现，在剃光腹部区域暴露于蓝光的小鼠在肝脏腹侧表面叶中表现出核易位的显著增加。这些结果提供了一种简单的方法来获得有用的信息，在组织中的光传输没有任何转基因动物或熟练的程序。
Optical manipulations are widely used to analyze neuronal functionsin vivo. Blue light is frequently used to activate channelrhodopsins or LOV domains, although the degrees of its absorption and scattering are higher than those of longer wavelength light. High spatial resolution of optical manipulation is easily achievedin vitro, while the light is unevenly scattered and absorbed in tissues due to many factors. It is difficult to spatially measure a blue light transmission areain vivo. Here, we propose a genetic method to visualize blue light transmission in the brain and other organs using light-induced nuclear translocation of fluorescent proteins with a LOV domain. A light-inducible nuclear localization signal (LINuS) consists of a LOV2 domain fused with a nuclear localization signal (NLS). We confirmed that blue light illumination induced reversible translocation of NES-tdTomato-LINuS from the cytosol to the nucleus within 30 min in HEK293 cells. By employing a PHP.eb capsid that can penetrate the blood-brain barrier, retro-orbital sinus injection of adeno-associated virus (AAV) vectors induced scattered expression of nuclear export signal (NES)-tdTomato-LINuS in the brain. We confirmed that 30-min transcranial blue light illumination induced nuclear translocation of NES-tdTomato-LINuS in the cortex, the hippocampus, and even the paraventricular nucleus of the thalamus. We also found that mice exposed to blue light in a shaved abdominal area exhibited a substantial increase in nuclear translocation in the ventral surface lobe of the liver. These results provide a simple way to obtain useful information on light transmission in tissues without any transgenic animals or skillful procedures.