Imaging Endogenous Synaptic Proteins in Primary Neurons at Single-Cell Resolution Using CRISPR/Cas9
Imaging Endogenous Synaptic Proteins in Primary Neurons at Single-Cell Resolution Using CRISPR/Cas9
复制标题
使用 CRISPR/Cas9 以单细胞分辨率对原代神经元中的内源突触蛋白进行成像
DOI：
10.1091/mbc.e19-04-0223
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发表时间：
2019
影响因子：
3.3
通讯作者：
Takahiko Matsuda & Izumi Oinuma
中科院分区：
文献类型：
--
作者：
Miura M;Makita S;Azuma S,Yasuno Y;Sugiyama S;Mino M;Yamaguchi T;Agawa T;Iwasaki T;Usui Y;Rao NA;Goto H;Takahiko Matsuda & Izumi Oinuma
单细胞分辨率荧光成像是分析复杂神经网络中单个细胞内蛋白质时空调节的重要方法。在这里，我们提出了一种非病毒策略，可以通过CRISPR/ cas9介导的基因组编辑结合细胞核转染技术来标记内源性位点。该方法允许在内源性水平上表达荧光标记的蛋白，我们成功地在培养的有丝分裂后神经元中标记了突触前蛋白synaptophysin （Syp）和突触后蛋白PSD-95。固定神经元的超分辨荧光显微镜证实了标记蛋白与免疫组织化学证实的内源性蛋白相同的定位模式。该系统也适用于多路标记和活细胞成像。用全内反射荧光显微镜实时成像Syp-mCherry和PSD-95-EGFP双标记的神经元的单个树突过程揭示了先前未描述的沿树突轴同步移动的蛋白质的动态定位。我们方便和通用的策略是有效的分析蛋白质的异位表达扰乱细胞功能。
Fluorescence imaging at single-cell resolution is a crucial approach to analyzing the spatiotemporal regulation of proteins within individual cells of complex neural networks. Here we present a nonviral strategy that enables the tagging of endogenous loci by CRISPR/Cas9-mediated genome editing combined with a nucleofection technique. The method allowed expression of fluorescently tagged proteins at endogenous levels, and we successfully achieved tagging of a presynaptic protein, synaptophysin (Syp), and a postsynaptic protein, PSD-95, in cultured postmitotic neurons. Superresolution fluorescence microscopy of fixed neurons confirmed the identical localization patterns of the tagged proteins to those of endogenous ones verified by immunohistochemistry. The system is also applicable for multiplexed labeling and live-cell imaging. Live imaging with total internal reflection fluorescence microscopy of a single dendritic process of a neuron double-labeled with Syp-mCherry and PSD-95-EGFP revealed the previously undescribed dynamic localization of the proteins synchronously moving along dendritic shafts. Our convenient and versatile strategy is potent for analysis of proteins whose ectopic expressions perturb cellular functions.