Intrabodies enable targeting of proteins in live cells, but generating specific intrabodies against the thousands of proteins in a proteome poses a challenge. We leverage the widespread availability of fluorescently labelled proteins to visualize and manipulate intracellular signalling pathways in live cells by using nanobodies targeting fluorescent protein tags. We generated a toolkit of plasmids encoding nanobodies against red and green fluorescent proteins (RFP and GFP variants), fused to functional modules. These include fluorescent sensors for visualization of Ca2+, H+ and ATP/ADP dynamics; oligomerising or heterodimerising modules that allow recruitment or sequestration of proteins and identification of membrane contact sites between organelles; SNAP tags that allow labelling with fluorescent dyes and targeted chromophore-assisted light inactivation; and nanobodies targeted to lumenal sub-compartments of the secretory pathway. We also developed two methods for crosslinking tagged proteins: a dimeric nanobody, and RFP-targeting and GFP-targeting nanobodies fused to complementary hetero-dimerizing domains. We show various applications of the toolkit and demonstrate, for example, that IP3 receptors deliver Ca2+ to the outer membrane of only a subset of mitochondria and that only one or two sites on a mitochondrion form membrane contacts with the plasma membrane. This toolkit greatly expands the utility of intrabodies and will enable a range of approaches for studying and manipulating cell signalling in live cells.

中文翻译：

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一种针对荧光蛋白的功能化纳米抗体的基因编码工具包，用于可视化和操纵细胞内信号传导

体内抗体能够靶向活细胞中的蛋白质，但针对蛋白质组中的数千种蛋白质生成特定的体内抗体却构成了挑战。我们利用荧光标记蛋白的广泛可用性，通过使用靶向荧光蛋白标签的纳米抗体来可视化和操纵活细胞中的细胞内信号通路。我们生成了一个质粒工具包，该工具包编码针对红色和绿色荧光蛋白（RFP 和 GFP 变体）的纳米抗体，并与功能模块融合。其中包括用于 Ca2+、H+ 和 ATP/ADP 动态可视化的荧光传感器；寡聚或异二聚模块，允许招募或隔离蛋白质并识别细胞器之间的膜接触位点；SNAP 标签允许用荧光染料进行标记并进行定向发色团辅助光灭活；以及针对分泌途径的腔亚区室的纳米抗体。我们还开发了两种交联标记蛋白的方法：二聚体纳米抗体，以及与互补异源二聚化结构域融合的 RFP 靶向和 GFP 靶向纳米抗体。我们展示了该工具包的各种应用，并证明，例如，IP3 受体仅将 Ca2+ 传递到线粒体子集的外膜，并且线粒体上只有一两个位点与质膜形成膜接触。该工具包极大地扩展了体内的实用性，并将实现一系列研究和操纵活细胞中细胞信号传导的方法。