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Background  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.    Results  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 Ca 2+ , 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 IP 3  receptors deliver Ca 2+  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.    Conclusions  This toolkit greatly expands the utility of intrabodies and will enable a range of approaches for studying and manipulating cell signalling in live cells.    Electronic supplementary material  The online version of this article (10.1186/s12915-019-0662-4) contains supplementary material, which is available to authorized users.

A genetically encoded toolkit of functionalized nanobodies against fluorescent proteins for visualizing and manipulating intracellular signalling