A genetically encoded probe for imaging nascent and mature HA-tagged proteins in vivo

Abstract

To expand the toolbox of imaging in living cells, we have engineered a single-chain variable fragment binding the linear HA epitope with high affinity and specificity in vivo. The resulting probe, called the HA frankenbody, can light up in multiple colors HA-tagged nuclear, cytoplasmic, membrane, and mitochondrial proteins in diverse cell types. The HA frankenbody also enables state-of-the-art single-molecule experiments in living cells, which we demonstrate by tracking single HA-tagged histones in U2OS cells and single mRNA translation dynamics in both U2OS cells and neurons. Together with the SunTag, we also track two mRNA species simultaneously to demonstrate comparative single-molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the HA frankenbody to precisely quantify the expression of HA-tagged proteins in developing zebrafish embryos. The versatility of the HA frankenbody makes it a powerful tool for imaging protein dynamics in vivo.

Protein manipulation using single copies of short peptide tags in cultured cells and inDrosophila melanogaster

“…The residual GFP signal not localizing at the mitochondrial membrane was also higher for these anti- HAscFvs than the anti-GCN4scFv signal in equivalent cotransfection conditions (Figure 2, panels of B). This may reflect a lower binding affinity of the scFvs to the HA epitope and consequently a lower efficiency of recruitment with only one epitope copy; this interpretation is in agreement with the lower signal to noise ratio of the Mito_mCherry_1xHA versus Mito_mCherry_smHA, containing 10xHA, reported by Zhao (Zhao et al, 2019).…”

“…The expression pattern of the two frankenbodies in the single transfection conditions in the absence of any bait was somewhat similar and equivalent to the intracellular distribution of the anti-GCN4scFv; uniform expression in both nucleus and cytoplasm, with stronger GFP signal in the nucleoplasm (see Figure 4, panels of A and Suppl. Figure 5, panels of A for p_frankenbody_anti-HA scFvX15F11_mEGFP and p_frankenbody_anti-HA scFvX2E2_mEGFP, respectively), confirming the expression of these scFvs reported by Zhao et al in a different cell line (U2OS)(Zhao et al, 2019).…”

“…We used transient transfection in HeLa cells as a model system to test the binding properties of these protein binders (Brauchle et al, 2014; Moutel et al, 2016; Vigano et al, 2018). We therefore generated mammalian expression constructs for the anti- GCN4 (SunTag) scFv (Tanenbaum et al, 2014), the anti-gp41(MoonTag) nanobody 2H10 (Boersma et al, 2019), the anti-HA scFvs frankenbodies (Zhao et al, 2019) and the anti-ALFA nanobody (Gotzke et al, 2019) fused to either sfGFP or mEGFP for intracellular visualization. All the binders were expressed under the control of the strong CMV promoter/enhancer (for a summary of binder constructs, see Figure 1).…”

“…The HA peptide derived from the influenza virus hemagglutinin has been extensively used in biochemical studies due to the availability of high-affinity monoclonal antibodies (Field et al, 1988; Wilson et al, 1984). Recently, two different anti- HAscFvs derived from the monoclonal anti-HA antibody 12CA5 were generated and called frankenbodies (Zhao et al, 2019). The two frankenbodies anti-HA- scFvX15F11 and anti-HA-scFvX2E2 were made by grafting the complementarity determining regions (CDRs) of the 12CA5 monoclonal antibody into two different scFv scaffolds with a demonstrated solubility in vivo .…”

“…We focused our study on short peptide tags for which specific, high-affinity binders, which are soluble and functional in the intracellular milieu have been characterized. Therefore, we selected the following systems: SunTag (Tanenbaum et al, 2014), MoonTag (Boersma et al, 2019), HA (Zhao et al, 2019) and ALFA (Gotzke et al, 2019). For other commonly used tags such as FLAG® (Hopp et al, 1988) or Myc (Evan et al, 1985), we are unaware of specific binders derived from the corresponding monoclonal antibodies that could perform as intrabodies (Fujiwara et al, 2002; Marschall et al, 2015; Moutel et al, 2016; Worn et al, 2000).…”