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.