Boosting the toolbox for live imaging of translation

Abstract

AbstractLive imaging of translation based on a tag recognition by a single chain antibody is a powerful technique to assess the fine tuning of translation regulation in living cells. However, especially in a multicellular organism, this approach is challenging and requires optimization in terms of expression level and detection sensitivity of the system. Here, we improved existing fluorescent tools and developed new ones to image and quantify nascent translation in the livingDrosophilaembryo and in mammalian cells. We tested and characterized five different Green Fluorescent Protein variants fused to the single chain fragment variable (scFv) and uncover photobleaching, aggregation and intensity disparities. Using different strengths of germline and somatic drivers, we determined that the availability of the scFv is critical in order to detect translation throughout development. Finally, we introduced a new translation imaging method, based on a nanobody/tag system, named ALFA_array, allowing the sensitive and simultaneous detection of the translation of several distinct mRNA species.