Boosting the toolbox for live imaging of translation
- Maelle Bellec1,
- Ruoyu Chen2,
- Jana Dhayni3,
- Antonio Trullo4,
- Damien Avinens5,
- Hussein Karaki6,
- Flavia Mazzarda6,
- Helene Lenden4,
- Cyril Favard5,
- Ruth Lehmann7,
- Edouard Bertrand6,
- Mounia Lagha4 and
- Jeremy Dufourt5,8
- 1 Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany.;
- 2 Vilcek Institute of Graduate Studies, NYU School of Medicine, New York, United States, Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.;
- 3 Institut de Genetique Humaine, University of Montpellier, CNRS, 34396 Montpellier, France;
- 4 Institut de Genetique Moleculaire de Montpellier, Univ Montpellier, CNRS, Montpellier, France.;
- 5 Institut de Recherche en Infectiologie de Montpellier, CNRS UMR 9004, University of Montpellier, 1919 Route de Mende, Montpellier, 34293, Cedex 5, France.;
- 6 Institut de Genetique Humaine, University of Montpellier, CNRS, 34396 Montpellier, France.;
- 7 Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
- ↵* Corresponding author; email: jeremy.dufourt{at}irim.cnrs.fr
Abstract
Live imaging of translation based on tag recognition by a single chain antibody is a powerful technique to assess 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 living Drosophila embryo 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. 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. Finally, we developed a largely improved RNA imaging system based on an MCP-tdStaygold fusion.
Keywords
- Received June 21, 2024.
- Accepted June 30, 2024.
- Published by Cold Spring Harbor Laboratory Press for the RNA Society
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