Abstract
Ribosomes are responsible for protein synthesis in all living systems. Determining their cellular organization, movement, and translational activity is crucial for dissecting ribosomes’ complex functions. In this study, we describe the development of a selective fluorescent probe for eukaryotic ribosomes — RiboBright. Using C-H activation, the natural product cycloheximide was aminated at the C13-position and fluorescently modified to afford RiboBright. We employ RiboBright for the quantification of ribosome content in 10 cell lines through microscopy and flow cytometry. RiboBright is applicable in live cells for tracking and quantification of ribosome movement and in fixed cells for visualization of sub-micrometer-sized spots, at the single-cell level. RiboBright reveals lineage-specific ribosome content, organization, and movement upon differentiation into either extraembryonic endoderm or ectoderm-like cells. Thus, RiboBright provides a versatile and convenient approach for imaging the cellular dynamics of ribosomes.
Data availability
All relevant data supporting the key findings of this study are available within the article and its Supplementary Information files. Due to the large number of images taken, these files, as well as any additional information needed to reanalyze the data, are available from the lead contact upon request. MaPseq sequencing data have been deposited at Gene Expression Omnibus (GEO) and are publicly available as of the date of publication at GSE316112. Source data are provided with this paper.
Code availability
All code is publicly available on GitHub at https://github.com/XinyuHuRU/RiboBright-project.git.
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Acknowledgements
We thank Dr. Hendrik Marks (Radboud University) for the mESC-E14 cells; Prof. Leor Weinberger (University of Miami) for the NANOG-GFP mESC-E14 cells; Prof. Ger Pruijn (Radboud University) for providing SH-SY5Y cells, Dr. Klaas Mulder (Radboud University) for providing HCT 116 and U2OS cells, Prof. Wilhelm Huck (Radboud University) for providing HeLa cells and Dr. Wenny Peeters (Radiotherapy & OncoImmunology Laboratory, Department of Radiation Oncology, Radboudumc) for providing MCF 10A cells. This work has received funding from the European Research Council under the European Union’s Horizon Europe research and innovation program under grant agreement number 101041939 (ChOICE) to M.M.K.H and 101041938 (RIBOCHEM) to W.A.V. M.M.K.H. further acknowledges generous support from Oncode Institute, which is partly financed by the Dutch Cancer Society.
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Conceptualization: W.A.V., M.M.K.H. Methodology: G.P., C.G., X.H., W.A.V., M.M.K.H. Investigation: G.P., C.G., X.H., P.Y. Visualization: G.P., C.G., X.H. Funding acquisition: W.A.V., M.M.K.H. Project administration: G.P., C.G., X.H., W.A.V., M.M.K.H. Supervision: W.A.V., M.M.K.H. Writing – original draft: G.P., C.G., X.H., W.A.V., M.M.K.H. Writing – review & editing: G.P., C.G., X.H., P.Y., W.A.V., M.M.K.H.
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A patent related to this work has been filed with: Applicant: STICHTING RADBOUD UNIVERSITEIT. Inventors: Maike Hansen, Willem Velema. Application Number: NL2025/150006. Status of the application: PCT application filed Specific aspects of the manuscript covered in the patent application: Probe for single cell ribosome imaging and tracking; RiboBright probe. Application claims priority from: NL 2039232 (NL application). All other authors declare no competing interests.
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Poulladofonou, G., Grandi, C., Hu, X. et al. RiboBright reveals cell-type-specific differences in ribosome organization and movement. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68947-7
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DOI: https://doi.org/10.1038/s41467-026-68947-7
