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A photoconvertible reporter of the ubiquitin-proteasome system in vivo

Nature Methods volume 7, pages 473–478 (2010)Cite this article

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A Corrigendum to this article was published on 01 July 2010

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Abstract

The ubiquitin-proteasome system (UPS) orchestrates many cellular and tissue-specific processes by degrading damaged and key regulatory proteins. To enable investigation of UPS activity in different cell types in a living animal, we developed a photoconvertible fluorescent UPS reporter system for live imaging and quantification of protein degradation in Caenorhabditis elegans. Our reporter consists of the photoconvertible fluorescent protein Dendra2 targeted for proteasomal degradation by fusion to the UbG76V mutant form of ubiquitin. In contrast to previous reporters, this system permits quantification of UPS activity independently of protein synthesis. Our reporter revealed that UPS-mediated protein degradation varies in a cell type–specific and age-dependent manner in C. elegans.

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Figure 1: UPS-mediated protein degradation in body-wall muscle cells.
Figure 2: Knockdown of the proteasome decreases degradation of the UPS reporter proteins.
Figure 3: UPS activity in GABAergic dorsorectal neurons.
Figure 4: UPS activity in dopaminergic neurons.
Figure 5: Aging decreases UPS activity in dorsorectal neurons but not in body-wall muscle cells.

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  • 17 June 2010

    In the version of this paper originally published, a reference to previous work on the use of Dendra2 as a reporter for protein stability in cultured cells should have been included (ref. 35). The error has been corrected in the PDF and HTML versions of the article.

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Acknowledgements

This study was supported by grants to C.I.H. from the Academy of Finland (113485 and 118450), the International Human Frontier Science Program Organization, Biocentrum Helsinki, the Sigrid Jusélius Foundation and the Magnus Ehrnrooth Foundation. G.H. was additionally supported by the University of Helsinki and O.M. by the Helsinki Biomedical Graduate School. We thank members of the Caenorhabditis Genetics Center for providing the N2 wild-type worms, S. Mitani (National Bioresource Project for the Nematode, Japan) for the rpn-10 mutant worms, R.I. Morimoto (Northwestern University) for the PF25B3.3::yfp expression vector and members of the Biomedicum Helsinki Molecular Imaging Unit for their help with confocal microscopy and imaging.

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Author notes

  1. Geert Hamer and Olli Matilainen: These authors contributed equally to this work.

Authors and Affiliations

  1. Molecular Cancer Biology Program and Institute for Biomedicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland

    Geert Hamer, Olli Matilainen & Carina I Holmberg

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  1. Geert Hamer
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Contributions

G.H. and O.M. developed the method, performed the experiments, analyzed the data, made the figures and wrote the paper. C.I.H. developed the method, analyzed the data, wrote the paper and supported the project.

Corresponding author

Correspondence to Carina I Holmberg.

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The authors declare no competing financial interests.

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Supplementary Figures 1–9 and Supplementary Tables 1–2 (PDF 2945 kb)

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Hamer, G., Matilainen, O. & Holmberg, C. A photoconvertible reporter of the ubiquitin-proteasome system in vivo. Nat Methods 7, 473–478 (2010). https://doi.org/10.1038/nmeth.1460

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