Abstract
The ubiquitin-proteasome system (UPS) plays a key role in maintaining proteostasis by degrading most of the cellular proteins. Traditionally, UPS activity is studied in vitro, in yeast, or in mammalian cell cultures by using short-lived GFP-based UPS reporters. Here, we present protocols for two fluorescent tools facilitating real-time imaging of UPS activity in living animals. We have generated transgenic Caenorhabditis elegans (C. elegans) expressing a photoconvertible UbG76V-Dendra2 UPS reporter, which permits measurement of reporter degradation by the proteasome independently of reporter protein synthesis, and a fluorescent polyubiquitin-binding reporter for detection of the endogenous pool of Lys48-linked polyubiquitinated proteasomal substrates. These reporter systems facilitate cell- and tissue-specific analysis of UPS activity especially in young adult animals, but can also be used for studies during development, aging, and for example stress conditions.
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Acknowledgement
We thank the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440), for strains and the Biomedicum Imaging Unit, University of Helsinki, for imaging services. This work was supported by grants to C.I.H. from the Academy of Finland (131408 and 259797), University of Helsinki Funds, International Human Frontier Science Program Foundation, and the Sigrid Juselius Foundation. The authors would like to acknowledge networking support by the Proteostasis COST Action (BM1307).
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Matilainen, O., Jha, S., Holmberg, C.I. (2016). Fluorescent Tools for In Vivo Studies on the Ubiquitin-Proteasome System. In: Matthiesen, R. (eds) Proteostasis. Methods in Molecular Biology, vol 1449. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3756-1_12
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DOI: https://doi.org/10.1007/978-1-4939-3756-1_12
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Online ISBN: 978-1-4939-3756-1
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