Fluorescent Tools for In Vivo Studies on the Ubiquitin-Proteasome System.
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.
Research Programs Unit, Translational Cancer Biology, University of Helsinki, Haartmaninkatu 8, 63, Helsinki, 00014, Finland.
Olli
O
Matilainen
Research Programs Unit, Translational Cancer Biology, University of Helsinki, Haartmaninkatu 8, 63, Helsinki, 00014, Finland.
Sweta
S
Jha
Research Programs Unit, Translational Cancer Biology, University of Helsinki, Haartmaninkatu 8, 63, Helsinki, 00014, Finland. carina.holmberg@helsinki.fi.
Carina I
CI
Holmberg
D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D000074702:Proteostasis / Q000235:genetics / Q000502:physiology; D025801:Ubiquitin / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex
C. elegans; Live imaging; Photoconversion; Polyubiquitin-binding reporter; Proteostasis; UbG76V-Dendra2; Ubiquitin-proteasome system (UPS)