Monitoring the distribution and dynamics of proteasomes in living cells.
The proteasome is a large protease complex present in the cytoplasm and the nucleus of eukaryotic cells. This chapter describes how proteasomes in living cells can be visualized using fluorescently tagged subunits. The use of noninvasive fluorescent tags like the green fluorescent protein enables visualization of various subunits of the ubiquitin-proteasome system and prevents possible artefacts like disruption by microinjection or altered fluorescence distribution caused by fixation. Once quantitative incorporation of tagged subunits into proteasomes is ensured, the distribution of proteasome complexes can be visualized in vivo. In addition, different bleaching techniques can be applied to study the dynamics of proteasomes within the cell. Finally, we describe how proteasomes can be recruited to particular sites of degradation during various cellular conditions like aggregate formation and virus infection.
Groothuis
Tom A M
TA
Department of Tumor Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands.
Reits
Eric A J
EA
D045744:Cell Line, Tumor; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*
D049452:Green Fluorescent Proteins; D046988:Proteasome Endopeptidase Complex