Use of Dendra2 to monitor degradation of the transcription factor Nrf2 in promyelocytic leukemia‐nuclear bodies in living cells (594.1)

Using a cellular fractionation approach, we have shown that the transcription factor Nrf2 can be degraded in the nucleus [Malloy et al. (2013) J. Biol. Chem. 288, 14569‐14583]. The use of photoconvertible proteins, which undergo irreversible change in color upon photoactivation, is a novel approach to monitor real‐time protein movement at the single cell level. In order to use this approach to assess degradation of Nrf2 within the nuclear compartment, we cloned Nrf2 into a plasmid expressing Dendra2, a photoconvertible protein derived from the soft coral species Dendronephthya. When expressed in cells, Dendra2 is a green‐colored fluorescent protein. Upon exposure to a high intensity laser pulse at 405 or 488 nm, its color changes irreversibly from green to red. Photoconversion of the Nrf2‐Dendra2 fusion protein within a region of interest such as within PML‐NB domains facilitates tracking the fate of the fusion protein via time‐lapse imaging. To locate such domains, we also constructed a plasmid expressing the fusion protein Crimson‐PML‐I to identify co‐localization of PML and Nrf2‐Dendra2 within PML‐NB domains. By measuring fluorescence intensity of Nrf2‐Dendra2 in PML‐NBs, we can determine the involvement of these nuclear bodies in the degradation of Nrf2 in the nucleus. Grant Funding Source : Supported by NIH grants SC1CA143985, T32GM007628‐36 and by CTSA award No. UL1TR000445