ArticleScientific reports2025
A fluorescence lifetime-based FLIM-timer for measuring the protein turnover of transcription factor Nrf2 in live cells.
Article in Scientific reports, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Measuring protein turnover in cells has been greatly assisted by fluorescent timers (FT). However, FT quantification requires relatively high fluorescence intensity samples, prohibiting their use for proteins with low or non-uniform expression like transcription factor Nrf2, the master regulator of redox homeostasis. To visualise changes in stability/turnover of Nrf2, we constructed a genetically encoded tag combining sfGFP and mCherry and used intensity-independent Fluorescence Lifetime Imaging (FLIM) to measure Förster Resonance Energy Transfer (FRET) within the tag (named FLIM-timer). We show that the ability of mCherry to act as a FRET-acceptor develops as the protein matures, allowing the use of FLIM-FRET as a readout of the FLIM-timer. FLIM-timer-tagged Nrf2 allowed to observe differences in its turnover between cellular compartments with equal precision in regions of high and low brightness. The reduction in fluorescence lifetime of FLIM-timer-Nrf2 confirmed its stabilisation by sulforaphane. Depletion of a degron for either Keap1-Cul3 or SCF
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