A fluorescence lifetime-based FLIM-timer for measuring the protein turnover of transcription factor Nrf2 in live cells

TY - JOUR

T1 - A fluorescence lifetime-based FLIM-timer for measuring the protein turnover of transcription factor Nrf2 in live cells

AU - Dikovskaya, Dina

AU - Bento-Pereira, Claudia

AU - Shiga, Kanade

AU - Corno, Andrea

AU - Higgins, Maureen

AU - Toth, Rachel

AU - Saurin, Adrian T

AU - Dinkova-Kostova, Albena

N1 - Copyright: © The Author(s) 2025.

PY - 2025/8/14

Y1 - 2025/8/14

N2 - 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 β-TrCP-mediated degradation decreased the fluorescence lifetime of Nrf2-FLIM-timer. FLIM-timer labelled cyclin B was also successfully used to track its destabilisation during mitotic exit. Thus, FLIM-timer methodology increases the FT applicability for visualisation and quantification of protein turnover, expanding it to cells with low and variable levels of any protein of interest.

AB - 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 β-TrCP-mediated degradation decreased the fluorescence lifetime of Nrf2-FLIM-timer. FLIM-timer labelled cyclin B was also successfully used to track its destabilisation during mitotic exit. Thus, FLIM-timer methodology increases the FT applicability for visualisation and quantification of protein turnover, expanding it to cells with low and variable levels of any protein of interest.

UR - https://www.scopus.com/pages/publications/105013463208

U2 - 10.1038/s41598-025-14721-6

DO - 10.1038/s41598-025-14721-6

M3 - Article

C2 - 40804104

SN - 2045-2322

VL - 15

JO - Scientific Reports

JF - Scientific Reports

M1 - 29772

ER -