ORCID

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 SCFβ-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.

Publication Date

2025-08-14

Publication Title

Scientific Reports

Volume

15

Issue

1

ISSN

2045-2322

Acceptance Date

2025-08-01

Deposit Date

2025-11-13

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