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

## TL;DR

A new FLIM-timer method allows measuring protein turnover of low-expression proteins like Nrf2 in live cells using fluorescence lifetime imaging.

## Contribution

A genetically encoded FLIM-timer tag enables intensity-independent measurement of protein turnover for low-abundance proteins.

## Key findings

- FLIM-timer accurately measures Nrf2 turnover in different cellular compartments with equal precision.
- FLIM-timer confirmed Nrf2 stabilization by sulforaphane and destabilization during mitotic exit in cyclin B.
- The FLIM-timer expands fluorescent timer applicability to proteins with low or variable expression.

## 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.

The online version contains supplementary material available at 10.1038/s41598-025-14721-6.