Dikovskaya Dina, Bento-Pereira Claudia, Shiga Kanade, Corno Andrea, Higgins Maureen, Toth Rachel, Saurin Adrian T, Dinkova-Kostova Albena T
Division of Cellular Medicine, University of Dundee School of Medicine, Dundee, DD1 9SY, UK.
MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, DD1 5EH, UK.
Sci Rep. 2025 Aug 14;15(1):29772. doi: 10.1038/s41598-025-14721-6.
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-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.
荧光定时器（FT）极大地助力了细胞内蛋白质周转的测量。然而，FT定量需要相对高荧光强度的样本，这使得它们无法用于表达水平低或不均匀的蛋白质，如氧化还原稳态的主要调节因子转录因子Nrf2。为了可视化Nrf2稳定性/周转的变化，我们构建了一个结合了超折叠绿色荧光蛋白（sfGFP）和单体红色荧光蛋白（mCherry）的基因编码标签，并使用强度无关的荧光寿命成像（FLIM）来测量标签内的荧光共振能量转移（FRET）（命名为FLIM定时器）。我们发现，随着蛋白质成熟，mCherry作为FRET受体的能力逐渐发展，这使得FLIM-FRET能够作为FLIM定时器的读数。带有FLIM定时器标签的Nrf2能够在高亮度和低亮度区域以相同的精度观察其在细胞区室之间周转的差异。FLIM定时器-Nrf2荧光寿命的缩短证实了萝卜硫素对其的稳定作用。去除Keap1-Cul3或SCF介导降解的降解结构域会降低Nrf2-FLIM定时器的荧光寿命。FLIM定时器标记的细胞周期蛋白B也成功用于追踪其在有丝分裂退出期间的不稳定过程。因此，FLIM定时器方法提高了FT在蛋白质周转可视化和定量方面的适用性，将其扩展到任何感兴趣的蛋白质表达水平低且变化的细胞。
