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Updated: Dec 12, 2025
Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques
Published on: November 2, 2018
Measuring Changes in Keap1-Nrf2 Protein Complex Conformation in Individual Cells by FLIM-FRET
Dina Dikovskaya1, Albena T Dinkova-Kostova1,2
1Jacqui Wood Cancer Centre, Division of Cellular Medicine, School of Medicine, University of Dundee, Dundee, United Kingdom.
Abstract:
The nuclear factor-erythroid 2 p45-related factor 2 (Nrf2)-mediated stress response is a major cellular defense mechanism against endogenous and exogenous oxidants, electrophiles, and pro-inflammatory agents. A number of Nrf2 inducers are being developed to therapeutically stimulate this pathway. Inducers are typically sensed by Kelch-like ECH-associated protein 1 (Keap1), a negative regulator and a binding partner of Nrf2. Modifications of Keap1 by oxidants or electrophiles, or its targeting by compounds that disrupt its interaction with Nrf2, alter the conformation of the Keap1-Nrf2 protein complex, which initiates the accumulation of Nrf2 required for mounting a stress response. To detect conformational changes in the Keap1-Nrf2 complex in live cells, we have developed a procedure based on Fluorescence Lifetime Imaging-Förster Resonance Energy Transfer (FLIM-FRET). The procedure includes a FLIM time course in cells expressing fluorescently-tagged Nrf2 and Keap1, followed by an extended analysis pipeline that quantifies changes in fluorescence lifetime of labeled Nrf2. The analysis visualizes and removes intensity-dependent bias in fluorescence lifetime measured with the Time-Correlated Single Photon Counting (TCSPC) approach, thereby improving the accuracy of quantification. The throughput is increased by the whole-experiment analysis within the newly developed FLIM dataset tool (FLIMDAST) and by the time-lapse FLIM described here. This pipeline is also suitable for applications beyond the Nrf2 field that assess small changes in fluorescence lifetime of objects with variable fluorescence intensities measured using TCSPC-based FLIM. © 2020 The Authors. Basic Protocol 1: Lipofectamine 2000 transfection Alternate Protocol 1: Calcium phosphate transfection Basic Protocol 2: Time course with individual FLIM Alternate Protocol 2: Time course with time-lapse FLIM Support Protocol: Measuring Instrument Response Function (IRF) Basic Protocol 3: Data analysis in SPCImage Basic Protocol 4: Data processing in ImageJ/FIJI Basic Protocol 5: Experiment analysis in FLIMDAST.
Insights
Researchers developed a new Fluorescence Lifetime Imaging-Förster Resonance Energy Transfer (FLIM-FRET) method to detect conformational changes in the Keap1-Nrf2 complex. This technique enhances the accuracy and throughput for studying cellular stress responses and Nrf2 inducers.
Area of Science:
- Cellular Biology
- Biochemistry
- Molecular Biology
Background:
- The nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) pathway is a critical cellular defense mechanism against oxidative and inflammatory stress.
- Kelch-like ECH-associated protein 1 (Keap1) negatively regulates Nrf2. Disrupting the Keap1-Nrf2 interaction triggers Nrf2 accumulation and the stress response.
- Current methods for detecting these conformational changes in live cells are limited.
Purpose of the Study:
- To develop and validate a novel Fluorescence Lifetime Imaging-Förster Resonance Energy Transfer (FLIM-FRET) procedure to detect conformational changes in the Keap1-Nrf2 complex in live cells.
- To improve the accuracy and throughput of measurements assessing cellular stress responses.
- To provide a versatile pipeline applicable to other biological systems studying fluorescence lifetime changes.
Main Methods:
- Development of a FLIM-FRET based procedure involving fluorescently tagged Nrf2 and Keap1.
- Implementation of a time-course FLIM acquisition, including time-lapse imaging.
- Utilized an extended analysis pipeline incorporating FLIM dataset tool (FLIMDAST) to quantify Nrf2 fluorescence lifetime changes, correcting for intensity-dependent bias and improving throughput.
Main Results:
- Successfully established a FLIM-FRET based method for live-cell detection of Keap1-Nrf2 complex conformational alterations.
- The developed analysis pipeline accurately quantifies fluorescence lifetime changes, mitigating intensity-dependent bias inherent in Time-Correlated Single Photon Counting (TCSPC) measurements.
- The FLIMDAST tool and time-lapse FLIM significantly increased experimental throughput.
Conclusions:
- The novel FLIM-FRET procedure offers a sensitive and efficient approach to monitor Keap1-Nrf2 complex dynamics in real-time.
- This methodology enhances the study of Nrf2 pathway activation and the development of Nrf2-inducing therapeutics.
- The pipeline's adaptability makes it valuable for diverse research areas involving TCSPC-based FLIM analysis of dynamic biological processes.
