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.
Highlights
The purpose of this article is to provide a detailed protocol for measuring changes in the protein-protein interactions between Nrf2 and Kelch-like ECH-associated protein 1 (Keap1) in live cells using time-resolved Fluorescence Lifetime Imaging (FLIM).This procedure monitors a crucial early step in the initiation of the oxidative stress response transcriptional program mediated by transcription factor nuclear factor−erythroid 2 p45-related factor 2 (Nrf2)
The state of the Keap1-Nrf2 complex can be observed in live cells co-transfected with GFP-labeled Nrf2 and mCherry-labeled Keap1 by assessing Förster Resonance Energy Transfer (FRET) between the fluorescent tags (Baird, Swift, Lleres, & Dinkova-Kostova, 2014)
We exploited the inbuilt capability of one such commonly used commercial instrument assembly, the Zeiss LSM 710 microscope equipped with the Becker & Hickl TCSPC module, to Current Protocols in Toxicology establish a procedure for automatic time-lapse FLIM across multiple stage positions, which we describe here
Summary
The purpose of this article is to provide a detailed protocol for measuring changes in the protein-protein interactions between Nrf and Keap in live cells using time-resolved Fluorescence Lifetime Imaging (FLIM). For sample data (images), please see Figure 2 This protocol describes measurements of fluorescence lifetime using a TCSPC-based approach that works by recording the exact time of emission of individual photons after fluorophores are excited by pulsed laser illumination (Becker, 2015). Reduce laser intensity and press “reset” on the DCC-100 panel
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