Measuring Changes in Keap1‐Nrf2 Protein Complex Conformation in Individual Cells by FLIM‐FRET

TY - JOUR

T1 - Measuring Changes in Keap1‐Nrf2 Protein Complex Conformation in Individual Cells by FLIM‐FRET

AU - Dikovskaya, Dina

AU - Dinkova-Kostova, Albena

N1 - Funding Information: We thank Paul Appleton and Iain Porter (University of Dundee Imaging Facility) for expert technical help, and Vladislav Shcheslavskiy (Becker‐Hickl GmbH) for help with time‐lapse FLIM setup, Marek Gierlinski (University of Dundee School of Life Sciences) and Graeme Ball (University of Dundee Imaging Facility) for advice on the data analysis algorithm and software publishing, and the Biotechnology and Biological Sciences Research Council (BB/L01923X/1), Tenovus Scotland (T17/14), and Cancer Research UK (C20953/A18644) for financial support. Publisher Copyright: © 2020 The Authors.

PY - 2020/9

Y1 - 2020/9

N2 - 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.

AB - 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.

KW - Nrf2

KW - Keap1

KW - time-lapse FLIM

KW - image analysis

UR - https://www.scopus.com/pages/publications/85089408125

U2 - 10.1002/cptx.96

DO - 10.1002/cptx.96

M3 - Article

C2 - 32786061

SN - 1934-9262

VL - 85

JO - Current Protocols in Toxicology

JF - Current Protocols in Toxicology

IS - 1

M1 - e96

ER -