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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
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Measuring Changes in Keap1-Nrf2 Protein Complex Conformation in Individual Cells by FLIM-FRET.

Dina Dikovskaya1, Albena T Dinkova-Kostova1,2

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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.

Keywords:
Keap1Nrf2image analysistime-lapse FLIM

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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.