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Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor
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MagFRET: the first genetically encoded fluorescent Mg2+ sensor.

Laurens H Lindenburg1, Jan L Vinkenborg, Jorn Oortwijn

  • 1Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands.

Plos One
|December 7, 2013
PubMed
Summary

Researchers developed MagFRET-1, the first genetically encoded fluorescent sensor for real-time imaging of magnesium ions (Mg2+) in cells. This tool offers a new way to study cellular magnesium homeostasis and signaling dynamics.

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Imaging

Background:

  • Magnesium ions (Mg2+) play crucial roles in cellular processes, including structural, catalytic, and signaling functions.
  • Existing tools for real-time, subcellular imaging of intracellular Mg2+ are limited, hindering research into Mg2+ homeostasis and signaling.

Purpose of the Study:

  • To develop and characterize the first genetically encoded fluorescent sensor for real-time imaging of intracellular Mg2+.
  • To create a sensor with physiologically relevant Mg2+ affinity and a measurable response to Mg2+ binding.

Main Methods:

  • Genetically encoded sensor (MagFRET-1) construction by fusing Cerulean and Citrine fluorescent proteins to the Mg2+-binding domain of human centrin 3 (HsCen3).
  • Characterization of Mg2+ binding affinity (Kd) and FRET efficiency changes upon Mg2+ binding.
  • Development of MagFRET variants with altered Mg2+ affinities.
  • In situ experiments in HEK293 cells to assess sensor localization and response to Mg2+ concentration changes.

Main Results:

  • MagFRET-1 exhibits a physiologically relevant Mg2+ affinity (Kd = 148 µM) and a 50% increase in emission ratio upon Mg2+ binding.
  • MagFRET variants with 2- to 100-fold attenuated Mg2+ affinities were generated, covering a wider range of Mg2+ concentrations.
  • The sensor can be targeted to the cytosol and nucleus in HEK293 cells.
  • Demonstrated responsiveness to extracellular Mg2+ changes in permeabilized cells, with minimal cross-reactivity to Ca2+.

Conclusions:

  • MagFRET-1 represents the first genetically encoded, ratiometric fluorescent sensor for Mg2+ imaging.
  • These sensors offer a promising tool for investigating intracellular Mg2+ homeostasis and signaling.
  • Further studies are needed to fully establish the potential and limitations of MagFRET sensors for live-cell imaging.