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Extracellular Ca2+-Sensing Fluorescent Protein Biosensor Based on a Collagen-Binding Domain.

Irina A Okkelman1, Ryan McGarrigle2, Shane O'Carroll1

  • 1Metabolic Imaging Group, Laboratory of Biophysics and Bioanalysis, ABCRF, University College Cork, College Road, Cork T12 YN60, Ireland.

ACS Applied Bio Materials
|January 13, 2022
PubMed
Summary

Researchers developed a novel calcium (Ca2+) biosensor using Twitch-2B fluorescent protein. This scaffold-based sensor enables real-time Ca2+ monitoring in 3D tissue models, aiding tissue development and regeneration studies.

Keywords:
biosensorcalciumextracellular matrixfluorescence lifetime imaging microscopy (FLIM)intestinal organoids

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Area of Science:

  • Biomolecular Engineering
  • Cellular Biology
  • Biophysics

Background:

  • Extracellular gradients of biomolecules are crucial for tissue development and regeneration.
  • Understanding extracellular calcium (Ca2+) dynamics is vital but challenging.
  • Existing Ca2+ sensors have limitations in complex 3D tissue environments.

Purpose of the Study:

  • To develop a novel Ca2+ biosensor for live 3D tissue models.
  • To enable scaffold-based sensing of extracellular Ca2+ dynamics.
  • To investigate Ca2+ responses in intestinal organoids.

Main Methods:

  • Engineered a low-affinity Ca2+ biosensor (Twitch-2B) fused with cellulose- and collagen-binding peptides.
  • Utilized fluorescence lifetime imaging microscopy (FLIM) for Ca2+ measurements.
  • Applied the biosensor to live Lgr5-GFP mouse intestinal organoids and Nile red-stained lipid droplets.

Main Results:

  • The Twitch-2B biosensor successfully binds to collagen and cellulose scaffolds for localized Ca2+ sensing.
  • The biosensor is compatible with intensity-based and FLIM measurements under various imaging modes.
  • Extracellular Ca2+ depletion induced changes in lipid droplet composition within intestinal epithelial cells.

Conclusions:

  • Twitch-2B is a promising Ca2+ sensor for scaffold-based, multiplexed FLIM analysis in complex 3D tissue models.
  • The developed biosensor facilitates the study of extracellular Ca2+ dynamics in physiological and pathological processes.
  • This technology offers new insights into cellular responses to Ca2+ fluctuations in the intestinal epithelium.