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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
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.
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.
