The importance of extracellular gradients of biomolecules becomes increasingly appreciated in the processes of tissue development and regeneration, in health and disease. In particular, dynamics of extracellular calcium concentration is rarely studied. Here, we present low affinity Ca2+ biosensor based on Twitch-2B fluorescent protein fused with the cellulose- and collagen-binding peptides. These recombinant chimeric proteins can bind cellulose and collagen scaffolds and enable for scaffold-based biosensing of Ca2+ in proximity of live 3D tissue models. We found that the Twitch-2B mutant is compatible with intensity-based ratiometric and fluorescence lifetime imaging microscopy (FLIM) measurement formats, und... More
The importance of extracellular gradients of biomolecules becomes increasingly appreciated in the processes of tissue development and regeneration, in health and disease. In particular, dynamics of extracellular calcium concentration is rarely studied. Here, we present low affinity Ca2+ biosensor based on Twitch-2B fluorescent protein fused with the cellulose- and collagen-binding peptides. These recombinant chimeric proteins can bind cellulose and collagen scaffolds and enable for scaffold-based biosensing of Ca2+ in proximity of live 3D tissue models. We found that the Twitch-2B mutant is compatible with intensity-based ratiometric and fluorescence lifetime imaging microscopy (FLIM) measurement formats, under one- and two-photon excitation modes. Furthermore, the donor fluorescence lifetime of ColBD-Twitch displays response to [Ca2+] over a range of ∼2-2.5 ns, making it attractive biosensor for multiplexed FLIM microscopy assays. To evaluate performance of this biosensor in physiological measurements, we applied ColBD-Twitch to the live Lgr5-GFP mouse intestinal organoid culture and measured its responses to the changes in extracellular Ca2+ upon chelation with EGTA. When we combined it with spectrally resolved FLIM of lipid droplets using Nile Red dye, we observed changes in cytoplasmic and basal membrane-associated lipid droplet composition in response to the extracellular Ca2+ depletion, suggesting that intestinal epithelium can respond to and compensate such treatment. Altogether, our results demonstrate ColBD-Twitch as a prospective Ca2+ sensor for multiplexed FLIM analysis in a complex 3D tissue environment.
