|Title||A genetically encoded Ca indicator based on circularly permutated sea anemone red fluorescent protein eqFP578.|
|Publication Type||Journal Article|
|Year of Publication||2018|
|Authors||Shen, Yi, Hod Dana, Ahmed S. Abdelfattah, Ronak Patel, Jamien Shea, Rosana S. Molina, Bijal Rawal, Vladimir Rancic, Yu-Fen Chang, Lanshi Wu, Yingche Chen, Yong Qian, Matthew D. Wiens, Nathan Hambleton, Klaus Ballanyi, Thomas E. Hughes, Mikhail Drobizhev, Douglas S. Kim, Minoru Koyama, Eric R. Schreiter, and Robert E. Campbell|
|Date Published||2018 Jan 16|
BACKGROUND: Genetically encoded calcium ion (Ca) indicators (GECIs) are indispensable tools for measuring Ca dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs have inherent advantages relative to green fluorescent protein-based GECIs due to the longer wavelength light used for excitation. Longer wavelength light is associated with decreased phototoxicity and deeper penetration through tissue. Red GECI can also enable multicolor visualization with blue- or cyan-excitable fluorophores.
RESULTS: Here we report the development, structure, and validation of a new RFP-based GECI, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We have characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem-cell-derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo.
CONCLUSION: K-GECO1 is the archetype of a new lineage of GECIs based on the RFP eqFP578 scaffold. It offers high sensitivity and fast kinetics, similar or better than those of current state-of-the-art indicators, with diminished lysosomal accumulation and minimal blue-light photoactivation. Further refinements of the K-GECO1 lineage could lead to further improved variants with overall performance that exceeds that of the most highly optimized red GECIs.
|Alternate Journal||BMC Biol.|
|PubMed Central ID||PMC5771076|
|Grant List||RGPIN 288338-2010 / / Natural Sciences and Engineering Research Council of Canada / |
UO1 NS090565 / / National Institutes of Health /
U01 NS094246 / NS / NINDS NIH HHS / United States
RGPIN-2014-06484 / / Natural Sciences and Engineering Research Council of Canada /
U01 NS094246 / / National Institutes of Health /
U01 NS090565 / NS / NINDS NIH HHS / United States
MOP-123514 / / Canadian Institutes of Health Research / Canada