PUBLICATION
FGCaMP7, an Improved Version of Fungi-Based Ratiometric Calcium Indicator for In Vivo Visualization of Neuronal Activity
- Authors
- Barykina, N.V., Sotskov, V.P., Gruzdeva, A.M., Wu, Y.K., Portugues, R., Subach, O.M., Chefanova, E.S., Plusnin, V.V., Ivashkina, O.I., Anokhin, K.V., Vlaskina, A.V., Korzhenevskiy, D.A., Nikolaeva, A.Y., Boyko, K.M., Rakitina, T.V., Varizhuk, A.M., Pozmogova, G.E., Subach, F.V.
- ID
- ZDB-PUB-200430-2
- Date
- 2020
- Source
- International Journal of Molecular Sciences 21(8): (Journal)
- Registered Authors
- Keywords
- FGCaMP, FGCaMP7, calcium imaging, crystal structure, genetically encoded calcium indicator, protein engineering
- MeSH Terms
-
- Humans
- Action Potentials
- Visual Cortex/physiology
- Molecular Imaging*/methods
- Genes, Reporter
- Fungi/genetics
- Protein Engineering
- Mice
- Animals
- Calcium/metabolism*
- Models, Molecular
- Protein Conformation
- Gene Expression*
- Neurons/cytology
- Neurons/metabolism*
- HeLa Cells
- Calcium-Binding Proteins/genetics
- Calcium-Binding Proteins/metabolism
- Microscopy, Fluorescence
- Structure-Activity Relationship
- PubMed
- 32344594 Full text @ Int. J. Mol. Sci.
Citation
Barykina, N.V., Sotskov, V.P., Gruzdeva, A.M., Wu, Y.K., Portugues, R., Subach, O.M., Chefanova, E.S., Plusnin, V.V., Ivashkina, O.I., Anokhin, K.V., Vlaskina, A.V., Korzhenevskiy, D.A., Nikolaeva, A.Y., Boyko, K.M., Rakitina, T.V., Varizhuk, A.M., Pozmogova, G.E., Subach, F.V. (2020) FGCaMP7, an Improved Version of Fungi-Based Ratiometric Calcium Indicator for In Vivo Visualization of Neuronal Activity. International Journal of Molecular Sciences. 21(8):.
Abstract
Genetically encoded calcium indicators (GECIs) have become a widespread tool for the visualization of neuronal activity. As compared to popular GCaMP GECIs, the FGCaMP indicator benefits from calmodulin and M13-peptide from the fungi Aspergillus niger and Aspergillus fumigatus, which prevent its interaction with the intracellular environment. However, FGCaMP exhibits a two-phase fluorescence behavior with the variation of calcium ion concentration, has moderate sensitivity in neurons (as compared to the GCaMP6s indicator), and has not been fully characterized in vitro and in vivo. To address these limitations, we developed an enhanced version of FGCaMP, called FGCaMP7. FGCaMP7 preserves the ratiometric phenotype of FGCaMP, with a 3.1-fold larger ratiometric dynamic range in vitro. FGCaMP7 demonstrates 2.7- and 8.7-fold greater photostability compared to mEGFP and mTagBFP2 fluorescent proteins in vitro, respectively. The ratiometric response of FGCaMP7 is 1.6- and 1.4-fold higher, compared to the intensiometric response of GCaMP6s, in non-stimulated and stimulated neuronal cultures, respectively. We reveal the inertness of FGCaMP7 to the intracellular environment of HeLa cells using its truncated version with a deleted M13-like peptide; in contrast to the similarly truncated variant of GCaMP6s. We characterize the crystal structure of the parental FGCaMP indicator. Finally, we test the in vivo performance of FGCaMP7 in mouse brain using a two-photon microscope and an NVista miniscope; and in zebrafish using two-color ratiometric confocal imaging.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping