FGCaMP7, an Improved Version of Fungi-Based Ratiometric Calcium Indicator for In Vivo Visualization of Neuronal Activity
by
Natalia V. Barykina 1
, Vladimir P. Sotskov 2, Anna M. Gruzdeva 2,3,4, You Kure Wu 4, Ruben Portugues 4,5,6, Oksana M. Subach 3, Elizaveta S. Chefanova 7, Viktor V. Plusnin 3,7, Olga I. Ivashkina 1,2,3, Konstantin V. Anokhin 1,2, Anna V. Vlaskina 3, Dmitry A. Korzhenevskiy 3, Alena Y. Nikolaeva 3, Konstantin M. Boyko 8, Tatiana V. Rakitina 3,9, Anna M. Varizhuk 10,11, Galina E. Pozmogova 10,11 and Fedor V. Subach 3,*
Natalia V. Barykina 1, Vladimir P. Sotskov 2, Anna M. Gruzdeva 2,3,4, You Kure Wu 4, Ruben Portugues 4,5,6, Oksana M. Subach 3, Elizaveta S. Chefanova 7, Viktor V. Plusnin 3,7, Olga I. Ivashkina 1,2,3, Konstantin V. Anokhin 1,2, Anna V. Vlaskina 3, Dmitry A. Korzhenevskiy 3, Alena Y. Nikolaeva 3, Konstantin M. Boyko 8, Tatiana V. Rakitina 3,9, Anna M. Varizhuk 10,11, Galina E. Pozmogova 10,11 and Fedor V. Subach 3,*
1
Laboratory for Neurobiology of Memory, P.K. Anokhin Research Institute of Normal Physiology, 125315 Moscow, Russia
2
Institute for Advanced Brain Studies, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia
3
Complex of NBICS Technologies, National Research Center “Kurchatov Institute”, 123182 Moscow, Russia
4
Sensorimotor Control Research Group, Max Planck Institute of Neurobiology, 82152 Martinsried, Germany
5
Institute of Neuroscience, Technical University of Munich, 80802 Munich, Germany
6
Munich Cluster for Systems Neurology (SyNergy), Munich, Germany
7
Department of NBIC-technologies, Moscow Institute of Physics and Technology, 123182 Moscow, Russia
8
Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia
9
Laboratory of Hormonal Regulation Proteins, M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, 117997 Moscow, Russia
10
Department of Biophysics, Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 119435 Moscow, Russia
11
Department of Biophysics, Center for Precision Genome Editing and Genetic Technologies for Biomedicine, 119435 Moscow, Russia
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(8), 3012; https://doi.org/10.3390/ijms21083012
Received: 12 April 2020 / Revised: 22 April 2020 / Accepted: 23 April 2020 / Published: 24 April 2020
(This article belongs to the Special Issue Advanced Fluorescent Probes for Structural and Functional Imaging of Biological Systems)
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.
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Keywords:
calcium imaging; genetically encoded calcium indicator; protein engineering; crystal structure; FGCaMP7; FGCaMP
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited
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MDPI and ACS Style
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. FGCaMP7, an Improved Version of Fungi-Based Ratiometric Calcium Indicator for In Vivo Visualization of Neuronal Activity. Int. J. Mol. Sci. 2020, 21, 3012.
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