A new design for a green calcium indicator with a smaller size and a reduced number of calcium-binding sites.
Genetically encoded calcium indicators (GECIs) are mainly represented by two- or one-fluorophore-based sensors. One type of two-fluorophore-based sensor, carrying Opsanus troponin C (TnC) as the Ca2+-binding moiety, has two binding sites for calcium ions, providing a linear response to calcium ions. One-fluorophore-based sensors have four Ca2+-binding sites but are better suited for in vivo experiments. Herein, we describe a novel design for a one-fluorophore-based GECI with two Ca2+-binding sites. The engineered sensor, called NTnC, uses TnC as the Ca2+-binding moiety, inserted in the mNeonGreen fluorescent protein. Monomeric NTnC has higher brightness and pH-stability in vitro compared with the standard GECI GCaMP6s. In addition, NTnC shows an inverted fluorescence response to Ca2+. Using NTnC, we have visualized Ca2+dynamics during spontaneous activity of neuronal cultures as confirmed by control NTnC and its mutant, in which the affinity to Ca2+is eliminated. Using whole-cell patch clamp, we have demonstrated that NTnC dynamics in neurons are similar to those of GCaMP6s and allow robust detection of single action potentials. Finally, we have used NTnC to visualize Ca2+neuronal activity in vivo in the V1 cortical area in awake and freely moving mice using two-photon microscopy or an nVista miniaturized microscope.
NBICS Department, Moscow Institute of Physics and Technology, Moscow 123182, Russia.|Department of Systems Neurobiology and Functional Neurochemistry, P.K. Anokhin Institute of Normal Physiology of RAMS, Moscow 125009, Russia.
Natalia V
NV
Barykina
NBICS Department, Moscow Institute of Physics and Technology, Moscow 123182, Russia.|National Research Center "Kurchatov Institute", Moscow 123182, Russia.
Oksana M
OM
Subach
NBICS Department, Moscow Institute of Physics and Technology, Moscow 123182, Russia.|National Research Center "Kurchatov Institute", Moscow 123182, Russia.
Danila A
DA
Doronin
NBICS Department, Moscow Institute of Physics and Technology, Moscow 123182, Russia.
Vladimir P
VP
Sotskov
National Research Center "Kurchatov Institute", Moscow 123182, Russia.
Marina A
MA
Roshchina
National Research Center "Kurchatov Institute", Moscow 123182, Russia.
Tatiana A
TA
Kunitsyna
Laboratory of Cellular Neurobiology of Learning, Institute of Higher Nervous Activity and Neurophysiology of RAS, Moscow 117485, Russia.
Aleksey Y
AY
Malyshev
Laboratory of Cellular Neurobiology of Learning, Institute of Higher Nervous Activity and Neurophysiology of RAS, Moscow 117485, Russia.|Medico-Biological Faculty, N.I. Pirogov Russian National Research Medical University, Moscow 117997, Russia.
Ivan V
IV
Smirnov
National Research Center "Kurchatov Institute", Moscow 123182, Russia.
Asya M
AM
Azieva
National Research Center "Kurchatov Institute", Moscow 123182, Russia.
Ilya S
IS
Sokolov
MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Kiryl D
KD
Piatkevich
National Research Center "Kurchatov Institute", Moscow 123182, Russia.
Mikhail S
MS
Burtsev
Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow 119435, Russia.|Engelhardt Institute of Molecular Biology RAS, Moscow 119991, Russia.
Anna M
AM
Varizhuk
Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow 119435, Russia.
Galina E
GE
Pozmogova
NBICS Department, Moscow Institute of Physics and Technology, Moscow 123182, Russia.|Department of Systems Neurobiology and Functional Neurochemistry, P.K. Anokhin Institute of Normal Physiology of RAMS, Moscow 125009, Russia.|National Research Center "Kurchatov Institute", Moscow 123182, Russia.
Konstantin V
KV
Anokhin
NBICS Department, Moscow Institute of Physics and Technology, Moscow 123182, Russia.
Fedor V
FV
Subach
NBICS Department, Moscow Institute of Physics and Technology, Moscow 123182, Russia.|Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.|Department of Anesthesiology, Stony Brook University Medical Center, NY 11794, USA.|Center for Developmental Genetics, Stony Brook University, NY 11794, USA.
Grigori N
GN
Enikolopov
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