PMID- 27677952
OWN - NLM
STAT- Publisher
LR  - 20191120
IS  - 2045-2322 (Electronic)
IS  - 2045-2322 (Linking)
VI  - 6
DP  - 2016 Sep 28
TI  - A new design for a green calcium indicator with a smaller size and a reduced 
      number of calcium-binding sites.
PG  - 34447
LID - 10.1038/srep34447 [doi]
LID - 34447
AB  - 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 Ca(2+)-binding moiety, has two binding sites for 
      calcium ions, providing a linear response to calcium ions. One-fluorophore-based 
      sensors have four Ca(2+)-binding sites but are better suited for in vivo 
      experiments. Herein, we describe a novel design for a one-fluorophore-based GECI 
      with two Ca(2+)-binding sites. The engineered sensor, called NTnC, uses TnC as 
      the Ca(2+)-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 Ca(2+). Using NTnC, we have visualized Ca(2+) dynamics during spontaneous 
      activity of neuronal cultures as confirmed by control NTnC and its mutant, in 
      which the affinity to Ca(2+) 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 Ca(2+) neuronal activity in vivo in the V1 cortical area in awake and 
      freely moving mice using two-photon microscopy or an nVista miniaturized 
      microscope.
FAU - Barykina, Natalia V
AU  - Barykina NV
AD  - NBICS Department, Moscow Institute of Physics and Technology, Moscow 123182, 
      Russia.
AD  - Department of Systems Neurobiology and Functional Neurochemistry, P.K. Anokhin 
      Institute of Normal Physiology of RAMS, Moscow 125009, Russia.
FAU - Subach, Oksana M
AU  - Subach OM
AD  - NBICS Department, Moscow Institute of Physics and Technology, Moscow 123182, 
      Russia.
AD  - National Research Center "Kurchatov Institute", Moscow 123182, Russia.
FAU - Doronin, Danila A
AU  - Doronin DA
AD  - NBICS Department, Moscow Institute of Physics and Technology, Moscow 123182, 
      Russia.
AD  - National Research Center "Kurchatov Institute", Moscow 123182, Russia.
FAU - Sotskov, Vladimir P
AU  - Sotskov VP
AD  - NBICS Department, Moscow Institute of Physics and Technology, Moscow 123182, 
      Russia.
FAU - Roshchina, Marina A
AU  - Roshchina MA
AD  - National Research Center "Kurchatov Institute", Moscow 123182, Russia.
FAU - Kunitsyna, Tatiana A
AU  - Kunitsyna TA
AD  - National Research Center "Kurchatov Institute", Moscow 123182, Russia.
FAU - Malyshev, Aleksey Y
AU  - Malyshev AY
AD  - Laboratory of Cellular Neurobiology of Learning, Institute of Higher Nervous 
      Activity and Neurophysiology of RAS, Moscow 117485, Russia.
FAU - Smirnov, Ivan V
AU  - Smirnov IV
AD  - Laboratory of Cellular Neurobiology of Learning, Institute of Higher Nervous 
      Activity and Neurophysiology of RAS, Moscow 117485, Russia.
AD  - Medico-Biological Faculty, N.I. Pirogov Russian National Research Medical 
      University, Moscow 117997, Russia.
FAU - Azieva, Asya M
AU  - Azieva AM
AD  - National Research Center "Kurchatov Institute", Moscow 123182, Russia.
FAU - Sokolov, Ilya S
AU  - Sokolov IS
AD  - National Research Center "Kurchatov Institute", Moscow 123182, Russia.
FAU - Piatkevich, Kiryl D
AU  - Piatkevich KD
AD  - MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
FAU - Burtsev, Mikhail S
AU  - Burtsev MS
AD  - National Research Center "Kurchatov Institute", Moscow 123182, Russia.
FAU - Varizhuk, Anna M
AU  - Varizhuk AM
AD  - Federal Research and Clinical Center of Physical-Chemical Medicine of Federal 
      Medical Biological Agency, Moscow 119435, Russia.
AD  - Engelhardt Institute of Molecular Biology RAS, Moscow 119991, Russia.
FAU - Pozmogova, Galina E
AU  - Pozmogova GE
AD  - Federal Research and Clinical Center of Physical-Chemical Medicine of Federal 
      Medical Biological Agency, Moscow 119435, Russia.
FAU - Anokhin, Konstantin V
AU  - Anokhin KV
AD  - NBICS Department, Moscow Institute of Physics and Technology, Moscow 123182, 
      Russia.
AD  - Department of Systems Neurobiology and Functional Neurochemistry, P.K. Anokhin 
      Institute of Normal Physiology of RAMS, Moscow 125009, Russia.
AD  - National Research Center "Kurchatov Institute", Moscow 123182, Russia.
FAU - Subach, Fedor V
AU  - Subach FV
AD  - NBICS Department, Moscow Institute of Physics and Technology, Moscow 123182, 
      Russia.
FAU - Enikolopov, Grigori N
AU  - Enikolopov GN
AD  - NBICS Department, Moscow Institute of Physics and Technology, Moscow 123182, 
      Russia.
AD  - Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
AD  - Department of Anesthesiology, Stony Brook University Medical Center, NY 11794, 
      USA.
AD  - Center for Developmental Genetics, Stony Brook University, NY 11794, USA.
LA  - eng
GR  - R01 AG040209/AG/NIA NIH HHS/United States
PT  - Journal Article
DEP - 20160928
PL  - England
TA  - Sci Rep
JT  - Scientific reports
JID - 101563288
PMC - PMC5039633
EDAT- 2016/09/30 06:00
MHDA- 2016/09/30 06:00
PMCR- 2016/09/28
CRDT- 2016/09/29 06:00
PHST- 2016/05/19 00:00 [received]
PHST- 2016/09/14 00:00 [accepted]
PHST- 2016/09/29 06:00 [entrez]
PHST- 2016/09/30 06:00 [pubmed]
PHST- 2016/09/30 06:00 [medline]
PHST- 2016/09/28 00:00 [pmc-release]
AID - srep34447 [pii]
AID - 10.1038/srep34447 [doi]
PST - epublish
SO  - Sci Rep. 2016 Sep 28;6:34447. doi: 10.1038/srep34447.