PMID- 33374320
OWN - NLM
STAT- MEDLINE
DCOM- 20210906
LR  - 20210906
IS  - 1422-0067 (Electronic)
IS  - 1422-0067 (Linking)
VI  - 22
IP  - 1
DP  - 2020 Dec 24
TI  - FRCaMP, a Red Fluorescent Genetically Encoded Calcium Indicator Based on 
      Calmodulin from Schizosaccharomyces Pombe Fungus.
LID - 10.3390/ijms22010111 [doi]
LID - 111
AB  - Red fluorescent genetically encoded calcium indicators (GECIs) have expanded the 
      available pallet of colors used for the visualization of neuronal calcium 
      activity in vivo. However, their calcium-binding domain is restricted by 
      calmodulin from metazoans. In this study, we developed red GECI, called FRCaMP, 
      using calmodulin (CaM) from Schizosaccharomyces pombe fungus as a calcium binding 
      domain. Compared to the R-GECO1 indicator in vitro, the purified protein FRCaMP 
      had similar spectral characteristics, brightness, and pH stability but a 1.3-fold 
      lower DeltaF/F calcium response and 2.6-fold tighter calcium affinity with K(d) of 
      441 nM and 2.4-6.6-fold lower photostability. In the cytosol of cultured HeLa 
      cells, FRCaMP visualized calcium transients with a DeltaF/F dynamic range of 5.6, 
      which was similar to that of R-GECO1. FRCaMP robustly visualized the spontaneous 
      activity of neuronal cultures and had a similar DeltaF/F dynamic range of 1.7 but 
      2.1-fold faster decay kinetics vs. NCaMP7. On electrically stimulated cultured 
      neurons, FRCaMP demonstrated 1.8-fold faster decay kinetics and 1.7-fold lower 
      DeltaF/F values per one action potential of 0.23 compared to the NCaMP7 indicator. 
      The fungus-originating CaM of the FRCaMP indicator version with a deleted 
      M13-like peptide did not interact with the cytosolic environment of the HeLa 
      cells in contrast to the metazoa-originating CaM of the similarly truncated 
      version of the GCaMP6s indicator with a deleted M13-like peptide. Finally, we 
      generated a split version of the FRCaMP indicator, which allowed the simultaneous 
      detection of calcium transients and the heterodimerization of bJun/bFos 
      interacting proteins in the nuclei of HeLa cells with a DeltaF/F dynamic range of 9.4 
      and a contrast of 2.3-3.5, respectively.
FAU - Subach, Oksana M
AU  - Subach OM
AD  - Complex of NBICS Technologies, National Research Center "Kurchatov Institute", 
      123182 Moscow, Russia.
FAU - Barykina, Natalia V
AU  - Barykina NV
AUID- ORCID: 0000-0001-5835-8388
AD  - Laboratory for Neurobiology of Memory, P.K. Anokhin Research Institute of Normal 
      Physiology, 125315 Moscow, Russia.
FAU - Chefanova, Elizaveta S
AU  - Chefanova ES
AD  - Department of NBIC-Technologies, Moscow Institute of Physics and Technology, 
      123182 Moscow, Russia.
FAU - Vlaskina, Anna V
AU  - Vlaskina AV
AD  - Complex of NBICS Technologies, National Research Center "Kurchatov Institute", 
      123182 Moscow, Russia.
FAU - Sotskov, Vladimir P
AU  - Sotskov VP
AUID- ORCID: 0000-0002-1729-5744
AD  - Institute for Advanced Brain Studies, Lomonosov Moscow State University, 119991 
      Moscow, Russia.
FAU - Ivashkina, Olga I
AU  - Ivashkina OI
AD  - Complex of NBICS Technologies, National Research Center "Kurchatov Institute", 
      123182 Moscow, Russia.
AD  - Laboratory for Neurobiology of Memory, P.K. Anokhin Research Institute of Normal 
      Physiology, 125315 Moscow, Russia.
AD  - Institute for Advanced Brain Studies, Lomonosov Moscow State University, 119991 
      Moscow, Russia.
FAU - Anokhin, Konstantin V
AU  - Anokhin KV
AD  - Laboratory for Neurobiology of Memory, P.K. Anokhin Research Institute of Normal 
      Physiology, 125315 Moscow, Russia.
AD  - Institute for Advanced Brain Studies, Lomonosov Moscow State University, 119991 
      Moscow, Russia.
FAU - Subach, Fedor V
AU  - Subach FV
AUID- ORCID: 0000-0003-2720-7821
AD  - Complex of NBICS Technologies, National Research Center "Kurchatov Institute", 
      123182 Moscow, Russia.
LA  - eng
GR  - numero sign1056 small o, Cyrillicsmall te, Cyrillic 02.07.2020/internal grant of the National Research Center Kurchatov 
      Institute/
GR  - 19-04-00395/Russian Foundation for Basic Research/
GR  - 075-15-2020-801/the Ministry of Science and Higher Education of the Russian 
      Federation/
PT  - Journal Article
DEP - 20201224
PL  - Switzerland
TA  - Int J Mol Sci
JT  - International journal of molecular sciences
JID - 101092791
RN  - 0 (Calmodulin)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 0 (Schizosaccharomyces pombe Proteins)
RN  - 147336-22-9 (Green Fluorescent Proteins)
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - Animals
MH  - Calcium/*metabolism
MH  - *Calmodulin/biosynthesis/genetics
MH  - *Green Fluorescent Proteins/biosynthesis/genetics
MH  - HeLa Cells
MH  - Humans
MH  - Mice
MH  - Neurons/*metabolism
MH  - *Recombinant Fusion Proteins
MH  - Schizosaccharomyces/*genetics
MH  - *Schizosaccharomyces pombe Proteins/genetics/metabolism
PMC - PMC7794825
OTO - NOTNLM
OT  - FRCaMP
OT  - GECI
OT  - Schizosaccharomyces pombe
OT  - bJun/bFos
OT  - calcium imaging
OT  - fluorescent protein
OT  - genetically encoded calcium indicator
OT  - protein engineering
OT  - red fluorescent
OT  - split
COIS- The authors declare no conflict of interest. The funders had no role in the 
      design of the study; in the collection, analyses, or interpretation of data; in 
      the writing of the manuscript; or in the decision to publish the results.
EDAT- 2020/12/31 06:00
MHDA- 2021/09/07 06:00
PMCR- 2020/12/24
CRDT- 2020/12/30 01:01
PHST- 2020/12/04 00:00 [received]
PHST- 2020/12/22 00:00 [revised]
PHST- 2020/12/22 00:00 [accepted]
PHST- 2020/12/30 01:01 [entrez]
PHST- 2020/12/31 06:00 [pubmed]
PHST- 2021/09/07 06:00 [medline]
PHST- 2020/12/24 00:00 [pmc-release]
AID - ijms22010111 [pii]
AID - ijms-22-00111 [pii]
AID - 10.3390/ijms22010111 [doi]
PST - epublish
SO  - Int J Mol Sci. 2020 Dec 24;22(1):111. doi: 10.3390/ijms22010111.