PMID- 28837632 OWN - NLM STAT- MEDLINE DCOM- 20171023 LR - 20240326 IS - 1932-6203 (Electronic) IS - 1932-6203 (Linking) VI - 12 IP - 8 DP - 2017 TI - Green fluorescent genetically encoded calcium indicator based on calmodulin/M13-peptide from fungi. PG - e0183757 LID - 10.1371/journal.pone.0183757 [doi] LID - e0183757 AB - Currently available genetically encoded calcium indicators (GECIs) utilize calmodulins (CaMs) or troponin C from metazoa such as mammals, birds, and teleosts, as calcium-binding domains. The amino acid sequences of the metazoan calcium-binding domains are highly conserved, which may limit the range of the GECI key parameters and cause undesired interactions with the intracellular environment in mammalian cells. Here we have used fungi, evolutionary distinct organisms, to derive CaM and its binding partner domains and design new GECI with improved properties. We applied iterative rounds of molecular evolution to develop FGCaMP, a novel green calcium indicator. It includes the circularly permuted version of the enhanced green fluorescent protein (EGFP) sandwiched between the fungal CaM and a fragment of CaM-dependent kinase. FGCaMP is an excitation-ratiometric indicator that has a positive and an inverted fluorescence response to calcium ions when excited at 488 and 405 nm, respectively. Compared with the GCaMP6s indicator in vitro, FGCaMP has a similar brightness at 488 nm excitation, 7-fold higher brightness at 405 nm excitation, and 1.3-fold faster calcium ion dissociation kinetics. Using site-directed mutagenesis, we generated variants of FGCaMP with improved binding affinity to calcium ions and increased the magnitude of FGCaMP fluorescence response to low calcium ion concentrations. Using FGCaMP, we have successfully visualized calcium transients in cultured mammalian cells. In contrast to the limited mobility of GCaMP6s and G-GECO1.2 indicators, FGCaMP exhibits practically 100% molecular mobility at physiological concentrations of calcium ion in mammalian cells, as determined by photobleaching experiments with fluorescence recovery. We have successfully monitored the calcium dynamics during spontaneous activity of neuronal cultures using FGCaMP and utilized whole-cell patch clamp recordings to further characterize its behavior in neurons. Finally, we used FGCaMP in vivo to perform structural and functional imaging of zebrafish using wide-field, confocal, and light-sheet microscopy. FAU - Barykina, Natalia V AU - Barykina NV AD - Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia. AD - P.K. Anokhin Institute of Normal Physiology of RAMS, Moscow, Russia. FAU - Subach, Oksana M AU - Subach OM AD - Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia. AD - National Research Center "Kurchatov Institute", Moscow, Russia. FAU - Piatkevich, Kiryl D AU - Piatkevich KD AD - MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA, United States of America. FAU - Jung, Erica E AU - Jung EE AD - MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA, United States of America. FAU - Malyshev, Aleksey Y AU - Malyshev AY AD - Institute of Higher Nervous Activity and Neurophysiology of RAS, Moscow, Russia. FAU - Smirnov, Ivan V AU - Smirnov IV AD - Institute of Higher Nervous Activity and Neurophysiology of RAS, Moscow, Russia. AD - Medico-Biological Faculty, N.I. Pirogov Russian National Research Medical University, Moscow, Russia. FAU - Bogorodskiy, Andrey O AU - Bogorodskiy AO AD - Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia. FAU - Borshchevskiy, Valentin I AU - Borshchevskiy VI AD - Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia. FAU - Varizhuk, Anna M AU - Varizhuk AM AD - Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow, Russia. AD - Engelhardt Institute of Molecular Biology RAS, Moscow, Russia. FAU - Pozmogova, Galina E AU - Pozmogova GE AD - Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow, Russia. FAU - Boyden, Edward S AU - Boyden ES AD - MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA, United States of America. AD - MIT McGovern Institute for Brain Research, MIT, Cambridge, MA, United States of America. FAU - Anokhin, Konstantin V AU - Anokhin KV AD - P.K. Anokhin Institute of Normal Physiology of RAMS, Moscow, Russia. AD - National Research Center "Kurchatov Institute", Moscow, Russia. AD - Lomonosov Moscow State University, Moscow, Russia. FAU - Enikolopov, Grigori N AU - Enikolopov GN AD - Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia. AD - Department of Anesthesiology, Stony Brook University Medical Center, Stony Brook, NY, United States of America. AD - Center for Developmental Genetics, Stony Brook University, Stony Brook, NY, United States of America. FAU - Subach, Fedor V AU - Subach FV AUID- ORCID: 0000-0003-2720-7821 AD - Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia. LA - eng GR - DP1 NS087724/NS/NINDS NIH HHS/United States GR - R01 AG040209/AG/NIA NIH HHS/United States GR - R01 DA029639/DA/NIDA NIH HHS/United States GR - R01 GM104948/GM/NIGMS NIH HHS/United States PT - Journal Article DEP - 20170824 PL - United States TA - PLoS One JT - PloS one JID - 101285081 RN - 0 (Calmodulin) RN - 0 (Fungal Proteins) RN - 0 (Peptide Fragments) RN - 0 (enhanced green fluorescent protein) RN - 147336-22-9 (Green Fluorescent Proteins) RN - SY7Q814VUP (Calcium) SB - IM MH - Animals MH - Calcium/*metabolism MH - Calmodulin/*metabolism MH - Fungal Proteins/chemistry/genetics/*metabolism MH - Green Fluorescent Proteins/*metabolism MH - HeLa Cells MH - Humans MH - Mutagenesis, Site-Directed MH - Neurons/metabolism MH - Patch-Clamp Techniques MH - Peptide Fragments/*metabolism MH - Spectrometry, Fluorescence MH - Zebrafish/growth & development/physiology PMC - PMC5570312 COIS- Competing Interests: The authors have declared that no competing interests exist. EDAT- 2017/08/25 06:00 MHDA- 2017/10/24 06:00 PMCR- 2017/08/24 CRDT- 2017/08/25 06:00 PHST- 2017/05/10 00:00 [received] PHST- 2017/08/10 00:00 [accepted] PHST- 2017/08/25 06:00 [entrez] PHST- 2017/08/25 06:00 [pubmed] PHST- 2017/10/24 06:00 [medline] PHST- 2017/08/24 00:00 [pmc-release] AID - PONE-D-17-18026 [pii] AID - 10.1371/journal.pone.0183757 [doi] PST - epublish SO - PLoS One. 2017 Aug 24;12(8):e0183757. doi: 10.1371/journal.pone.0183757. eCollection 2017.