title: Fluorescent ratiometric pH indicator SypHer2: Applications in neuroscience and regenerative biology.
abstract: BACKGROUND
  SypHer is a genetically encoded fluorescent pH-indicator with a ratiometric readout, suitable for measuring fast intracellular pH shifts. However, the relatively low brightness of the indicator limits its use.
  
  
  METHODS
  Here we designed a new version of pH-sensor called SypHer-2, which has up to three times brighter fluorescence in cultured mammalian cells compared to the SypHer.
  
  
  RESULTS
  Using the new indicator we registered activity-associated pH oscillations in neuronal cell culture. We observed prominent transient neuronal cytoplasm acidification that occurs in parallel with calcium entry. Furthermore, we monitored pH in presynaptic and postsynaptic termini by targeting SypHer-2 directly to these compartments and revealed marked differences in pH dynamics between synaptic boutons and dendritic spines. Finally, we were able to reveal for the first time the intracellular pH drop that occurs within an extended region of the amputated tail of the Xenopus laevis tadpole before it begins to regenerate.
  
  
  CONCLUSIONS
  SypHer2 is suitable for quantitative monitoring of pH in biological systems of different scales, from small cellular subcompartments to animal tissues in vivo.
  
  
  CONCLUSIONS
  The new pH-sensor will help to investigate pH-dependent processes in both in vitro and in vivo studies.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia; Nizhny Novgorod State Medical Academy, 603005 Nizhny Novgorod, Russia.
authors.forename: Mikhail E
authors.initials: ME
authors.lastname: Matlashov
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia; Faculty of Biology, Moscow State University, 119991 Moscow, Russia.
authors.forename: Yulia A
authors.initials: YA
authors.lastname: Bogdanova
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia.
authors.forename: Galina V
authors.initials: GV
authors.lastname: Ermakova
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia; Nizhny Novgorod State Medical Academy, 603005 Nizhny Novgorod, Russia.
authors.forename: Natalia M
authors.initials: NM
authors.lastname: Mishina
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia.
authors.forename: Yulia G
authors.initials: YG
authors.lastname: Ermakova
authors.affiliation: Institute of Higher Nervous Activity and Neurophysiology, 117485 Moscow, Russia.
authors.forename: Evgeny S
authors.initials: ES
authors.lastname: Nikitin
authors.affiliation: Institute of Higher Nervous Activity and Neurophysiology, 117485 Moscow, Russia.
authors.forename: Pavel M
authors.initials: PM
authors.lastname: Balaban
authors.affiliation: Department of Cellular Neurobiology, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan.
authors.forename: Shigeo
authors.initials: S
authors.lastname: Okabe
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia; Nizhny Novgorod State Medical Academy, 603005 Nizhny Novgorod, Russia.
authors.forename: Sergey
authors.initials: S
authors.lastname: Lukyanov
authors.affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; Department of Anesthesiology, Stony Brook School of Medicine, Stony Brook, NY 11794, USA; Center for Developmental Genetics, Stony Brook University, Stony Brook, NY 11794, USA; NBIC, Moscow Institute of Physics and Technology, 123182 Moscow, Russia. Electronic address: enik@cshl.edu.
authors.forename: Grigori
authors.initials: G
authors.lastname: Enikolopov
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia. Electronic address: azaraisky@yahoo.com.
authors.forename: Andrey G
authors.initials: AG
authors.lastname: Zaraisky
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia; Nizhny Novgorod State Medical Academy, 603005 Nizhny Novgorod, Russia. Electronic address: vsevolod.belousov@gmail.com.
authors.forename: Vsevolod V
authors.initials: VV
authors.lastname: Belousov
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008810:Mice, Inbred C57BL; D041681:NIH 3T3 Cells; D009488:Neurosciences; D011874:Radiometry; D012038:Regeneration / Q000502:physiology*; D014982:Xenopus laevis / Q000502:physiology
chemical_list: D002118:Calcium
keywords: Genetically encoded sensors; Imaging; Neurons; Regeneration; Synapse; SypHer2; pH