Format

Send to

Choose Destination
Biochim Biophys Acta. 2015 Nov;1850(11):2318-28. doi: 10.1016/j.bbagen.2015.08.002. Epub 2015 Aug 8.

Fluorescent ratiometric pH indicator SypHer2: Applications in neuroscience and regenerative biology.

Author information

1
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia; Nizhny Novgorod State Medical Academy, 603005 Nizhny Novgorod, Russia.
2
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia; Faculty of Biology, Moscow State University, 119991 Moscow, Russia.
3
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia.
4
Institute of Higher Nervous Activity and Neurophysiology, 117485 Moscow, Russia.
5
Department of Cellular Neurobiology, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan.
6
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.
7
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia. Electronic address: azaraisky@yahoo.com.
8
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia; Nizhny Novgorod State Medical Academy, 603005 Nizhny Novgorod, Russia. Electronic address: vsevolod.belousov@gmail.com.

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.

GENERAL SIGNIFICANCE:

The new pH-sensor will help to investigate pH-dependent processes in both in vitro and in vivo studies.

KEYWORDS:

Genetically encoded sensors; Imaging; Neurons; Regeneration; Synapse; SypHer2; pH

PMID:
26259819
PMCID:
PMC4587288
DOI:
10.1016/j.bbagen.2015.08.002
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center