Fluorescent ratiometric pH indicator SypHer2: Applications in neuroscience and regenerative biology.
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.
['Matlashov|Mikhail E|ME|', 'Bogdanova|Yulia A|YA|', 'Ermakova|Galina V|GV|', 'Mishina|Natalia M|NM|', 'Ermakova|Yulia G|YG|', 'Nikitin|Evgeny S|ES|', 'Balaban|Pavel M|PM|', 'Okabe|Shigeo|S|', 'Lukyanov|Sergey|S|', 'Enikolopov|Grigori|G|', 'Zaraisky|Andrey G|AG|', 'Belousov|Vsevolod V|VV|']
D000818:Animals D002118:Calcium D005453:Fluorescence D006863:Hydrogen-Ion Concentration D051379:Mice D008810:Mice, Inbred C57BL D041681:NIH 3T3 Cells D009488:Neurosciences D011874:Radiometry D012038:Regeneration D014982:Xenopus laevis
pH SypHer2 Neurons Regeneration Genetically encoded sensors Imaging Synapse