Biochimica et Biophysica Acta (BBA) - General Subjects
Fluorescent ratiometric pH indicator SypHer2: Applications in neuroscience and regenerative biology
Introduction
A broad range of synthetic and genetically encoded indicators, together with active development of fluorescent microscopy in recent years, yielded powerful tools to image many different parameters in living cells. One of the key parameters in biological cells is pH. Not surprisingly, a number of indicators for pH exist, either synthetic or genetically encoded [1], [2], [3]. These pH probes are of different nature (synthetic [4], [5] vs. protein [6], [7], [8], [9], [10]), color (mostly green [4], [6], [7] and red [9], [10]), pKa and readout (ratiometric [4], [5], [7], [8], [9] vs. intensiometric [6], [10]). Of those, genetically encoded sensors provide many benefits, including a vast range of options for targeting tissues, cells, or organelles [3] and the possibility of expression in transgenic animals [11]. Ratiometric readout offers quantitative measurements devoid of potential artifacts caused by object movement or different expression levels among parts of the object.
One such pH sensor, SypHer, is a genetically encoded fluorescent ratiometric pH probe [12]. SypHer is a product of C199S mutation of HyPer, an indicator for H2O2 [13]. The mutation makes HyPer insensitive to hydrogen peroxide while preserving its sensitivity to changes in pH. SypHer has been successfully applied in vitro; however, its fluorescence is relatively dim, making the use of the indicator somewhat problematic in tissue culture and live animal studies.
Recently an A406V substitution was described for HyPer that significantly increased the dynamic range of the biosensor [14]. For yet unknown reason, the resulting probe, HyPer-2, demonstrates a two to three-fold brighter signal when expressed in mammalian cells (unpublished observation), although purified HyPer and HyPer-2 demonstrate similar brightness [14]. Based on this finding, we decided to improve the reporter features of SypHer using the same mutation. For that, we have introduced C199S substitution into HyPer-2 and tested its performance in various models, ranging from individual synaptic termini in neurons to regenerating Xenopus leavis tadpole fins.
During neuronal activity, large amounts of ions move across the cellular membranes. Within milliseconds intracellular concentrations of Na+, K+, Ca2 + and Cl− change significantly [15], [16], [17] and maintaining normal homeostasis requires a complex system and high metabolic rate [18]. Not surprisingly, intense neuronal activity causes changes in intracellular pH in neurons. Numerous studies have shown that prolonged membrane depolarization and calcium entry are accompanied by significant cytoplasm acidification [19], [20], [21], [22]. The mechanism of such change in cytoplasmic pH is yet to be determined. One possible explanations relates to the activity of the ATP-dependent Ca2 +/H+ and Na+/H+ antiport [21]. Another explanation for intracellular acidification would invoke, as the main cause, lactate accumulation resulting from an accelerated glycolysis [20]. In turn, changes in the intracellular pH affect neuronal activity. For instance, activities of voltage-gated calcium channels, NMDA and GABA receptors, and other key components of the neuronal machinery depend on pH [23], [24], [25]. In addition, neurotransmitter transport is dependent on the H+ gradient across the synaptic vesicular membrane [26]. Finally, in some models of epileptiform activity it was shown that spontaneous seizure-like electric activity duration may be shortened by intracellular acidification [22], [27]. Therefore, pH changes may play a significant role as a feedback mechanism for regulating neuronal activity.
Neuronal cells have complex morphological and functional structure and measuring intracellular pH is not a simple task. The use of chemical indicators such as SNARF and BCECF does not allow effective differentiation between, for example, cytoplasm of a dendrite and the content of a dendritic spine, in which critical processes of electrical input modulation occur [28]. However, it is possible that pH changes in fine synaptic structures differ from those in the dendrite given partial isolation of the spines' content from the rest of the cytoplasm.
Changes in pH and concentration of H+ ions may also play an important role in early development and during regeneration. Transport of H+ ions across cell membrane attracts special attention because in amphibians it is known to regulate critical processes such as neural induction and tadpole tail regeneration. For example, it has been demonstrated in Xenopus laevis that tail regeneration requires the activity of the V-ATPase H+ pump and that enhancing V-ATPase activity induces H+ export across cell plasma membranes in regenerating tissues starting 6 h post-amputation [29], [30]. So far, however, pH dynamics during tail regeneration had been studied, to our knowledge, only by indirect approaches, using membrane-voltage-reporter dyes [29], [30], [31]. Therefore, detailed data on pH dynamics during regeneration are lacking. Here, we analyze early pH dynamics in the Xenopus laevis tails using SypHer-2 pH sensor and reveal a new phenomenon: a rapid drop of the intracellular pH occurring within a broad region adjacent to the site of tail amputation.
Section snippets
Reagents used
DMEM, glutamine, HBSS, trypsin-EDTA, and penicillin/streptomycin solutions were from PanEko (Russia); heat inactivated FBS, Neurobasal medium, Tyrode solution, B27 supplement, Gluta-Max solution, poly d-lysine and Ca-phosphate transfection kit were from Invitrogen (USA); potassium gluconate, sodium gluconate, BCECF, BAPTA-AM, bicuculline, nigericin, monensin, tetrodotoxin, and bafilomycin were from Sigma; X-tremeGene 9 was from Roche (France). Glass bottom dishes were from Mattek. NIH/3T3 cells
Investigating SypHer-2 pH sensitivity and brightness in cell cultures
Brightness is a crucial characteristic of fluorescent proteins used for live cell imaging because higher brightness allows for lowering the excitation light intensity and exposure time, thereby minimizing phototoxic effects. We have noticed that despite similar brightness of purified HyPer [13] and HyPer-2 [14] H2O2 sensors, HyPer-2 is visibly brighter than HyPer when expressed in mammalian cell cultures. HyPer-2 differs from HyPer by a single amino acid (A406V) residing in the dimerization
Discussion
Here we describe SypHer-2, a genetically encoded ratiometric pH indicator of improved brightness. SypHer-2 shows 2–3 times higher fluorescence in mammalian cells than the previous version, SypHer. SypHer-2 has a ratiometric readout that makes measurements independent of the indicator concentration and cytoplasm volume change, which is particularly useful for protracted measurements. The indicator works in a broad pH range with a pKa value of 8.1, which is slightly more alkaline compared to
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Acknowledgements
This work was supported by the Russian Foundation for basic research (13-04-40333-H to V.V.B., 13-04-40332-Н to P.M.B., 13-04-01516-A and 13-04-40194-H to A.G.Z., 13-04-00352 to E.S.N.); Molecular and Cell Biology Program of Russian Academy of Sciences (V.V.B. & E.S.N.); CREST, JST and Grants-in-Aid for Scientific Research (26250014), JAPAN to S.O.; National Institute of Aging (R01AG040209 to G.E.); NYSTEM (to G.E.); Russian Science Foundation (14-14-00557 to A.G.Z., experiments with time-lapse
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