Green fluorescent protein based pH indicators for in vivo use: a review

Abstract

Green fluorescent protein (GFP) and its variants have been used as fluorescent reporters in a variety of applications for monitoring dynamic processes in cells and organisms, including gene expression, protein localization, and intracellular dynamics. GFP fluorescence is stable, species-independent, and can be monitored noninvasively in living cells by fluorescence microscopy, flow cytometry, or macroscopic imaging techniques. Owing to the presence of a phenol group on the chromophore, most GFP variants display pH-sensitive absorption and fluorescence bands. Such behavior has been exploited to genetically engineer encodable pH indicators for studies of pH regulation within specific intracellular compartments that cannot be probed using conventional pH-sensitive dyes. These pH indicators contributed to shedding light on a number of cell functions for which intracellular pH is an important modulator. In this review we discuss the photophysical properties that make GFPs so special as pH indicators for in vivo use and we describe the probes that are utilized most by the scientific community.

Neuronal hyperactivity causes Na+/H+ exchanger-induced extracellular acidification at active synapses

“…We asked whether the optical properties of E 2 GFP were affected by exposure to the extracellular environment. Primary cortical neurons were infected with ex.E 2 GFP and fluorescence emission was evaluated at two extracellular pH values (5.8 and 7.4) using 405 and 488 nm as excitation wavelengths (λ ex 405 and λ ex 488, respectively) (Bizzarri et al, 2006(Bizzarri et al, , 2009 (Fig. 2A).…”

GFP-Based Biosensors

“…Biosensors may function in vivo or in vitro. GFP variants that exhibit analyte-sensitive properties are genetically encoded biosensors, acting in vivo.GFP biosensors that contain amino acid substitutions that enable detection of pH changes, specific ions (Cl -or Ca 2+ ), reactive oxygen species, redox state, and specific peptides have been reported [39,[58][59][60]. In addition, modifications have been reported that enable selective activation (irreversible or reversible) of the fluorescence [61,62].Genetically encoded GFP biosensors may be single GFP domains or FRET pairs.In the following subsections we describe selected examples of GFP-based biosensors used in vivo or in vitro, with special emphasis on computationally designed biosensors.…”

“…Many GFP variants show sensitivity to pH which results from protonation and deprotonation of the chromophore (see Maturation of the GFP Chromophore)(reviewed in [58]). The rapid and reversible response of EGFP to pH changes in the cells enabled EGFP to be used as an intracellular pH indicator [63] in place of chemical pH indicators such as fluorescein.A range of GFP based pH biosensors have been generated from modification of wtGFP and EGFP which resulted from amino acid substitutions primarily in and around the region of the chromophore.…”

“…Two classes of GFP pH indicators have been described: ratiometric and nonratiometric [58,64].In the ratiometric pH indicators, the chromophore environment is such that the GFP biosensor has two sets of excitation/ emission spectra, one that varies with pH and another that does not.For these GFP variants, a calibration curve can be generated for the ratio of the spectra versus the pH.Nonratiometric GFP variants,such as EGFP [63] or ecliptic GFP [64], have pH dependent emission from the anionic chromophore (deprotonated) but almost no fluorescence of the neutral chromophore (protonated). These variants are used for reporting pH changes within cells when used as single molecule pH sensor or used in tandem with pH insensitive fluorescent partner (described below).…”

Sustained accurate recording of intracellular acidification in living tissues with a photo-controllable bioluminescent protein

“…However, the challenge remains of analyzing acidification in intracellular microenvironments in living cells and tissues. In contrast, genetically encoded fluorescent probes can be targeted into a specific intracellular organelle; the probes for pH are now applied for single-cell analysis (6)(7)(8). The fluorescence protein itself is, however, sensitive to pH, and intensity-based imaging techniques remain difficult to apply for in vivo imaging because of bleaching of chromophores by excitation light and low penetration of the emitting light.…”