pHlameleons: A Family of FRET-Based Protein Sensors for Quantitative pH Imaging

Abstract

Intracellular pH is an important indicator for cellular metabolism and pathogenesis. pH sensing in living cells has been achieved using a number of synthetic organic dyes and genetically expressible sensor proteins, even allowing the specific targeting of intracellular organelles. Ideally, a class of genetically encodeable sensors need to cover relevant cellular pH ranges. We present a FRET-based pH sensor platform, based on the pH modulation of YFP acceptor fluorophores in a fusion construct with ECFP. The concurrent loss of the overlap integral upon acidification results in a proportionally reduced FRET coupling. The readout of FRET over the sensitized YFP fluorescence lifetime yields a highly sensitive and robust pH measurement that is self-calibrated. The principle is demonstrated in the existing high-efficiency FRET fusion Cy11.5, and tunability of the platform design is demonstrated by genetic alteration of the pH sensitivity of the acceptor moiety.

Quantitative imaging of endosome acidification and single retrovirus fusion with distinct pools of early endosomes

“…The nearly complete quenching of the ecliptic pHluorin signal at pH below 6.2 marked the ASLV entry into mildly acidic compartments but did not allow quantitative measurements of endosomal pH. To enable simultaneous measurements of endosomal pH and ASLV fusion, we used a modified version of the FRET-based pH sensor, pHlameleon (16). The CFP (donor) module of pHlameleon was replaced with the monomeric teal fluorescent protein, mTFP1 (Fig.…”

“…From several FRET-based sensors designed to measure endosomal pH (16,(30)(31)(32), we used a derivative of pHlameleon (16) incorporated into the viral membrane. Unlike other approaches, this strategy avoided chemical modifications of the virus, which could compromise its ability to undergo fusion.…”

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

“…concentration range of these cameleons ranges from 10 -8 to 10 -2 M. Many variants of the cameleon sensors are currently available and are continuously being improved (Nagai et al 2004). Other FRET biosensors have been developed for measuring chloride ('clomeleon') (Jose et al 2007) and pH ('pHlameleon') (Esposito et al 2008).…”

Advances in cellular, subcellular, and nanoscale imaging in vitro and in vivo

“…The lifetime of yellow emission upon donor excitation falls with acidification as the FRET contribution is lost. These sensors are extremely sensitive due to the nonlinear nature of FRET, and are intrinsically calibrated due to the concentration independence of FRET (22). This assay holds promise for molecular imaging of malignant tumor tissue since the microenvironment of tumor tissue is more acidic that the surrounding healthy tissue-a sign of their increased anaerobic glucose metabolism, which affects the efficacy of some therapeutic regimens (23).…”