pHlameleons: a family of FRET-based protein sensors for quantitative pH imaging.
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.
Esposito
Alessandro
A
Laboratory for Molecular and Cellular Systems, Department of Neuro- and Sensory Physiology, University Medicine Gottingen, Humboldtalee 23, 37073 Gottingen, Germany. aesposito@quantitative-microscopy.org
Gralle
Matthias
M
Dani
Maria Angela C
MA
Lange
Dirk
D
Wouters
Fred S
FS
D000818:Animals; D001709:Biotechnology / Q000379:methods*; D002460:Cell Line; D002478:Cells, Cultured; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007091:Image Processing, Computer-Assisted; D008164:Luminescent Proteins / Q000032:analysis / Q000737:chemistry*; D012680:Sensitivity and Specificity
D008164:Luminescent Proteins