pHlash: a new genetically encoded and ratiometric luminescence sensor of intracellular pH.
We report the development of a genetically encodable and ratiometic pH probe named "pHlash" that utilizes Bioluminescence Resonance Energy Transfer (BRET) rather than fluorescence excitation. The pHlash sensor-composed of a donor luciferase that is genetically fused to a Venus fluorophore-exhibits pH dependence of its spectral emission in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification in vivo. Its spectral ratio response is H(+) specific; neither Ca(++), Mg(++), Na(+), nor K(+) changes the spectral form of its luminescence emission. Moreover, it can be used to image pH in single cells. This is the first BRET-based sensor of H(+) ions, and it should allow the approximation of pH in cytosolic and organellar compartments in applications where current pH probes are inadequate.
Departments of Biological Science and Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, United States of America.
Yunfei
Y
Zhang
Qiguang
Q
Xie
J Brian
JB
Robertson
Carl Hirschie
CH
Johnson
D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004735:Energy Transfer; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D049449:Luminescence; D008164:Luminescent Proteins; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012969:Sodium Fluoride / Q000494:pharmacology; D013997:Time Factors
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D012969:Sodium Fluoride