title: The enhanced cyan fluorescent protein: a sensitive pH sensor for fluorescence lifetime imaging.
abstract: pH is an important parameter that affects many functions of live cells, from protein structure or function to several crucial steps of their metabolism. Genetically encoded pH sensors based on pH-sensitive fluorescent proteins have been developed and used to monitor the pH of intracellular compartments. The quantitative analysis of pH variations can be performed either by ratiometric or fluorescence lifetime detection. However, most available genetically encoded pH sensors are based on green and yellow fluorescent proteins and are not compatible with multicolor approaches. Taking advantage of the strong pH sensitivity of enhanced cyan fluorescent protein (ECFP), we demonstrate here its suitability as a sensitive pH sensor using fluorescence lifetime imaging. The intracellular ECFP lifetime undergoes large changes (32 %) in the pH 5 to pH 7 range, which allows accurate pH measurements to better than 0.2 pH units. By fusion of ECFP with the granular chromogranin A, we successfully measured the pH in secretory granules of PC12 cells, and we performed a kinetic analysis of intragranular pH variations in living cells exposed to ammonium chloride.
authors.affiliation: Université of Paris Sud, Orsay, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Poëa-Guyon
authors.forename: Hélène
authors.initials: H
authors.lastname: Pasquier
authors.forename: Fabienne
authors.initials: F
authors.lastname: Mérola
authors.forename: Nicolas
authors.initials: N
authors.lastname: Morel
authors.forename: Marie
authors.initials: M
authors.lastname: Erard
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods; D053379:Chromogranin A / Q000032:analysis / Q000235:genetics; D003594:Cytoplasmic Granules / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D018613:Microscopy, Confocal; D016716:PC12 Cells; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics
chemical_list: D053379:Chromogranin A; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; C527701:enhanced cyan fluorescent protein; D049452:Green Fluorescent Proteins