title: In vivo determination of organellar pH using a universal wavelength-based confocal microscopy approach.
abstract: Many essential cellular processes are affected by transmembrane H(+) gradients and intracellular pH (pHi). The research of such metabolic events calls for a non-invasive method to monitor pHi within individual subcellular compartments. We present a novel confocal microscopy approach for the determination of organellar pHi in living cells expressing pH-dependent ratiometric fluorescent proteins. Unlike conventional intensity-based fluorometry, our method relies on emission wavelength scans at single-organelle resolution to produce wavelength-based pH estimates both accurate and robust to low-signal artifacts. Analyses of Ato1p-pHluorin and Ato1p-mCherry yeast cells revealed previously unreported wavelength shifts in pHluorin emission which, together with ratiometric mCherry, allowed for high-precision quantification of actual physiological pH values and evidenced dynamic pHi changes throughout the different stages of yeast colony development. Additionally, comparative pH quantification of Ato1p-pHluorin and Met17p-pHluorin cells implied the existence of a significant pHi gradient between peripheral and internal cytoplasm of cells from colonies occurring in the ammonia-producing alkali developmental phase. Results represent a step forward in the study of pHi regulation and subcellular metabolic functions beyond the scope of this study.
authors.affiliation: Department of Genetics and Microbiology, Charles University, Prague, Czech Republic.
authors.forename: Albert
authors.initials: A
authors.lastname: Pineda Rodó
authors.forename: Libuše
authors.initials: L
authors.lastname: Váchová
authors.forename: Zdena
authors.initials: Z
authors.lastname: Palková
mesh_terms: D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D015388:Organelles / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D000097573:Red Fluorescent Protein; C520069:ATO1 protein, S cerevisiae; C485187:PHluorin