title: Ca²⁺ regulation of mitochondrial ATP synthesis visualized at the single cell level.
abstract: Intracellular Ca(2+) levels play a crucial role in the control of ATP synthesis. However, the spatiotemporal correlation between ATP and Ca(2+) remains unclear due to the inability to visualize these factors within same individual cells. A Förster resonance energy transfer (FRET)-based fluorescent ATP probe, named ATeam, was recently developed for ATP imaging in single living cells. However, the spectra of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) used as the FRET donor and the acceptor, respectively, significantly overlap with the ultraviolet-excitable Ca(2+) probe, fura-2. In the present work, we developed new red-shifted ATP probes, GO-ATeams, in which green fluorescent protein (GFP) and orange fluorescent protein (OFP) was used as the FRET pair to minimize spectral overlap with the fura-2 emission. The dynamics of intracellular Ca(2+) and mitochondrial ATP levels in single histamine-stimulated HeLa cells were successfully visualized by using fura-2 and GO-ATeam. The experiments showed that histamine induced increases of both intracellular Ca(2+) and mitochondrial ATP levels. The increment of mitochondrial ATP levels was proportional to that of Ca(2+). This finding suggests that cellular Ca(2+) levels might precisely control mitochondrial ATP synthesis in response to the increased ATP consumption triggered by Ca(2+). In addition, GO-ATeam has several advantages over the original ATeam. The GO-ATeam signal was more stable against acidification, which would allow ATP imaging inside acidic intracellular compartments. Also, the GO-ATeam excitation wavelength is much less phototoxic to cells, making the probe suitable for long-time observation.
authors.affiliation: Institute of Scientific and Industrial Research, Osaka University, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nakano
authors.forename: Hiromi
authors.initials: H
authors.lastname: Imamura
authors.forename: Takeharu
authors.initials: T
authors.lastname: Nagai
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Noji
mesh_terms: D000255:Adenosine Triphosphate / Q000032:analysis* / Q000096:biosynthesis* / Q000378:metabolism; D000963:Antimetabolites / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D003847:Deoxyglucose / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism; D006367:HeLa Cells; D006632:Histamine / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008027:Light; D008274:Magnesium / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*
chemical_list: D000963:Antimetabolites; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006632:Histamine; D000255:Adenosine Triphosphate; D003847:Deoxyglucose; D008274:Magnesium; D002118:Calcium; D016257:Fura-2