title: Exploring dynamics of molybdate in living animal cells by a genetically encoded FRET nanosensor.
abstract: Molybdenum (Mo) is an essential trace element for almost all living organisms including animals. Mo is used as a catalytic center of molybdo-enzymes for oxidation/reduction reactions of carbon, nitrogen, and sulfur metabolism. Whilst living cells are known to import inorganic molybdate oxyanion from the surrounding environment, the in vivo dynamics of cytosolic molybdate remain poorly understood as no appropriate indicator is available for this trace anion. We here describe a genetically encoded Förester-resonance-energy-transfer (FRET)-based nanosensor composed of CFP, YFP and the bacterial molybdate-sensor protein ModE. The nanosensor MolyProbe containing an optimized peptide-linker responded to nanomolar-range molybdate selectively, and increased YFP:CFP fluorescence intensity ratio by up to 109%. By introduction of the nanosensor, we have been able to successfully demonstrate the real-time dynamics of molybdate in living animal cells. Furthermore, time course analyses of the dynamics suggest that novel oxalate-sensitive- and sulfate-resistant- transporter(s) uptake molybdate in a model culture cell.
authors.affiliation: Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan. nakanish@agr.nagoya-u.ac.jp
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Nakanishi
authors.forename: Syuntaro
authors.initials: S
authors.lastname: Iida
authors.forename: Hanayo
authors.initials: H
authors.lastname: Ueoka-Nakanishi
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Niimi
authors.forename: Rie
authors.initials: R
authors.lastname: Tomioka
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Maeshima
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D015374:Biosensing Techniques / Q000295:instrumentation*; D004058:Diffusion; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry; D008982:Molybdenum / Q000494:pharmacology*; D036103:Nanotechnology / Q000379:methods*; D010070:Oxalates / Q000737:chemistry; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D012680:Sensitivity and Specificity; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; C101050:ModE protein, bacteria; D010070:Oxalates; D010455:Peptides; D014157:Transcription Factors; C054562:yellow fluorescent protein, Bacteria; C044659:molybdate; D049452:Green Fluorescent Proteins; D008982:Molybdenum