Exploring dynamics of molybdate in living animal cells by a genetically encoded FRET nanosensor

PLoS One. 2013;8(3):e58175. doi: 10.1371/journal.pone.0058175. Epub 2013 Mar 5.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism*
  • Biosensing Techniques / instrumentation*
  • Diffusion
  • Fluorescence Resonance Energy Transfer / methods*
  • Green Fluorescent Proteins / chemistry
  • HEK293 Cells
  • Humans
  • Luminescent Proteins / chemistry
  • Molybdenum / pharmacology*
  • Nanotechnology / methods*
  • Oxalates / chemistry
  • Oxidation-Reduction
  • Peptides / chemistry
  • Sensitivity and Specificity
  • Time Factors
  • Transcription Factors / metabolism*

Substances

  • Bacterial Proteins
  • Cyan Fluorescent Protein
  • Luminescent Proteins
  • ModE protein, bacteria
  • Oxalates
  • Peptides
  • Transcription Factors
  • yellow fluorescent protein, Bacteria
  • molybdate
  • Green Fluorescent Proteins
  • Molybdenum

Grants and funding

This work was supported by Grant-in-Aid for Scientific Research on Innovative Areas (20200031) from The Ministry of Education, Culture, Sports, Science and Technology (to Y.N.), and by Grant-in-Aid for Young Scientists B (22780089) from Japan Society for the Promotion of Science (to Y.N.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.