A genetically encoded single-wavelength sensor for imaging cytosolic and cell surface ATP

Nat Commun. 2019 Feb 12;10(1):711. doi: 10.1038/s41467-019-08441-5.

Abstract

Adenosine 5' triphosphate (ATP) is a universal intracellular energy source and an evolutionarily ancient, ubiquitous extracellular signal in diverse species. Here, we report the generation and characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of F0F1-ATPase from Bacillus PS3. On the cell surface and within the cytosol, iATPSnFR1.0 responds to relevant ATP concentrations (30 μM to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. After careful consideration of their modest pH sensitivity, iATPSnFRs represent promising reagents for imaging ATP in the extracellular space and within cells during a variety of settings, and for further application-specific refinements.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATPase Inhibitory Protein
  • Adenosine Triphosphate / chemistry*
  • Adenosine Triphosphate / genetics
  • Bacillus / cytology
  • Bacillus / genetics
  • Bacillus / metabolism
  • Bacterial Proteins / genetics
  • Cell Membrane / chemistry*
  • Cytosol / chemistry*
  • Fluorescence Resonance Energy Transfer / methods*
  • Gene Expression
  • HEK293 Cells
  • Humans
  • Hydrogen-Ion Concentration
  • Image Processing, Computer-Assisted
  • Kinetics
  • Luminescent Proteins / genetics
  • Microscopy, Fluorescence
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Protein Conformation
  • Proteins / metabolism*
  • Red Fluorescent Protein

Substances

  • Bacterial Proteins
  • Luminescent Proteins
  • Proteins
  • Adenosine Triphosphate