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

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Adenosine 5' triphosphate (ATP) serves as a primary intracellular energy currency.
• ATP also functions as a critical extracellular signaling molecule across numerous species.
• Existing methods for ATP imaging present limitations in scope and application.

Purpose of the Study:

• To develop and characterize novel genetically encoded fluorescent sensors for imaging extracellular and cytosolic ATP.
• To assess the sensitivity, specificity, and applicability of these sensors in biological systems.
• To provide a new tool for real-time ATP monitoring in diverse cellular environments.

Main Methods:

• Generation of genetically encoded fluorescent sensors (iATPSnFRs) by inserting circularly permuted superfolder GFP into the epsilon subunit of F0F1-ATPase.
• Characterization of sensor response to ATP concentrations (30 μM to 3 mM) in both extracellular and cytosolic compartments.
• Testing sensor specificity against other nucleotides and nucleosides, and assessing pH sensitivity.

Main Results:

• iATPSnFRs exhibit fast fluorescence increases in response to relevant ATP concentrations.
• Sensors can be genetically targeted to specific cell types and subcellular locations.
• iATPSnFRs demonstrate specificity for ATP over other nucleotides and nucleosides, with modest pH sensitivity.
• Fusion with red fluorescent proteins enables ratiometric ATP imaging.

Conclusions:

• iATPSnFRs are effective tools for imaging both extracellular and cytosolic ATP.
• These sensors are suitable for use with standard light microscopy and can be targeted to specific cellular compartments.
• iATPSnFRs offer a promising platform for advancing ATP research in various biological settings, with potential for further optimization.