Imaging extracellular ATP with a genetically-encoded, ratiometric fluorescent sensor.

Area of Science:

• Cell Biology
• Biochemistry
• Neuroscience

Background:

• Extracellular adenosine triphosphate (ATP) is a crucial signaling molecule in purinergic communication.
• Existing methods for measuring extracellular ATP are often invasive or require exogenous substrates.
• There is a need for robust, minimally invasive techniques to monitor extracellular ATP dynamics.

Purpose of the Study:

• To engineer a genetically-encoded sensor for real-time measurement of extracellular ATP.
• To develop a cell-surface expressed sensor for improved accessibility and reduced invasiveness.
• To enable ratiometric quantitation of extracellular ATP fluxes for accurate signaling analysis.

Main Methods:

• Re-engineering the ATeam ATP sensor for cell-surface expression.
• Utilizing live-cell microscopy to image extracellular ATP dynamics.
• Characterizing sensor performance in cultured Neuro2A cells.
• Measuring stimulated ATP release and ectonucleotidase-mediated clearance.

Main Results:

• Successfully expressed a genetically-encoded ATP sensor on the cell surface.
• Demonstrated real-time imaging of extracellular ATP levels without exogenous substrates.
• Achieved ratiometric quantitation of extracellular ATP, allowing flux measurements.
• Validated sensor performance by observing ATP release and clearance dynamics.

Conclusions:

• This proof-of-principle study presents a first-generation cell-surface sensor for extracellular ATP.
• The sensor facilitates robust and minimally invasive monitoring of purinergic signaling.
• This tool holds potential for studying extracellular ATP dynamics in living specimens and various biological contexts.