An engineered biosensor enables dynamic aspartate measurements in living cells

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Intracellular aspartate levels fluctuate with cellular metabolism, impacting cell function.
• Current methods like mass spectrometry (MS) for aspartate measurement lack throughput and temporal resolution.
• Robust tools are needed to comprehensively understand aspartate metabolism.

Purpose of the Study:

• To develop a novel, high-throughput sensor for measuring intracellular aspartate abundance.
• To overcome the limitations of existing static, low-throughput aspartate measurement techniques.
• To enable real-time monitoring of aspartate dynamics in mammalian cells.

Main Methods:

• Development of a genetically encoded aspartate sensor (jAspSnFR3) based on green fluorescent protein (GFP).
• Characterization of the sensor's fluorescence properties, including dynamic range and specificity, using purified protein.
• Validation of the sensor's performance in mammalian cell lines by correlating fluorescence intensity with MS-measured aspartate levels.

Main Results:

• The jAspSnFR3 sensor exhibits a 20-fold increase in fluorescence upon aspartate saturation.
• Sensor fluorescence changes are dose-dependent and cover physiologically relevant aspartate concentrations.
• jAspSnFR3 successfully tracked real-time intracellular aspartate changes induced by genetic, pharmacological, and nutritional interventions.

Conclusions:

• jAspSnFR3 provides a sensitive and dynamic tool for quantifying intracellular aspartate.
• This sensor facilitates high-throughput and temporally resolved studies of aspartate metabolism.
• The developed sensor opens new avenues for investigating cellular processes influenced by aspartate levels.