High-performance genetically-encoded green and red fluorescent biosensors for pyruvate

Area of Science:

• Biochemistry
• Cell Biology
• Metabolomics

Background:

• Pyruvate is a critical metabolite linking glycolysis to various cellular pathways.
• Investigating pyruvate's physiological roles is hampered by the absence of high-performance fluorescent protein (FP)-based biosensors.
• Existing biosensors often lack the required sensitivity (ΔF/F₀ > 10) for robust pyruvate detection.

Purpose of the Study:

• To develop novel, high-performance FP-based biosensors for accurate pyruvate detection.
• To create both green fluorescent protein (GFP)-based and red fluorescent protein (RFP)-based biosensor variants.
• To enable advanced imaging techniques for studying pyruvate dynamics in live cells.

Main Methods:

• Development of GreenPy1 (GFP-based) and ApplePy1 (RFP-based) pyruvate biosensor series.
• Characterization of biosensor performance, including fluorescence intensity change (ΔF/F₀) and affinity (Kd).
• Application of biosensors for multicolor imaging of pyruvate concentration changes in mammalian cells.

Main Results:

• GreenPy1 and ApplePy1 biosensors demonstrate significant fluorescence intensity changes (ΔF/F₀ ~ 20 to >40).
• The biosensors offer a range of affinities, suitable for detecting pyruvate concentrations from tens of μM to several mM.
• Successful multicolor imaging of dynamic pyruvate level changes in mammalian cells was achieved.

Conclusions:

• The GreenPy1 and ApplePy1 biosensor series represent a significant advancement in pyruvate detection technology.
• These biosensors provide high sensitivity and versatility for studying pyruvate metabolism.
• Their utility in multicolor imaging opens new avenues for understanding cellular metabolic regulation.