New biosensors and transgenic mice for multiplex cGMP imaging

Area of Science:

• Cellular signaling
• Molecular imaging
• Biotechnology

Background:

• Cyclic guanosine monophosphate (cGMP) is a crucial second messenger in human physiology and pharmacotherapy.
• Live-cell biosensors are vital for real-time elucidation of cGMP dynamics.
• Spectral compatibility is essential for monitoring multiple cGMP pools or cGMP/cAMP crosstalk.

Purpose of the Study:

• To develop and characterize novel Förster/fluorescence resonance energy transfer (FRET)-based cGMP biosensors.
• To enable spectrally compatible imaging of cGMP and cAMP within the same cell.
• To establish transgenic mouse models for advanced cGMP research.

Main Methods:

• Development of membrane-targeted mcGi500 and red-shifted cytosolic red-cGi500 FRET-based cGMP indicators.
• Creation of transgenic mouse lines for global and cell-specific mcGi500 expression.
• Performance characterization in primary vascular smooth muscle cells and comparison with existing sensors.

Main Results:

• mcGi500 and red-cGi500 exhibit high selectivity for cGMP over cAMP.
• New sensors demonstrate approximately 3-fold higher sensitivity than the gold standard cGi500.
• Red-cGi500 enables robust cGMP detection in intact cells, and co-imaging with Epac1-camps allows simultaneous cGMP/cAMP visualization.

Conclusions:

• Red-cGi500 and mcGi500 are valuable tools for advanced cGMP imaging.
• These biosensors facilitate multiplex imaging of cGMP compartments and signaling crosstalk.
• Transgenic mouse lines provide powerful models for in vivo cGMP studies.