A genetically encoded far-red fluorescent calcium ion biosensor derived from a biliverdin-binding protein

Area of Science:

• Biophysics
• Molecular Biology
• Neuroscience

Background:

• Genetically encoded calcium ion (Ca2+) indicators (GECIs) are vital for visualizing neural activity and cell signaling.
• Far-red and near-infrared (NIR) GECIs are crucial for deep-tissue and multiplexed imaging due to compatibility with common laser lines.
• Previous engineering efforts focused on developing NIR fluorescent proteins from biliverdin (BV)-binding proteins.

Purpose of the Study:

• To engineer a novel far-red fluorescent GECI, named iBB-GECO1, from a previously reported NIR GECI.
• To characterize the performance of iBB-GECO1, including its brightness, Ca2+ response, and dissociation constant.
• To demonstrate the utility of iBB-GECO1 in multicolor imaging applications in mammalian cells.

Main Methods:

• Protein engineering of a biliverdin-binding NIR fluorescent protein to create a far-red GECI.
• Characterization of iBB-GECO1's photophysical properties and Ca2+ sensitivity (Kd).
• Demonstration of iBB-GECO1 in four-color and five-color multiplexed imaging experiments in MIN6 and HEK293T cells.

Main Results:

• Developed iBB-GECO1, a far-red excitable GECI with high molecular brightness.
• iBB-GECO1 exhibits an inverse Ca2+ response (ΔF/Fmin = -13) and a near-optimal Kd of 105 nM.
• Successfully demonstrated iBB-GECO1 in four-color and five-color multiplexed imaging in cultured mammalian cells.
• Preliminary in vivo imaging in mice showed potential, guiding future development.

Conclusions:

• iBB-GECO1 is a promising far-red fluorescent GECI suitable for multiplexed imaging in mammalian cells.
• The engineered GECI possesses desirable characteristics for advanced cellular and potentially deep-tissue imaging applications.
• Further optimization is needed for robust in vivo neural activity imaging, but initial results are encouraging.