Calcium Indicators with Fluorescence Lifetime-Based Signal Readout: A Structure-Function Study

Area of Science:

• Cellular biology
• Neuroscience
• Biochemistry

Background:

• Calcium ions (Ca2+) are vital signaling molecules in cellular processes.
• Genetically encoded calcium indicators (GECIs) are essential for monitoring Ca2+ dynamics in living cells.
• Existing GECIs face limitations in temporal resolution and absolute concentration measurements.

Purpose of the Study:

• To engineer novel GECIs based on the GCaMP6 backbone and BrUSLEE fluorescent protein.
• To address challenges in calcium imaging, specifically temporal resolution and static concentration measurements.
• To investigate structural determinants of calcium sensitivity in GECIs.

Main Methods:

• Design and construction of two GECI variants: GCaMP6s-BrUS and GCaMP6s-BrUS-145.
• Utilizing fluorescence lifetime measurements to assess indicator properties.
• Employing structural determination and comparative analysis to understand calcium sensitivity.
• Optimizing GCaMP6s-BrUS for enhanced molecular brightness and temporal dynamics.

Main Results:

• GCaMP6s-BrUS exhibits reduced, calcium-insensitive fluorescence lifetime for high temporal resolution imaging.
• GCaMP6s-BrUS-145 shows flexible, calcium-sensitive fluorescence lifetime for absolute concentration determination via FLIM.
• Structural analysis identified key determinants for calcium sensitivity.
• Optimized GCaMP6s-BrUS variant demonstrates improved brightness and calcium-sensitive temporal behavior.

Conclusions:

• The developed GECIs offer distinct advantages for different calcium imaging applications.
• These engineered indicators provide enhanced capabilities for studying cellular calcium dynamics.
• The findings provide a foundation for future development of advanced GECIs with tailored properties.