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Updated: Jun 4, 2025

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

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

Tatiana R Simonyan¹, Larisa A Varfolomeeva², Anastasia V Mamontova¹

¹Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997, Russia.

International Journal of Molecular Sciences

|December 17, 2024

Summary

This summary is machine-generated.

Researchers developed novel genetically encoded calcium indicators (GECIs) by modifying the GCaMP6 backbone. These new tools offer improved temporal resolution and capabilities for measuring absolute calcium concentrations in living cells.

Keywords:

FLIM fluorescence lifetime genetically encoded indicators quantitative calcium imaging structural analysis

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Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Published on: February 13, 2012

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.