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Published on: February 7, 2018
Genetically-encoded yellow fluorescent cAMP indicator with an expanded dynamic range for dual-color imaging
Haruki Odaka1, Satoshi Arai2, Takafumi Inoue3
1Cell Signaling Group, Waseda Bioscience Research Institute in Singapore (WABIOS), Waseda University, Singapore, Singapore; Department of Life Science and Medical Bioscience, School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan.
Researchers developed Flamindo2, an improved yellow fluorescent cAMP indicator with enhanced dynamic range and brightness. This tool enables better live-cell imaging of cyclic AMP (cAMP) signaling dynamics in various cellular compartments.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Cyclic AMP (cAMP) is a crucial second messenger regulating numerous cellular processes.
- Existing Förster resonance energy transfer (FRET)-based cAMP indicators have limitations in dynamic range and brightness.
- Development of advanced single fluorescent protein-based indicators is needed for precise cAMP monitoring.
Purpose of the Study:
- To develop an improved yellow fluorescent protein-based cyclic AMP (cAMP) indicator with enhanced performance.
- To assess the dynamic range, brightness, and applicability of the new indicator in live-cell imaging.
Main Methods:
- Optimization of linker peptides in a yellow fluorescent protein-based cAMP indicator (Flamindo) to create Flamindo2.
- Characterization of Flamindo2's dynamic range and brightness.
- Live-cell imaging of cAMP dynamics in cytosol and nucleus using Flamindo2 and nlsFlamindo2.
- Dual-color imaging of cAMP and Ca2+ dynamics using Flamindo2 and R-GECO.
Main Results:
- Flamindo2 exhibits a 2-fold expanded dynamic range and 8-fold increased brightness compared to Flamindo.
- Fluorescence intensity of Flamindo2 decreased by 75% in response to cAMP.
- Forskolin elevated cAMP levels in cytosol and nucleus with distinct kinetics.
- Noradrenaline induced simultaneous elevation of cAMP and Ca2+ in single HeLa cells.
Conclusions:
- Flamindo2 is a significantly improved cAMP indicator offering enhanced dynamic range and brightness.
- Flamindo2 and its nuclear variant (nlsFlamindo2) are effective for live-cell imaging of cAMP dynamics in different cellular compartments.
- Flamindo2 is suitable for multi-color imaging, providing a valuable tool for studying intracellular signaling crosstalk.
