A Monochromatically Excitable Green-Red Dual-Fluorophore Fusion Incorporating a New Large Stokes Shift Fluorescent...

Area of Science:

• Biotechnology
• Cell Biology
• Molecular Imaging

Background:

• Genetically encoded sensors are crucial for quantitative live-cell imaging of analytes.
• Single fluorescent protein (FP) sensors can suffer from artifacts due to expression level or distribution changes.
• Dual-FP Matryoshka sensors offer ratiometric quantification capabilities by nesting a reference FP within a reporter FP.

Purpose of the Study:

• To engineer a novel genetically encoded calcium sensor with enhanced imaging properties.
• To develop a sensor utilizing green apple (GA) Matryoshka technology with a new red large Stokes shift (LSS) fluorophore.
• To enable high-throughput quantitative calcium imaging in yeast.

Main Methods:

• Construction of a dual-FP Matryoshka sensor incorporating a red LSSmApple fluorophore and cpEGFP reporter.
• Characterization of LSSmApple maturation time and spectral properties for monochromatic coexcitation.
• Development of an image analysis pipeline for yeast (Saccharomyces cerevisiae) timelapse imaging.

Main Results:

• A new red LSSmApple fluorophore was engineered with faster maturation and optimized spectral overlap with cpEGFP.
• The LSSmApple fluorophore demonstrated improved emission spectrum separation, minimizing bleed-through for clearer imaging.
• The developed GA-MatryoshCaMP6s sensor successfully enabled quantitative calcium imaging in yeast.

Conclusions:

• The engineered LSSmApple fluorophore and GA-MatryoshCaMP6s sensor represent a significant advancement in genetically encoded calcium indicators.
• The sensor facilitates robust, high-throughput quantitative calcium imaging in yeast, overcoming limitations of single-FP sensors.
• This technology holds promise for advancing live-cell imaging and quantitative analysis across various biological systems.