Development of a FRET biosensor with high specificity for Akt

Area of Science:

• Cell Biology
• Molecular Signaling
• Biotechnology

Background:

• The serine/threonine kinase Akt is crucial for cell proliferation, survival, and cancer.
• While Akt's plasma membrane activation is known, its subcellular activity in living cells remains unclear.
• Fluorescence resonance energy transfer (FRET) biosensors enable visualization of signaling molecule activity.

Purpose of the Study:

• To develop and validate a specific FRET biosensor for monitoring Akt activity in living cells.
• To investigate the subcellular localization and dynamics of Akt activation.
• To compare Akt activation patterns in different cell lines upon epidermal growth factor (EGF) stimulation.

Main Methods:

• Development of a novel FRET biosensor, Eevee-iAkt, based on the Eevee backbone.
• Validation of Eevee-iAkt specificity using kinase inhibitors.
• Targeting the biosensor to various subcellular compartments (plasma membrane, mitochondria, nucleus) in HeLa and Cos7 cells.
• Live-cell imaging of Akt activity following EGF stimulation.

Main Results:

• Eevee-iAkt specifically monitored Akt activity in living cells.
• HeLa cells showed transient Akt activation across the plasma membrane, cytosol, and mitochondria upon EGF stimulation.
• Cos7 cells exhibited sustained and localized Akt activation at the plasma membrane, with low cytosolic and undetectable mitochondrial/nuclear activity.

Conclusions:

• The Eevee-iAkt biosensor is a valuable tool for studying subcellular Akt activity.
• Significant differences in Akt activation dynamics and localization exist between HeLa and Cos7 cells.
• This tool can advance research on the functional relationship between subcellular Akt activation and its role in cell survival.