A high-performance genetically encoded sensor for cellular imaging of PKC activity in vivo

Area of Science:

• Neuroscience
• Biochemistry
• Molecular Biology

Background:

• Protein kinase C (PKC) plays a crucial role in cellular signaling pathways.
• Existing sensors for PKC activity have limitations in dynamic range and in vivo application.
• Understanding PKC activity in specific neuronal populations is essential for deciphering brain function.

Purpose of the Study:

• To develop and validate a novel genetically encoded fluorescence lifetime sensor for monitoring protein kinase C (PKC) activity.
• To enhance the dynamic range of PKC sensors for improved sensitivity.
• To enable in vivo imaging of PKC activity in the brain during complex behaviors.

Main Methods:

• Development of CKAR3, a Förster resonance energy transfer (FRET)-based genetically encoded sensor.
• Characterization of CKAR3's dynamic range and performance compared to parental sensors.
• In vivo imaging of PKC activity in neuronal subsets within the motor cortex during rodent locomotion.

Main Results:

• CKAR3 demonstrated a 10-fold increased dynamic range compared to previous sensors.
• Robust and specific PKC activity was observed in a sparse neuronal subset in the motor cortex during locomotion.
• Muscarinic acetylcholine receptors were identified as partially mediating the observed PKC activity.

Conclusions:

• CKAR3 is a powerful new tool for studying PKC signaling in vivo.
• PKC activity in specific motor cortex neurons is dynamically regulated during locomotion.
• Muscarinic acetylcholine receptor signaling contributes to PKC activation in motor circuits.