Dynamic visualization of physiological CaMKII activity using sensitive FRET biosensors

Area of Science:

• Molecular Biology
• Neuroscience
• Biochemistry

Background:

• Calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) regulates vital physiological processes.
• Visualizing CaMKII activity in living systems is crucial for understanding health and disease.
• Current methods lack the sensitivity and specificity for robust CaMKII monitoring.

Purpose of the Study:

• To engineer novel Förster resonance energy transfer (FRET)-based biosensors for CaMKII.
• To achieve high specificity, sensitivity, and signal-to-noise ratio for CaMKII activity reporters.
• To enable quantitative visualization of endogenous CaMKII signaling in various cell types and tissues.

Main Methods:

• Leveraged a serine/threonine kinome-wide substrate atlas for rational biosensor design.
• Engineered a suite of FRET-based CaMKII kinase activity reporters.
• Utilized 2-photon fluorescence lifetime imaging (2pFLIM) on organotypic hippocampal slices.

Main Results:

• Developed highly specific and sensitive FRET biosensors for CaMKII.
• Successfully visualized endogenous CaMKII activity in cultured cell lines, cardiomyocytes, oocytes, and neurons.
• Quantitatively tracked LTP-induced CaMKII activity in single dendritic spines using 2pFLIM imaging.

Conclusions:

• The new FRET biosensors represent a significant advance for studying CaMKII signaling.
• These tools allow for robust and quantitative monitoring of CaMKII dynamics in physiologically relevant contexts.
• Enables deeper insights into the molecular regulation of CaMKII in health and disease.