Development of a Dual Reporter System to Simultaneously Visualize Ca²⁺ Signals and AMPK Activity

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• AMP-activated kinase (AMPK) is a crucial energy sensor involved in cellular metabolism.
• Monitoring AMPK activity in real-time is essential for understanding cellular responses to various stimuli.
• Existing methods for measuring AMPK activity can be complex and lack dynamic temporal resolution.

Purpose of the Study:

• To develop a novel reporter system, AMPK-SPARK, for visualizing AMP-activated kinase (AMPK) activation.
• To create a dual reporter system, GCaMP-AMPK-SPARK, for simultaneous monitoring of intracellular calcium (Ca2+) levels and AMPK activity.
• To investigate cell-to-cell heterogeneity in AMPK activation dynamics in response to Ca2+ signaling.

Main Methods:

• Development of a separation of phases-based activity reporter of kinase (SPARK) for AMPK.
• Incorporation of a Ca2+ biosensor (GCaMP6f) into the SPARK system to create a dual reporter.
• Utilizing single-channel fluorescence microscopy for simultaneous detection of Ca2+ and AMPK activity.
• Mapping endogenous AMPK activity and visualizing its dynamics in various cell types and in response to stimuli.

Main Results:

• AMPK-SPARK successfully reports AMPK activation through the formation of bright fluorescent clusters.
• GCaMP-AMPK-SPARK enables simultaneous, single-channel monitoring of Ca2+ and AMPK activity without spectral overlap issues.
• The study visualized the dynamics of AMPK activation in response to diverse stimuli.
• Cell-to-cell heterogeneities in AMPK activation, particularly in relation to Ca2+ mobilization, were revealed using the dual reporter.

Conclusions:

• The developed AMPK-SPARK and GCaMP-AMPK-SPARK systems provide powerful tools for studying kinase activity and cellular signaling.
• The dual reporter strategy facilitates the investigation of crosstalk between Ca2+ signaling and AMPK pathways.
• This approach can be broadly applied to explore complex interplays within cellular signaling networks.