Visualizing PINK1 Activity Dynamics in Single Cells with a Phase Separation-Based Kinase Activity Reporter.

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• Phosphatase and tensin homologue-induced kinase 1 (PINK1) is crucial for mitophagy, cell death, and cellular bioenergetics.
• Existing bulk techniques for studying PINK1 activity provide limited temporal resolution and miss cell-to-cell variations.
• A need exists for dynamic, single-cell methods to investigate PINK1 function.

Purpose of the Study:

• To develop a novel biosensor for real-time monitoring of PINK1 kinase activity.
• To characterize PINK1 activity dynamics and heterogeneity in single live cells.
• To enable deeper understanding of PINK1's role in cellular processes like mitophagy.

Main Methods:

• Adapted the separation of phases-based activity reporter of kinase (SPARK) design to create a PINK1-specific biosensor (PINK1-SPARK).
• Utilized PINK1-SPARK to observe real-time kinase activity in single cells under various conditions (mitochondrial depolarization, pharmacological activation).
• Developed a Halo Tag-based PINK1-SPARK for multiplexed imaging alongside mitochondrial damage markers.

Main Results:

• Successfully developed and validated the PINK1-SPARK biosensor for temporal measurement of PINK1 activity.
• Observed real-time PINK1 activity in single cells, revealing dynamic changes.
• Enabled simultaneous imaging of PINK1 activity and mitochondrial damage using the Halo Tag-SPARK variant.

Conclusions:

• PINK1-SPARK is a novel tool for precise, temporal measurement of PINK1 activity in individual live cells.
• This biosensor overcomes limitations of bulk assays, facilitating studies on PINK1 dynamics and heterogeneity.
• The tool will advance research into PINK1's roles in mitophagy, cell death, and bioenergetics.