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

Phosphatase and tensin homologue-induced kinase 1 (PINK1) is a serine/threonine kinase that plays roles in mitophagy, cell death, and regulation of cellular bioenergetics. Current approaches for studying PINK1 function depend on bulk techniques that can only provide snapshots of activity and could miss the dynamics and cell-to-cell heterogeneity of PINK1 activity. Therefore, we sought to develop a novel PINK1 kinase activity reporter to characterize PINK1 activity. Taking advantage of the separation of phase-based activity reporter of kinase (SPARK) design, we developed a phase separation-based PINK1 biosensor (PINK1-SPARK). With PINK1-SPARK, we observe real-time PINK1 activity in single cells treated with mitochondria-depolarizing agents or pharmacological activators. We then developed a HaloTag-based PINK1-SPARK for multiplexed imaging of PINK1 activity with live-cell markers of mitochondrial damage. Thus, PINK1-SPARK is a new tool that enables temporal measurement of PINK1 activity in single live cells, allowing for further elucidation of the role of PINK1 in mitophagy and cell function.