ACS sensorsFeb 3, 2026

Tracking PINK1 Enzyme Activity Over Time in Single Cells Using a Phase Separation Sensor

Longevity & Aging Weekly Brief ↗PubMed ↗DOI ↗

Updated Feb 4, 2026

Abstract

A novel PINK1 biosensor enables real-time measurement of PINK1 activity in single cells.

PINK1 is associated with crucial processes such as mitophagy and cellular energy regulation.
Current methods for studying PINK1 may overlook the dynamic and heterogeneous nature of its activity.
The newly developed PINK1-SPARK biosensor allows for the observation of PINK1 activity in real-time.
This tool facilitates the assessment of PINK1 activity in response to mitochondrial stress and pharmacological agents.
Multiplexed imaging with PINK1-SPARK can be achieved alongside live-cell markers of mitochondrial damage.

Simplified

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.

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Funding

Competing interests

The authors declare they have no competing interests. A preprint of this article was posted on October 9, 2025 as 2. Vineall K.G., Andrikopoulos A., Sun M.J., Yan A., Hartanto E.R., and Schmitt D.L. Visualizing PINK1 Activity Dynamics in Single Cells with a Phase Separation-Based Kinase Activity Reporter. BioRxiv, 2025 DOI: 10.1101/2025.10.08.681260.

PubMed

Tracking PINK1 Enzyme Activity Over Time in Single Cells Using a Phase Separation Sensor

Abstract

Full Text

Funding

Competing interests

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