Background

Phosphatase and tensin homolog-induced kinase 1 (PINK1) is a mitochondrial serine/threonine kinase involved in mitophagy, the selective degradation of damaged mitochondria. In healthy mitochondria, PINK1 is constitutively recruited to the outer mitochondrial membrane and imported into the inner membrane space, where it undergoes N-terminal cleavage by proteases and subsequent degradation in the cytosol [2]. Following mitochondrial damage, the inner mitochondrial membrane becomes depolarized, and PINK1 is stabilized in its full-length form. This results in the dimerization, trans-autophosphorylation, and activation of PINK1, allowing it to phosphorylate and activate the E3 ubiquitin ligase Parkin and ubiquitin [3]. Parkin catalyzes the ubiquitination of outer mitochondrial membrane proteins, tagging damaged mitochondria for degradation by autophagic machinery [4,5]. Loss-of-function autosomal recessive mutations in PINK1 are known to cause early onset Parkinson’s disease (EOPD) [6], leading to the onset of symptoms, including tremor, rigidity, and bradykinesia, at a mean age of 31 years [7]. While advances have been made in understanding the role of PINK1 in mitochondrial dynamics and involvement of PINK1 in the development of Parkinson’s disease, current tools available to study PINK1 typically only provide snapshots of PINK1 activity, provide indirect readouts of PINK1 activity, or rely on overexpression of PINK1 [8–10]. We therefore sought to develop a kinase activity reporter capable of characterizing endogenous PINK1 activity dynamics in live cells using both time-lapse microscopy and end-point imaging.

Fluorescent protein-based kinase activity reporters (KARs) offer a unique approach to study kinase activity dynamics in single cells with high spatiotemporal resolution [11]. KARs have been developed for several kinases, including AMP-activated protein kinase (AMPK), protein kinase A (PKA), and protein kinase C (PKC) [12]. One KAR design—separation of phase-based activity reporter of kinase (SPARK)—takes advantage of liquid–liquid phase separation for the detection of kinase activity [13]. In this KAR design, phosphorylation of the SPARK construct increases the local concentration of fluorophores, observed as puncta, resulting in a simple readout of kinase activity that can be multiplexed with other biosensors [14]. SPARK-based biosensors have been made for kinases, including PKA, ataxia-telangiectasia mutated (ATM), and AMPK [13–15]. As the SPARK design enables both time-lapse imaging and snapshots of kinase activity in single cells with high signal-to-noise, we sought to design a SPARK-based KAR for PINK1 (Figure 1).

While several methods exist to measure mitochondrial function, none are capable of directly reporting PINK1 activity in real time. Western blot analysis has so far been the standard for measuring PINK1 activity, but it only provides bulk readouts of activity at specific points in time. Other fluorescence protein–based tools have been created to provide readouts of mitophagy, for example, MT-Keima and Mito-QC [16]. Both rely on pH-dependent changes in fluorescent proteins to visualize mitochondria being taken up by autophagic machinery, but this is an indirect readout of PINK1 activity, as PINK1-independent mitophagy mechanisms have been described [17]. A more recent tool is MitoPain, which uses a mitochondrial PINK1 accumulation index to quantify mitochondrial stress by comparing PINK1-GFP and RFP-Omp25 signals [10]. While MitoPain allows for the quantification of mitochondrial stress, this reporter does not provide a direct readout of PINK1 activity and requires overexpression of PINK1. Our newly developed PINK1-SPARK utilizes the PINK1 phosphomotif from ubiquitin, a canonical PINK1 substrate, to overcome these limitations and measure endogenous PINK1 activity. Furthermore, we developed a HaloTag-based PINK1-SPARK for multiplexing of PINK1 activity with other markers of mitochondrial damage. We validated the use of PINK1-SPARK in a variety of cell lines, including PINK1 knockout HeLa cervical cancer cells, and found minimal response of PINK1-SPARK in PINK1 knockout HeLa cells. While PINK1-SPARK has a large dynamic range, its spatial resolution falls short of that of other KARS, notably Förster resonance energy transfer (FRET)-based KARS, which can measure kinase activity at distinct subcellular locations [11]. Additionally, we did not test the reversibility of PINK1-SPARK, as PINK1 inhibitors are nonspecific [18]; thus, PINK1-SPARK functions best as a “turn on” reporter of PINK1 activity. Nevertheless, PINK1-SPARK represents a significant advancement in the detection and quantification of endogenous PINK1 activity. In this protocol, we provide an overview of how to use PINK1-SPARK to study endogenous PINK1 activity at the single-cell level.

Figure 1. PINK1-SPARK protocol overview. (A) General scheme of reporter function. The first component consists of the tensin homolog-induced kinase 1 (PINK1)-specific ubiquitin consensus sequence, EGFP, and the hexameric homooligomeric tag, HOTag3. The phosphoamino acid binding domain, FHA2, and the tetrameric HOTag6 make up the second component. PINK1 phosphorylates Ser65 within its consensus sequence, allowing recognition and binding by FHA2. The resulting proximity of multivalent interactions promotes phase separation and allows for the visualization of PINK1 activity as green fluorescent droplets. (B) Time-lapse images of U2OS cells transiently expressing PINK1-SPARK following the addition of 10 μM CCCP. Adapted from Vineall et al. [1] under a CC BY-NC-ND 4.0 license.