Filtering of Transient and Low-Level Mitochondrial Damage Signals by the PINK1:Parkin Mitophagy Pathway

Abstract

Mitophagy describes a collection of pathways that direct the selective autophagic removal of damaged or superfluous mitochondria within eukaryotic cells. Of these, the PINK1 (PTEN-induced putative kinase 1):Parkin mitophagy pathway is perhaps the best known and is responsible for marking depolarized mitochondria for destruction. PINK1 continuously surveils mitochondrial membrane potential (MMP), an indicator of mitochondrial health, and is stabilized at mitochondria that exhibit a significant loss in MMP, and recruits cytosolic Parkin. Together PINK1 and Parkin assemble phospho-polyubiquitin (ppUB) chains on outer mitochondrial membrane (OMM) substrates, thereby tagging those mitochondria for removal by autophagy. This is accomplished through the binding of autophagy receptors, such as optineurin (OPTN), to ppUb chains which facilitates the recruitment of the autophagosome and subsequent degradation of the mitochondrion. The topology of the PINK1:Parkin pathway is complex and contains several feedback loops, including a coherent feed-forward loop between PINK1, Parkin, and ppUb. It is believed that this network motif may create a delay in ppUb chain assembly, such that only mitochondria which demonstrate a near-complete and continuous loss in MMP are successfully autophagized. However, mitochondria are likely to experience a range of insults and stresses in vivo, particularly in aged cells, and it is unclear how the PINK1:Parkin pathway might interpret the resultant time-varying changes in MMP. In this study we investigate this in detail by carefully manipulating MMP in live cells through titration of the reversible protonophore carbonyl cyanide m-chlorophenyl hydrazine (CCCP), and measuring the dynamics of PINK1 and Parkin mitochondrial recruitment and loss by fluorescence microscopy. These data show that PINK1 is highly sensitive to fluctuations in MMP and rapidly dissociates when MMP is even partially restored. Conversely, Parkin dissociation and ppUb chain disassembly from repolarized mitochondria is comparatively slow and thus allows pulses of mitochondrial PINK1 to drive a step-wise accumulation of Parkin and ppUb. This nuanced view of the pathway proposes a model whereby mitochondria that do not exhibit large and persistent losses in MMP, but still pose a threat to overall cellular health, can be removed by PINK1 and Parkin activity.