Abstract
BackgroundThe PINK1:Parkin pathway regulates the autophagic removal of damaged and dysfunctional mitochondria. While the response of this pathway to complete loss of ΔΨm, as caused by high concentrations of mitochondrial ionophores, has been well characterized, it remains unclear how the pathway makes coherent decisions about whether to keep or purge mitochondria in situations where ΔΨm is only partially lost or exhibits fluctuations, as has been observed in response to certain types of cellular stress.ResultsTo investigate the responses of the PINK1:Parkin pathway to mitochondrial insults of different magnitude and duration, controlled titration of the mitochondrial protonophore, CCCP, was used to manipulate ΔΨm in live cells, and the dynamics of PINK1 and Parkin recruitment was measured by fluorescence microscopy. In contrast to the stable accumulation of PINK1 and Parkin seen at completely depolarized mitochondria, partial depolarization produced a transient pulse of PINK1 stabilization and rapid loss, which was driven by small fluctuations in ΔΨm. As the rate of Parkin dissociation from the mitochondria and phospho-polyubiquitin chain removal was comparatively slow, repetitive pulses of PINK1 were able to drive a slow step-wise accumulation of Parkin and phospho-polyubiquitin leading to deferred mitophagy.ConclusionThese data suggest that the PINK1:Parkin mitophagy pathway is able to exhibit distinct dynamic responses to complete and partial mitochondrial depolarization. In this way, the pathway is able to differentiate between irretrievably damaged mitochondria and those showing signs of dysfunction, promoting either rapid or delayed autophagy, respectively.
Highlights
The PTEN-induced putative kinase 1 (PINK1):Parkin pathway regulates the autophagic removal of damaged and dysfunctional mitochondria
Titration of carbonyl cyanide m– chlorophenyl hydrazone (CCCP) to generate states of partial and complete loss of mitochondrial membrane potential In order to investigate how the PINK1:Parkin mitophagy pathway responds to complex stress signals – defined here as transient or continuous, complete or partial loss of mitochondrial membrane potential – we utilized the reversible inhibitor of oxidative phosphorylation, CCCP
In order to test whether a partial loss of Mitochondrial membrane potential (ΔΨm) could induce mitophagy, EYFP-Parkin expressing HeLa cells were incubated with 0, 5, or 10 μM CCCP for 24 h and immunofluorescently stained with anti-DNA antibodies to detect mtDNA nucleoids
Summary
The PINK1:Parkin pathway regulates the autophagic removal of damaged and dysfunctional mitochondria. The mitochondria are vitally important, multifunctional organelles, responsible for ATP production, the regulation of apoptosis, and other cell type-specific processes. While several pathways exist that regulate the autophagic removal of mitochondria from the cell [1,2,3,4], the PTEN-induced putative kinase 1 (PINK1):Parkin pathway is perhaps the most prominent and most closely associated with the destruction of mitochondria in response to stress or loss of mitochondrial membrane potential (ΔΨm) in normal, somatic cells [2, 5]. The PINK1 protein itself is a highly labile, nuclearencoded, mitochondrially-targeted kinase that essentially acts as a sensor of mitochondrial membrane potential (ΔΨm). There, the amino-terminus of PINK1 undergoes two rounds of proteolytic processing; the mitochondrial targeting sequence
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
