During aging, mitochondria—the cellular organelles responsible for respiration and the production of energy—become dysregulated. In Caenorhabditis elegans, mitophagy, a selective type of autophagy that targets mitochondria for degradation, can interface with mitochondrial biogenesis. Normally, mitophagy helps ensure that only the most healthy mitochondria stay around, but under certain conditions, the balance of removing damaged mitochondria and generating new ones can become dysregulated.
Recently, Palikaras et al. examined mitophagy in C. elegans during aging. The team was interested in understanding molecular interactions between mitochondrial phosphatase and tensin (PTEN)-induced kinase 1 (PINK-1), PDR-1 (another component of mitophagy), and DCT-1 protein, which is a key mediator of mitophagy and longevity assurance under stress in C. elegans.
The researchers generated transgenic animals expressing the mitochondria-targeted Rosella (mt Rosella) biosensor, which combines a fast-maturing pH-insensitive DsRed fused to a pH-sensitive green fluorescent protein variant. They also performed knockdown experiments in wild-type and long-lived mutants, which revealed that the depletion of DCT-1, PINK-1 or PDR-1 alters mitochondrial network morphology in body wall muscle cells. In addition, the team determined that dct-1 or pink-1 knockdown shortens the lifespan and makes worms more susceptible to stress. The team hypothesized that this diminished stress resistance may originate from the accumulation of dysfunctional mitochondria in mitophagy mutants.
Next, the team examined the molecular interaction between DCT-1, PINK-1 and PDR-1 by immunoprecipitating DCT-1 in mitophagy-stimulating conditions. After immunoprecipitating the proteins, the team digested them with trypsin and then analyzed them using mass spectrometry. The researchers used nanoflow liquid chromatography-electrospray ionization with tandem mass spectrometry (nLC-ESI–MS/ MS) using an EASY-nLC liquid chromatograph system (software version 2.7.6 #1) coupled with an LTQ-Orbitrap XL ETD hybrid ion trap-Orbitrap mass spectrometer, through a nES ion source. The team used Xcalibur software to acquire data. The nLC-MS/MS raw data were loaded in Proteome Discoverer software and run using both Mascot 2.3.02 (Matrix Science) and Sequest (Thermo Scientific).
As a result, they determined DCT-1 is ubiquitinated on lysine 26, a modification that becomes enriched under mitophagy-inducing conditions. They also discovered that DCT-1 co-localizes with PDR-1, revealing a homeostatic feedback loop. They found that by uncoupling metabolic signals, mitochondrial biogenesis during aging contributes to overproliferation of damaged mitochondria and decline of cellular function.The team also identified a SKN-1 transcription factor that regulates both mitochondrial biogenesis genes and mitophagy by enhancing DCT-1 expression. The team posits that the ubiquitous expression of regulatory factors involved in mitophagy could be conserved across other taxa.
1. Palikaras, K., Lionaki, E. and Tavernarakis, N. (2015) “Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans,” Nature, 521 (pp. 525–528), doi:10.1038/nature14300.