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Perturbation of mitochondrial function is a major hallmark of several pathological conditions and ageing, underlining the essential role of fine-tuned mitochondrial activity (Lopez-Otin et al., 2013). Mitochondrial selective autophagy, known as mitophagy, mediates the removal of dysfunctional and/or superfluous organelles, preserving cellular and organismal homeostasis (Palikaras and Tavernarakis, 2014; Pickrell and Youle, 2015; Scheibye-Knudsen et al., 2015). In this protocol, we describe a method for assessing mitophagy in the nematode Caenorhabditis elegans.
Mitochondria are characterized as cellular powerhouses of eukaryotic cells, since they are the major energy providers through oxidative phosphorylation (OXPHOS) and ATP generation. Moreover, their pivotal role in cellular homeostasis is highlighted by their contribution in the regulation of several fundamental cellular processes including calcium buffering, metabolite synthesis and apoptosis, among others. Deregulation of mitochondrial function is associated with the onset of several pathological conditions including ageing and age-related neurodegenerative diseases (Vafai and Mootha, 2012; Palikaras and Tavernarakis, 2014). Thus, eukaryotic organisms have evolved several complex and highly specialized molecular pathways to guard energy homeostasis (Pickrell and Youle, 2015; Scheibye-Knudsen et al., 2015). Mitophagy is a selective type of autophagy promoting the elimination of impaired mitochondria, and the major degradation pathway by which cells regulate mitochondrial content in response to intracellular and environmental signals (Palikaras et al., 2015; Schiavi et al., 2015; Fang et al., 2016). In this protocol, we describe two methods for monitoring mitophagy in C. elegans. We developed two composite, in vivo imaging systems to asses mitophagy based, first, on the Rosella biosensor (Rosado et al., 2008), which combines a fast-maturing pH-insensitive DsRed fused to a pH-sensitive GFP variant, and second, on a custom, dual-fluorescence reporter system that involves a mitochondria-targeted GFP, together with the autophagosomal marker LGG-1/LC3 fused to DsRed. These protocols facilitate non-invasive monitoring of mitophagy in live specimens.
This work was funded by grants from the European Research Council (ERC), the European Commission 7th Framework Programme and Bodossaki Foundation Postdoctoral Research Fellowship. The protocol has been adapted from Palikaras et al. (2015), Nature 521, 525-528.