In vivo imaging of mitophagy in Caenorhabditis elegans

Abstract

Mitochondrial dysfunction is a shared hallmark of many human pathologies and ageing. Mitochondrial selective autophagy mediates the removal of damaged or superfluous mitochondria preserving mitochondrial and cellular homeostasis. Deregulation of mitophagy is associated with the onset of several pathological conditions including ageing and age­related neurodegenerative diseases. The nematode Caenorhabditis elegans is a widely used model organism for studying the biology of ageing and neurodegeneration. Here, we describe tools and resources for monitoring mitophagy in C. elegans. We developed two composite, in vivo mitophagy imaging systems based, first, on the Rosella biosensor, which combines a fast­maturing pH­insensitive DsRed fused to a pH­sensitive GFP variant, and second, on a custom, dual­fluorescence reporter system that utilizes a mitochondria­targeted GFP, together with the autophagosomal marker LGG­1/LC3 fused to DsRed. We validated both systems in various cell types and under conditions known to induce mitophagy in the worm. These protocols facilitate non­ invasive monitoring of mitophagy in physiologically­relevant contexts.