Live imaging of mitophagy in Drosophila

Identification: Cornelissen, Tom


Description

Live imaging of mitophagy in Drosophila
 
Tom Cornelissen1, Sven Vilain2, Katlijn Vints2, Natalia Gounko2, Patrik Verstreken2, Wim Vandenberghe1, 3
1Laboratory for Parkinson Research, KU Leuven, Leuven, Belgium; 2VIB-KU Leuven Center for Brain & Disease Research, Leuven, Belgium; 3Department of Neurology, University Hospitals Leuven, Leuven, Belgium
 
PINK1 and parkin, two proteins involved in Parkinson's disease, are important regulators of selective autophagic mitochondrial degradation (mitophagy) in cultured cells. However, direct evidence for a role of these proteins in mitophagy in vivo is still scarce. PINK1- and parkin-deficient Drosophila accumulate abnormal mitochondria in their muscle cells, but whether this results from impaired mitophagy, is still unknown. To address this, we generated a fly model expressing mitochondrially targeted Keima (mito-Keima), a coral-derived protein that is resistant to lysosomal proteases and has a dual excitation peak dependent on environmental pH. Using live confocal ratiometric imaging we were able to demonstrate that mito-Keima was in part delivered to acidic compartments in flight muscles and in dopaminergic neurons in vivo in wild-type flies. Costaining with the lysosomal dye LysoTracker as well as correlative light electron microscopy (CLEM) confirmed that these acidic compartments were lysosomes, indicating that mitophagy occurs in vivo in basal conditions. Mitophagy increased with aging and was suppressed by overexpression of a dominant-negative variant of Atg1. We crossed the mito-Keima fly with PINK1- and parkin-deficient flies and found that loss of PINK1 and parkin impaired mitophagy in flight muscles, demonstrating that PINK1 and parkin mediate mitophagy in vivo. Knockdown of the fly homologues of the deubiquitinase USP15 and, to a lesser extent, USP30, rescued the mitophagy defect of the parkin-deficient flies. In conclusion, the mito-Keima fly is a novel model to assess mitophagy in vivo and to determine the impact of disease-linked mutations on this pathway.
 

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