3D quantification of autophagy activation and autophagosome-to-mitochondria recruitment in a Drosophila model of Parkinson's disease

Abstract

Here, we describe a protocol for comprehensive quantification of autophagosome recruitment to mitochondria as an early step in mitophagy. Data collected using this protocol can be useful in the study of neurodegenerative disease, cancer, and metabolism-related disorders using models in which co-expression of mito-GFP and mCherry-Atg8a is feasible. This protocol has the advantage of assessment in an in vivo model organism (Drosophila melanogaster), where tissue-specific mitophagy can be investigated. For complete details on the use and execution of this protocol, please refer to (Cackovic et al., 2018).

Keywords: Cell Biology; Microscopy; Model Organisms; Neuroscience.

Graphical abstract

Figure 1

Identifying soluble and lipidated mCherry-At8ga Blue represents TH-positive neurons and red represents mCherry-Atg8a. Bold arrows indicate diffusely distributed, soluble mCherry-Atg8a, while small arrows point to lipidated mCherry-Atg8a in autophagosomes. Scale bar represents 10 μm.

Figure 2

Quantification of autophagosome formation Top row images show raw data projections for a representative sample of (A) park^+/+ and (C) park^-/- PPL1 neurons expressing mCherry-Atg8a and TH-antibody staining (blue). Red puncta indicate lipidated mCherry-Atg8a. In the second row, red, blue and grey isosurfaces highlight mCherry-Atg8a puncta selected by the imaging software. We counted and compared the number of puncta located within TH-labeled region (bottom row) and did not detect an effect of the park-null mutation on autophagosome formation (B). Scale bar represents 10 μm. Data are represented as mean ± SEM. The effect of parkin loss of function was determined using a Mann-Whitney test. This figure has been modified from (Cackovic et al., 2018) and falls under terms of the Creative Commons Attribution License (CC BY).

Figure 3

3D colocalization of mCherry-Atg8a puncta with mito-GFP indicates level of autophagosome-to-mitochondria recruitment Raw data projections for representative samples of (A) park^+/+ and (C) park^-/- PPL1 neurons expressing mCherry-Atg8a, mito-GFP and blue TH label (top row). Red and green isosurfaces depict mCherry-Atg8a puncta and mitochondria selected by the imaging software (second row). Images in the third row illustrate distribution of puncta colocalized with mito-GFP (yellow) in the cell bodies; that is, the colocalized objects had positive Pearson’s coefficient. (B) We found that park-null flies had decreased autophagosome-to-mitochondria recruitment in dopaminergic neurons. Scale bar represents 10 μm. Data are represented as mean ± SEM. The effect of parkin loss of function was determined using a Mann-Whitney test. p < 0.001 for ∗∗∗. This figure has been modified from (Cackovic et al., 2018) and falls under terms of the Creative Commons Attribution License (CC BY).