Mitophagy intermediates in Vps13D depleted neurons lack endogenous soluble matrix proteins, but contain endogenous IMM protein.

Representative images of single motoneuron cell bodies which express Vps13D RNAi via the D42-Gal4 driver. A,C) VNC tissue was stained for stained for endogenous mitochondrial matrix proteins pyruvate dehydrogenase E1ɑ (PDH) (A) or Hsp60 (C) (cyan) and PolyUb or Ub/PolyUb (red). Arrowheads highlight examples of PolyUb+ objects lacking matrix staining, while the arrow highlights an example of a PolyUb+ object that contains PDH. Scale bars = 2μm. B,D) Distribution of mean intensities for PDH (B) and Hsp60 (D) from mitochondria in motoneurons. The right columns show objects selected based on positivity for PolyUb (n = 351 PolyUb+ objects from 5 larval VNCs for (B) and n = 227 PolyUb+ objects from 5 larval VNCs in (D)). The left columns show the population of conventional (PolyUb-) mitochondria (n = 789 mitochondria from 5 larval VNCs for (B); n = 744 mitochondria from 5 larval VNCs for (D)). Based on the criteria of intensity greater than 2.5% of conventional mitochondria (shading), 33.6% of the PolyUb+ objects contain PDH, while 13.7% contain Hsp60. E) VNC tissue was stained for endogenous mitochondrial IMM protein ATPβ (yellow) and Ref(2)p (red). Arrowheads highlight examples of Ref(2)p+ objects that lack mitoGFP but contain ATPβ. Scale bar = 2μm. F) Distribution of mean intensities for ATPβ from mitochondria in motoneurons. The right column shows objects selected based on positivity for PolyUb (n = 296 PolyUb+ objects from 5 larval VNCs). The left column shows the population of conventional (PolyUb-) mitochondria (PolyUb-) (n = 883 mitochondria from 5 larval VNCs). Based on the criteria of intensity greater than 2.5% of conventional mitochondria (shading), 94.3% of the PolyUb+ objects contain ATPβ.