Ref(2)P localizes in protein aggregates in old flies during normal aging. (A–D) Electron micrographs of adult brain of old wild-type flies. Ref(2)P localizes in electron-dense structures ranging from 50 nm to 1 μm in diameter that are surrounded (arrows) or not (arrowhead) by a limiting membrane. (E and F) Electron micrographs of wild-type brain of old flies demonstrating that Ref(2)P forms shell-like structures that surround aggregated filamentous material (arrows). Bars: (A–C) 50 nm; (D) 100 nm; (E and F) 200 nm.

Ref(2)P is a component of protein aggregates in flies defective in autophagy and D. melanogaster models of human neurodegenerative diseases. (A and B) Protein aggregates in atg8a^KG07569 mutants exhibit sequestosome morphology and are positively labeled for Ref(2)P (A) and ubiquitin (B). Protein aggregates are surrounded by small electron-dense vesicles (arrows). (C) Western blot analysis of cell lysates of wild type and atg8a^KG07569 young adult heads demonstrating a significant increase of Ref(2)P protein levels in atg8a^KG07569 flies. (D, left) Ref(2)P protein migration profile in ref(2)P loss-of-function alleles. Ref(2)P mutant protein for the PB1 domain has an apparent molecular mass of 85 kD (ref(2)P^od2/ref(2)^Pod2) and the Ref(2)P mutant protein for the UBA domain has an apparent molecular mass of 69 kD (ref(2)P^od3/ref(2)^Pod3). (D, right) Western blot analysis of insoluble protein fractions of adult heads of double mutants for atg8a and ref(2)P probed for ubiquitin. Genotypes: (1) atg8a^KG07569/Y; ref(2)P^od2/Cyo, (2) atg8a^KG07569/Y; ref(2)P^od2/ref(2)P^od2, (3) atg8a^KG07569/Y; ref(2)P^od3/CyO, and (4) atg8a^KG07569/Y; ref(2)P^od3/ref(2)P^od3. The insoluble ubiquitinated protein profile of the double mutants is diminished compared with the one of the respective control heterozygous for ref(2)P. (E and F) Neuronal cells mutant for atg1 (green) accumulate Ref(2)P- (E) and ubiquitin-positive (F) structures (arrows), whereas the normal surrounding cells do not exhibit positive staining. (G) Confocal micrographs of the optic lobe (medulla region) of young flies expressing a mutated form of human tau protein exhibiting Ref(2)P- and tau-positive aggregates (arrows). Genotypes: (E and F) yw/UAS-CD8-GFP, hs-flp; tubGal4/+; atg1^Δ3D, FRT80/tubGal80, FRT80, and (G) elav/+;UAS-E14/+. Bars: (A) 500 nm; (B) 200 nm; (E) 2 μm; (F) 5 μm; (G) 10 μm.

Ref(2)P localization and expression in the adult brain of wild-type flies. (A) Confocal micrographs of adult brain of a young (2 d old) wild-type fly. Positive staining for Ref(2)P and ubiquitin is not evident. (B–D) Confocal micrographs of adult brain of an old (8 wk old) wild-type fly. Ref(2)P accumulates in protein aggregates that often colocalize with ubiquitin (arrows). The white rectangle in C indicates the area shown in D. (E and F) Western blot analysis of total cell lysates (E) and insoluble protein fraction (F) of wild-type young and old adult heads probed with anti-ref(2)P and anti-ubiquitin antibodies, demonstrating a significant increase in Ref(2)P and insoluble ubiquitinated protein levels in old flies. wt, wild type. Bars: (A–C) 100 μm; (D) 10 μm.

Ref(2)P localizes to protein aggregates in flies that have impaired autophagy or proteasome function. The formation of these aggregates is inhibited in atg8a/ref(2)P double mutant flies. (A and B) atg8a mutants exhibiting a striking positive staining for Ref(2)P and ubiquitin staining in all the regions of the adult brain. As it is shown in high magnification of the optic lobe (medulla region; B), Ref(2)P- and ubiquitin-positive aggregates are observed (arrows). (C) Normal phenotype is observed in β2 and 6 dominant-negative temperature-sensitive mutants at 25°C. (D) Accumulation of Ref(2)P- and ubiquitin-positive aggregates are observed in β2 and 6 mutants at 29°C (arrows). (E and F) double mutants for atg8a and ref(2)P loss-of-function alleles lacking the PB1 (E) or UBA (F) domain do not form ubiquitinated protein aggregates. Ubiquitin exhibits a cytoplasmic staining pattern (arrows). Genotypes: (A and B) atg8a^KG07569/Y, (C and D) elavGal4/+; UAS-β6/+; UAS-β2/+, (E) atg8a^KG07569/Y; ref(2)P^od2/ref(2)P^od2, and (F) atg8a^KG07569/Y; ref(2)P^od3/ref(2)P^od3. Bars: (A) 100 μm; (B–F) 10 μm.