Figure 3. VAP^P58S forms ubiquitinated aggregates and induces aggregation of VAP^wt in vivo.

(A–C) Confocal images of 3^rd instar larval muscles stained with anti-VAP. (A) A control animal (G14-GAL4/+). VAP expression is observed at the neuromuscular junction and in underlying muscles 6 and 7. (B) G14-GAL4/UAS-VAP^wt. Increased VAP immunoreactivity is observed with expression of VAP^wt. (C) G14-GAL4/UAS-VAP^P58S. Punctate staining of ALS-associated mutant VAP is observed. (D) Staining with anti-VAP (red) and anti-ubiquitin (Ubi; green) of animals expressing VAP^P58S in the muscle. Mutant VAP forms aggregates that appeared as puncta through out the muscle (red). Ubiquitin immunoreactive puncta also are observed (green) that largely colocalized with the VAP aggregates (merged image). (E–G) Staining with anti-myc (green) and anti-HA (red) of animals expressing myc-VAP^wt and HA-VAP^P58S in the muscle. (E) Similar to untagged VAP^wt and VAP^P58S transgenes, expression of myc-VAP^wt appears diffuse and cytoplasmic. (F) HA-VAP^P58S forms aggregates that appear punctate throughout the muscle. (G) Muscle field of a 3^rd instar larva expressing both myc-VAP^wt and HA-VAP^P58S. Expression of myc-VAP^wt (green) appears punctate and colocalizes with HA-VAP^P58S aggregates (merged image). Scale bar, 20 µm.