(A) A GFP amino terminal tagged MyoVIIa construct, GFP-MyoVIIa, is expressed in wild type (control) or ubr3 mutant clones in larval eye-antennal discs. The lysate of eye-antenna discs and brains containing ubr3 mutant cells expressing GFP-MyoVIIa protein was immunoprecipitated with GFP nanobody-conjugated beads and examined on western blots. (B) Western blots with anti-GFP, anti-poly & mono-ubiquitin, anti-mono-ubiquitin and anti-MyoII antibodies. (C) Quantification of mono-ubiquitination of MyoII normalized by total amount of MyoII proteins from (B). (D–D’’) Immunolabeling of GFP (green), MyoVIIa (red) and HRP (neurons, in blue) in Johnston’s Organ from a myoII-GFP-myoII transgenic fly. D’ and D’’ show magnified images of the region shown by white box in D. (E) Distribution of MyoII proteins in Johnston’s organ. (F, G) Wild type MyoII over-expressing cells show normal apical structures of scolopidia, whereas ubr3B/B mutant cells expressing wild type MyoII exhibit enhanced detachment of scolopidia. (H) Quantification of detached scolopidia in ubr3B/B mutant cells, ubr3B/B mutant cells over-expressing MyoII and wild type cells over-expressing MyoII. Error bars show SEM. Numbers of flies quantified are shown in the columns. (I) Diagram shows HA-MyoII-Ub in which MyoII is fused to a Ub coding sequence on the carboxyl terminal. (J–K’) Johnston’s organ containing HA-MyoII-Ub expressing clones (labeled by GFP, green) is immunolabeled by anti-NompA (red) and phalloidin (actin, in blue). Arrow marks detached scolopidia. (K–K’) One MyoII-Ub expressing scolopidium exhibits accumulated NompA at the tips, but stays attached to the cuticle from the third segment (arrowheads). This may be a defective scolopidium just before detaching, suggesting that NompA mis-localization happens prior to apical detachment, as opposed to being a consequence of detachment.