FIGURE 1:
Past1 localizes to a postsynaptic tubular-reticular domain at the larval NMJ and is required for postsynaptic membrane assembly. (A) Localization of Past1 to type Ib and Is but not type II or III NMJs. Large image shows 2D projections of 60× spinning-disk confocal stack from an NMJ on muscles 12 and 13. Scale bar, 50 μm. Right, magnified single confocal slices from the area indicated by the dashed rectangle. (B) Large image shows 2D projection of 100× spinning-disk confocal stack from an NMJ on muscles 6 and 7. Right, magnified single confocal slices from the area indicated with the dashed lines; arrows indicate Past1-labeled tubules. (C) NMJ α-Past1 antibody staining is lost in past1 mutants and upon RNAi of Past1 using the muscle-specific driver BG57-GAL4. Images show 2D projections of 60× spinning-disk confocal stacks from muscle 4. (D) Postsynaptically expressed Past1-EGFP exhibits similar NMJ localization to endogenous Past1, whereas an ATP-binding mutant (contrast enhanced) is largely cytoplasmic. Large images show 2D projection of 60× spinning-disk confocal stack from an NMJ on muscles 6 and 7. Bottom, magnified single confocal slices from the area indicated by the dashed rectangle. (E) α-HRP staining showing aberrant bouton morphology in past1 mutants that is rescued by postsynaptic reexpression of Past1-EGFP. Overall bouton number per muscle area is normal in past1 mutants (muscles 4 and 6/7, segment A3). Image shows 2D projection of 60× spinning-disk confocal stacks from muscle 4. (F) past1-mutant NMJs exhibit ghost boutons. Images shows 2D projection of 60× spinning-disk confocal stacks from muscle 6/7, segment A3. (G) Quantification of ghost boutons. (H) Quantification of postsynaptic α-HRP debris from three-dimensional (3D) volumes surrounding presynaptic terminals. Number in bar graphs indicates number of NMJs measured; scale bars, 20 μm (B–F). Past1-EGFP rescue represents the genotype UAS-Past1-EGFP/+; BG57, past1110-1/past160-4.