Furthermore, transmembrane proteins known to cycle through endosomes, including synaptotagmin ( Takei et al., 1996) and APP ( Haass et al., 1992), also accumulate at these TBs and partially colocalize with anti-HRP ( Figure 3C). Together, these data

show that overexpression of p150G38S causes a marked accumulation of endosomal membranes and proteins at NMJ TBs. To determine whether the accumulation of endosomes at Glued mutant TBs is due to disruption of dynein/dynactin function, we asked whether similar phenotypes are present in mutant alleles of genes encoding components of the dynein/dynactin complex. Because most available alleles BMN 673 supplier are early larval or embryonic lethal, we knocked down dynein/dynactin subunits in motor neurons by using RNAi ( Figure S4A). As expected, knockdown of three dynactin subunits (Gl, cpa, and p62) and three dynein subunits (dhc, dic, and dlic) phenocopies the

TB accumulation of anti-HRP immunoreactivity and Syt:GFP that we observed in D42 > p150G38S animals ( Figures 3C, 3E, and S4B). These data demonstrate that disruption of the dynein/dynactin complex causes an accumulation of endosomes within TBs of the NMJ. In filamentus fungi, the dynactin complex is required for MT plus-end localization of dynein GS 1101 and for the interaction between dynein and endosomes (Xiang et al., second 2000 and Zhang et al., 2010). To determine whether dynein is mislocalized in Glued animals, we analyzed the expression of the cytoplasmic dynein heavy chain (cDhc64C, referred to here as Dhc). Surprisingly, GlG38S larvae reveal a striking accumulation of Dhc at NMJ TBs in all segments in 100% of GlG38S and GlG38S/GlΔ22 animals; this phenotype is never observed in wild-type animals ( Figures 4A–4C and Figures S5A and S5B). At wild-type synapses, Dhc is localized

to small puncta at the periphery of all boutons ( Figure 4A), and occasionally small Dhc(+) puncta are observed near the center of the TB ( Figure 4E, arrow). In GlG38S animals, however, the mean Dhc signal intensity is increased ∼10-fold within TBs, with no significant differences between proximal and distal segments ( Figure 4B). Interestingly, in GlG38S larvae, Dhc predominantly accumulates at TBs of the longest branch in synapses with multiple branches ( Figures S5A and S5B). These accumulations are not seen in axons or motor neuron cell bodies ( Figure S5B and data not shown). Microtubules do not appear to be altered at GlG38S NMJs; however, we did note that mutant TBs with observable microtubule bundles did not accumulate dynein ( Figure S5C, arrow), in contrast to those TBs with no significant tubulin staining. These data suggest that dynein accumulates in GlG38S TBs lacking stable microtubules.