UEV-1 and Glutamate Receptors Figure 4. UEV-1 is required for motoneuron synaptic bouton differentiation. (A–D) Fluorescence from animals transgenic for Punc-25::snb- 1::gfp (synaptobrevin). (A) Presynaptic boutons labeled with SNB-1::GFP are distinct along the dorsal and ventral cords of wild-type animals. Such boutons are missing, irregular in shape and size, and/or abnormally spaced in (C) uev-1(od10) mutants, a phenotype reminiscent of that observed in (B) rpm-1 mutants. (D) Mutations in pmk-3 do not alter the uev-1 phenotype. (E) The total number of dorsal cord SNB-1::GFP puncta is plotted for the indicated genotypes. (F) GLR-1::GFP accumulation in accretions in uev-1 pmk-3 double mutants is similar to that observed in uev-1(od10) single mutants. Bar, 5 microns. Error bars are SEM. N = 20–30 animals for each genotype. ***P,0.001 by ANOVA with Bonferroni multiple comparison tests. doi:10.1371/journal.pone.0014291.g004 baseline in wild type and uev-1 mutants is similar, but that the peaks of GLR-1::GFP fluorescence are higher and more frequent in uev-1 mutants compared to wild-type animals (Figure 5B, D, I). We also noticed a spatial asymmetry in the uev-1 mutant phenotype, with GLR-1 accretions occurring more anterior along the ventral cord and hence more proximal to the neuron cell bodies (Figure 5I, J; Figure S2 for overview of ventral cord spatial organization). As described above, mutations in ire-1 dramatically reduce the amount of GLR-1 that reaches ventral cord neurites (Figure 5F). Interestingly, while double mutants for both ire-1 and uev-1 have similar levels of GLR-1 in their cell bodies compared to ire-1 single mutants, they have elevated levels of GLR-1 in their ventral cord neurites along the same proximal region at which GLR-1 accretions accumulate in uev-1 single mutants, and decreased GLR-1 at more distal sites (Figure 5H, M). We also examined the levels of glr-1 mRNA and GLR-1::GFP protein in wild type and uev-1 mutants, and found no significant difference in their levels between the two genotypes (Figure S3). Taken together, our results indicate that UEV-1 regulates the spatial location at which GLR-1 receptors accumulate along the proximal-distal axis of the neurites rather than just affecting overall GLR-1 protein levels throughout the neuron. GLR-1 accumulates at RAB-10-containing endosomes in uev-1 mutants The GLR-1::GFP accretions that we observed in uev-1 mutants could represent receptor trapped at an intracellular location, perhaps endosomal in nature [55,56]. To test this possibility, we examined several endosomal markers, each labeled with mRFP and expressed under the glr-1 promoter. We coexpressed these with GLR-1::GFP in our wild-type and uev-1 animals, allowing us to check for colocalization. While these endosomal markers were too dim to observe colocalization in the neurites, we were able to examine colocalization in the neuron cell bodies [55,76]. We observed an increase in the amount of GLR-1::GFP that colocalized with mRFP::RAB-10 in neuron cell bodies from uev- 1 mutants compared to wild-type animals (Figure 6A–J); RAB-10 is a small Rab GTPase that associates with the early endosomes that are involved in recycling clathrin-independent cargo [55,77]. By contrast, we observed no significant difference in the amount of PLoS ONE | www.plosone.org 7 December 2010 | Volume 5 | Issue 12 | e14291