UEV-1 and Glutamate Receptors Figure 5. UEV-1 regulates both GLR-1 levels and spatial distribution. (A–H) GLR-1::GFP fluorescence in (A, C, E, G) neuron cell bodies and (B, D, F, H) along the ventral cord for (A, B) wild type, (C, D) uev-1(od10) mutants, (E, F) ire-1 mutants, and (G, H) ire-1 uev-1 double mutants. To quantify the unusual spatial distribution of GLR-1 in uev-1 mutants, the region 65 microns anterior (left in the figure) of the RIG/AVG cell bodies (these neurons contribute little GLR-1::GFP to the ventral cord, used here solely as a landmark) was considered as ‘‘proximal’’ to the neuron cell bodies of the command neurons in the head (AVA, AVB, AVD, AVE, and PVC, which contribute almost all of the GLR-1::GFP along the ventral and are outside of the images in this figure), whereas the region 65 microns posterior (right in the figure) of the RIG/AVG cell bodies were considered as ‘‘distal’’ to the command neuron cell bodies. The images in E–H were collected at several times the exposure as the images in A–D as no single exposure time possessed the dynamic range to precisely capture all four genotypes. (I) Line profiles for GLR-1::GFP fluorescent intensity along the ventral cord for a wild-type animal (blue) and a uev-1(od10) mutant (red) after removal of background autofluorescence. Baseline is a measure of unlocalized GLR- 1::GFP levels. Brighter GLR-1::GFP puncta are observed more proximal along the neurites to the cell bodies. This proximal-distal bias is increased in uev-1 mutants compared to wild type. (J) Mean GLR-1::GFP fluorescence (sum of pixel values) along the ventral cord for either the total cord, the proximal region, or the distal region for wild type (stippled) or uev-1 mutants (gray). (K, L) GLR-1::GFP fluorescence (sum of pixel values) observed in the PVC neuron cell bodies for the indicated genotypes. (M) Mean number of GLR-1::GFP puncta along the ventral cord (per 100 micron length) for either the total cord, the proximal region, or the distal region for wild type (stippled) or uev-1(od10) mutants (gray). Bar, 5 microns. Error bars are SEM. N = 20–30 animals for each genotype. (J, M) **P,0.01, ***P,0.001 by ANOVA with Bonferroni multiple comparison tests (only shown for wild type versus mutant for each reporter). (K, L) *P,0.01, ***P,0.0001 by t-test. doi:10.1371/journal.pone.0014291.g005 colocalization between GLR-1::GFP and SYN-13::mRFP (Figure 6L, M; Figure S4) or RME-1::mRFP (data not shown), which are associated with early and recycling endosomes, respectively, and are involved in recycling clathrin-dependent cargo [55,76,78]. Given that we see no increase in the levels of GLR-1 colocalization with SYN-13 or RME-1, it is unlikely that the increased colocalization of GLR-1 with RAB-10 observed in uev-1 mutants is simply due to elevated levels of GLR-1 in the cell body. We confirmed this by independently analyzing colocaliza- tion using randomization through a confined displacement algorithm (Figure S4) [79]. If the GLR-1::GFP accretions observed in uev-1 mutants indicate that receptors are becoming trapped at endosomes, then we would expect a decrease in GLR-1 function in uev-1 mutants [55,56]. Normally, GLR-1 functions to regulate changes in locomotion direction. As C. elegans move, they generally go forward, but will occasionally spontaneously reverse direction; GLR-1 signaling positively induces these spontaneous reversals in locomotion [49,51]. Mutants with reduced GLR-1 signaling or reduced levels of GLR-1 at the synapse have a lower frequency of spontaneous reversals, whereas mutants with increased GLR-1 signaling or higher levels of cell surface GLR-1 have a higher frequency of spontaneous reversal [21,47,48,51–53]. We measured the spontaneous reversal rate of uev-1 mutants, and found that the rate was significantly lower in uev-1 mutants than in wild type, similar to glr-1 mutants, which entirely lack GLR-1 (Figure 6K). Expression of a wild-type uev-1 minigene via the glr-1 promoter is sufficient to rescue this behavioral defect. Therefore, little receptor appears to be functioning at the postsynaptic membrane in uev-1 mutants. Instead, it is likely that a large amount of the receptor is in an intracellular compartment, possibly endosomal in nature. UEV-1 regulates GLR-1 trafficking independent of clathrin-mediated endocytosis GLR-1 is endocytosed by a combination of clathrin-dependent and clathrin-independent mechanisms, with the PDZ protein LIN- 10 mediating the recycling of receptors endocytosed by the former mechanism, and RAB-10 mediating the recycling of receptors endocytosed by the latter mechanism (Figure S6) [55,77]. In uev-1 mutants, GLR-1 is colocalized with RAB-10, suggesting that it might regulate GLR-1 recycling along the clathrin-independent pathway, and that the accumulation of GLR-1 that occurs in uev-1 mutants does not occur through clathrin-mediated endocytosis. To examine this possibility, we used the itsn-1 mutation, which impairs the function of the ITSN-1 Intersectin protein – an adaptor that helps mediate the clathrin-dependent endocytosis of GLR-1 [55,80–84]. We introduced an itsn-1 mutation into uev-1 mutants to determine whether it would suppress the accumulation of GLR- 1 in accretions. We found that itsn-1 did not have any significant effect on either the average size or number of accretions in uev-1 animals (Figure 7A–D, I–J). We had previously generated a transgene, Pglr-1::rfp::rab-5(GDP), which contains a mutated form of the rab-5 cDNA that can suppress the clathrin-dependent endocytosis of GLR-1 [56]. We expressed this transgene in both wild-type and uev-1 animals, but as with the itsn-1 experiment, this did not result in the suppression of the uev-1 mutant phenotype (data not shown), suggesting that UEV-1 regulates GLR-1 trafficking at a step independent of clathrin-mediated endocytosis. As LIN-10 and RAB-10 mediate the recycling of GLR-1 receptors along the clathrin-dependent and clathrin-independent endocytosis pathways, respectively, double mutants for lin-10 and rab-10 show an additive effect on GLR-1 accumulation by simultaneously blocking both pathways [55]. Given that UEV-1 does not appear to be downstream of clathrin-dependent endocytosis, and that GLR-1 colocalizes with RAB-10 in uev-1 mutants, we reasoned that UEV-1 might function in the same pathway as RAB-10. To test this possibility, we made double mutant strains of uev-1 with either lin-10 or rab-10 mutations. We found that in a lin-10 uev-1 double mutant, there was a significant increase in GLR-1 accretion size and number compared to either single mutant alone (Figure 7E, F, K, L), making it likely that LIN-10 and UEV-1 do not work in the same pathway. However, we found that in a rab-10 uev-1 double mutant, there was a significant increase in GLR-1 accretion size and number only compared to the rab-10 single mutant, but not compared to the uev-1 single mutant alone (Figure 7G, H, K, L). This suggests that UEV-1 and RAB-10 might function in the same pathway, with the more severe phenotype in uev-1 mutants indicating the possibility that UEV-1 also works in an additional pathway. UEV-1 and ubiquitin-mediated turnover of GLR-1 The ubiquitination of GLR-1, like many membrane receptors, is necessary for its endocytosis [19–22]. GLR-1 ubiquitination and hence turnover can be accelerated by the overexpression of ubiquitin monomers from a transgene containing the glr-1 promoter, resulting in reduced number and size of GLR-1::GFP puncta (Figure 8A, C) [21]. We introduced this Pglr-1::ubiquitin transgene into uev-1 mutants and found that overexpression of ubiquitin can decrease the number and size of GLR-1 accretions in uev-1 mutants (Figure 8D, G, H). However, uev-1 mutations partially block the turnover of GLR-1 caused by increased ubiquitin levels (Figure 8D,G), suggesting that UEV-1 is required for one or more ubiquitin-mediated processes that regulate GLR-1 trafficking. PLoS ONE | www.plosone.org 9 December 2010 | Volume 5 | Issue 12 | e14291