posterior face of the nerve ring and the dorsal face of the ventral cord are directed out of the page. The lateral ganglion and retrovesicular ganglion regions are indicated by brackets. Found at: doi:10.1371/journal.pone.0014291.s002 (3.25 MB TIF) Figure S3 GLR-1 mRNA and protein levels do not vary significantly in uev-1 mutants compared to wild type. (A) The levels of glr-1 mRNA relative to dlg-1 (a control adherens junction protein) mRNA as detected by qRT-PCR are shown for the indicated genotypes. N = 5 trials. (B) The levels of GLR-1::GFP protein relative to actin protein as detected by quantitative Western blotting are shown. (C) A sample Western blot for GLR- 1::GFP (top) and actin (bottom) is shown. N = 3 trials. Found at: doi:10.1371/journal.pone.0014291.s003 (1.00 MB TIF) Figure S4 GLR-1 colocalization with RAB-10 and SYN-13 as analyzed by a confinement displacement algorithm. (A,D) GLR- 1::GFP fluorescence and (B,E) SYN-13::RFP fluorescence from (A- C) wild type or (D-F) uev-1 mutants. (C,F) Merged images. The mean Manders colocalization coefficient is shown for (G) GLR-1 that colocalizes with RAB-10, (H) RAB-10 that colocalizes with GLR-1, (I) GLR-1 that colocalizes with SYN-13, and (J) SYN-13 that colocalizes with GLR-1 for the indicated genotypes. Gray bars indicate the coefficients determined from the original fluorescent images. Red bars indicate the coefficients determined from the same images randomized by a confinement displacement algorithm, thus measuring the probability that the correlation coefficients for colocalization are occurring my random chance within the small confined space of these cells [79]. *P,0.05, **P,0.01, ***P,0.001 by ANOVA with the indicated Bonferonni comparisons. N = 15-22 animals for each genotype. Found at: doi:10.1371/journal.pone.0014291.s004 (9.38 MB TIF) Figure S5 The ubc-13 transcription unit. (Top) The intron/exon structure of ubc-13 based on sequenced cDNAs is shown in the top panel. Gray boxes indicate exonic coding sequences. The arrow indicates the start of transcription. The purple line indicates the sequences that are removed by the tm3546 deletion. (Bottom) Genomic sequences in and around the ubc-13 transcription unit, from the start of translation until the final stop codon present in the mRNA, are shown. Capital letters highlighted in gray indicate coding sequences within exons. Numbers are based on amino acids starting from the ATG and containing only exonic sequences. The References 1. Dingledine R, Borges K, Bowie D, Traynelis SF (1999) The glutamate receptor ion channels. Pharmacol Rev 51: 7–61. 2. Malenka RC, Bear MF (2004) LTP and LTD: an embarrassment of riches. Neuron 44: 5–21. 3. Malenka RC (2003) Synaptic plasticity and AMPA receptor trafficking. Ann N Y Acad Sci 1003: 1–11. 4. Kessels HW, Malinow R (2009) Synaptic AMPA receptor plasticity and behavior. 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GLR-1 receptors (red) are being endocytosed and recycled by either the clathrin-independent endocytosis pathway (CIE) and RAB-10 (the synapse on the left) or the clathrin-dependent endocytosis pathway (CDE) and LIN-10 (the synapse on the right). (A) Trafficking in wild-type animals, based on our model and previously published results [55,56]. (B) In lin-10 mutants, GLR-1 is endocytosed by CDE, including RAB-5 and ITSN-1; however, receptors are not recycled and accumulate in internal endosomes. (C) In uev-1 mutants, rab-10 mutants, or uev-1 rab-10 double mutants, our findings suggest that GLR-1 is endocytosed by CIE; however, receptors are not recycled and accumulate in internal endosomes. (D) Since UEV-1 and LIN-10 are expected to regulate GLR-1 by these two separate pathways, we would expect an increase in the amount of internalized GLR-1 in uev-1 lin-10 double mutants. 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