which could have implications for synaptic plasticity and neurodegeneration in other organisms, including mammals. Most studies of UEV protein function to date have been in single celled organisms or have been in tissue culture. Our study indicates that the in vivo roles of UEV proteins in metazoans are likely to be even broader than originally expected. Materials and Methods Strains Animals were grown at 20uC on standard NGM plates seeded with OP50 E. coli. Some strains were provided by the Caenorhabditis Genetics Center. Most strains were backcrossed multiple times to our laboratory N2 strain to minimize other genetic variation. The following strains were used: alx-1(gk338), ire-1(ok799), itsn-1(ok268), juIs1[Punc-25::SNB-1::GFP], lin-10(e1439), nhl-1(gk15), nuIs108[GLR- 1(4KR)::GFP], nuIs24[Pglr-1::GLR-1::GFP], nuIs25[Pglr-1::GLR-1::GFP], nuIs68[Pglr-1::UNC-43::GFP], nuIs89[Pglr-1::MUb], odEx[F39B2 cosmid, rol-6dm], odEx[Pglr-1::mCherry::UEV-1, rol-6dm], odEx[Pglr-1::SYN-13:: RFP], odEx[Pglr-1::UEV-1(cDNA), rol-6dm], odEx[Pglr-1::UEV-1(F8A cDNA), rol-6dm], odEx[Pglr-1::UEV-1(F8A mini-gene)], odEx[Pglr-1::UEV- 1(mini-gene)], odEx[Pglr-1::UEV-1(mini-gene)], odEx[Pglr-1::UEV-1(geno- mic)], odEx[Pglr-1::UEV-1::GFP], odEx[Rab-5(S23N)], odEx[rfp::rme-1], odIs1[Pglr-1::SNB-1::GFP], odIs22[Pglr-1::LIN-10::GFP], odIs42[Pglr-1::RAB -10::RFP], pdr-1(tm395), pdr-1(tm598), pmk-3(ok169), stam-1(ok406), rab- 10(q373), rpm-1(js317), trf-1(nr2014), ubc-1(gk14), uev-1(od10), uev- 1(ok2610), ubc-13(tm3546), and the CB4856 Hawaiian strain. Transgenes and Germline Transformation Transgenic strains generated in this study were isolated after microinjecting various plasmids (5–50 ng/ml) using rol-6dm (a gift from C. Mello, UMass), ttx-3::rfp (a gift from O. Hobert, Columbia Univ.), or lin-15(+) (a gift from J. Mendel, CalTech) as a marker. Plasmids containing the glr-1 or uev-1 promoters, followed either by a uev-1 minigene (wild type or F8A mutant), uev-1 cDNA (wild type or F8A mutant), uev-1 genomic, or mCherry::uev-1, were generated using standard techniques. All resulting transgenes were introduced into the germline and followed as extrachromosomal arrays. Isolation and mapping of uev-1(od10) P0 nuIs25 nematodes were EMS mutagenized using standard procedures. F2 animals from individual plates were sampled (n = 30–50) by mounting on 2% agarose pads containing levamisole. Animals were scored by fluorescence microscopy for defects in GLR-1::GFP localization. Mutants were recovered either directly from the slide or by isolating siblings from the parental F1 plate. Mutants were further characterized after 4 rounds of backcrossing. The od10 mutation was closely linked to unc-54 (map position +27.4), and was three-factor mapped between SNP pkP1071 (map position +23.4) and SNP pkP1072 (map position +28.48) on the right arm of LGI by crossing marked uev-1 strains to the polymorphic strain CB4856. We detected 20 recombination events between pkP1071 and od10, and 2 recombination events between od10 and pkP1072, suggesting that od10 mapped to around +27.0. We injected genomic cosmids covering ,200 kb around the unc-54 locus, and found that cosmid F39B2 rescued the uev-1 mutant phenotype. This cosmid contains 12 genes, including uev-1. We sequenced several candidate genes from od10 mutant genomic DNA, and identified a nonsense mutation in the uev-1 gene. Expression of a uev-1 minigene containing uev-1 coding sequences and 2 kilobases of promoter was also sufficient to rescue the uev-1 mutant phenotype. UEV-1 and Glutamate Receptors Fluorescence Microscopy GFP- and RFP-tagged fluorescent proteins were visualized in nematodes by mounting larvae on 2% agarose pads with levamisole. Fluorescent images were observed using a Zeiss Axioplan II. A 100X (N.A. = 1.4) PlanApo objective was used to detect GFP and RFP signals. Imaging was done with an ORCA charge-coupled device (CCD) camera (Hamamatsu, Bridgewater, NJ) using IPLab software (Scanalytics, Inc, Fairfax, VA) or iVision v4.0.11 (Biovision Technologies, Exton, PA) software. Exposure times were chosen to fill the 12-bit dynamic range without saturation. In most cases, maximum intensity projections of z- series stacks were obtained and out-of-focus light was removed with a constrained iterative deconvolution algorithm (Vaytek). For most images, we captured the ventral cord neurites in the retrovesicular ganglion region surrounding the RIG and AVG cell bodies. The quantification of ventral nerve cord fluorescent objects (i.e., puncta and accretions) was done using ImageJ [96] to automat- ically threshold the images and then determine the outlines of fluorescent objects in ventral cord neurites. ImageJ was used to quantify both the shape and the size of all individual fluorescent objects along the ventral cord. This allowed us to distinguish between the small GLR-1::GFP puncta in wild-type animals and the large, aberrant accretions (which have an elongated shape not observed in wild type) in uev-1 and other mutants. Object size was measured as the maximum diameter for each outlined cluster. Object number was calculated by counting the average number of clusters per 100 microns of dendrite length. To quantify the fluorescent intensities of individual GLR- 1::GFP puncta and accretions along the ventral cord neurites, a 20X objective was used to capture images of animals, and a median filter was used to subtract away the background nematode and coverslip autofluorescence. Line profiles were drawn along the length of the ventral cord starting from the anterior end of the retrovesicular ganglion and moving posterior for approximately 250 microns to show the fluorescent intensity peaks for individual puncta and accretions. For quantification of ventral cord fluorescence, ImageJ was used to measure the integrated fluorescent density (the sum of all detectable pixel intensities along a given ventral cord process) normalized to the length of that process. The quantification of PVC cell body fluorescence was done using ImageJ to measure the integrated fluorescent density (the sum of all detectable pixel intensities per cell body) for each neuron. For the quantification of GLR-1::GFP and mRFP::RAB- 10 colocalization, we fixed animals with ice cold 1% paraformal- dehyde in PBS for 10 minutes and imaged them using a previously published protocol [56,76]. Images for neuronal cell bodies were taken using a Carl Zeiss confocal microscope equipped with the BD CARV IITM Confocal Imager and a Carl Zeiss 100X Plan- Apochroma objective (N.A. = 1.4). For quantitative colocalization analysis, all image manipulations were performed with iVision v4.0.11 (Biovision Technologies, Exton, PA) software using the FCV colocalization function. We applied an empirically derived threshold to all images for both the GLR-1::GFP channel and the mRFP::RAB-10 channel to eliminate background coverslip fluorescence and other noise (typically, 5% of pixels for each channel). The fluorescent intensity values for both the GLR-1::GFP and mRFP::RAB-10 channels were then scatter plotted for each pixel. Pixels with similar intensity values for both channels (within an empirically established tolerance factor) were counted as colocalized. To acquire the fraction of GLR-1::GFP colocalized with mRFP::RAB-10, the number of colocalized pixels was normalized PLoS ONE | www.plosone.org 17 December 2010 | Volume 5 | Issue 12 | e14291