Supplementary Methods:
C. elegans strains and phenotypic analysis
Strains were maintained as described by Brenner 97 at 20°C, except for when noted. OP50 E. coli was used for feeding. The wild type reference strain was N2 Bristol. The mutant strains used were:LGI: nab-1(ok943), wwp-1(ok1102), gska-3(ok970), egl-30(ad806), fer-1(hc1ts), pqn-19(ok406), smg-1(r861), eat-5(ad1402), unc-13(s69), unc-57(e406), eat-16(sa768), goa-1(sa734). LGII: cam-1(ks52), unc-104(e1265), rab-3(js49), fkh-8(tm292), hum-1(ok634) . LGIII: mpk-1(oz140), mat-3(or180ts), rbf-1(js232).LGIV: eri-1(mg366), nlp12(ok335), unc-31(e298), osm-9(ky10), inx-8(gk42), unc-43(n498n1186), daf-7(m62), daf-7(e1372). LGV: egl-10 (n692), egl-8(sa47), sbt-1(ok901), egl-3(nr2090), sos-1(cs41ts), ric-4(md1088), snb-1(md249), pkc-1(nu448), fshr-1(ok778), skr-3(ok365), tam-1(cc567). LGX: lin-15B(n744), dgk-1(nu62), tap-1(gk202), erp-1(ok463), arr-1(ok401), unc-115(mn481), unc-78(e1217), hpk-1(pk1393), unc-10(e102), egl-15(n484ts), sad-1(ky289), syd-2(ju37), unc-18(e81).
Allele lesions can be found at nu448 is a nonsense mutation at residue W643 of pkc-1 (D.S. unpublished results). All aldicarb resistant mutants obtained from the C. elegans knockout consortia were outcrossed into N2 at least 4 times and genotyped by PCR before analysis.
Aldicarb (1mM, Chem Service) assays were preformed blind on young adults in triplicate as described 98. Full time courses were performed on all mutants as well (data not shown). For temperature sensitive mutants, fer-1, mat-3, sos-1, egl-15, assays were done after incubation at 25°C over night. Mutants with wild type aldicarb responses were unc-78, hpk-1, fkh-8, tam-1, hum-1, and daf-7. The 18% non-resistant mutants could reflect the false positive rate for our screen; however, it is also possible that some of these alleles (particularly deletions isolated by reverse genetics) do not eliminate the activity of these genes. For phorbol ester assays, animals were pre-incubated for 2 hours on PMA (0.25 mg/ml, sigma) and then transferred to plates containing PMA and aldicarb (0.8mM).
RNAi feeding experiments and functional profiling
RNAi feeding screens were preformed as described 99100. Bacteria containing each RNAi clone were cultured in 400 l LB media containing 50 g/ml ampicillin for 10-12 hrs. 40 l of each culture was spotted in a single well of a 24-well plate containing NGM agar, 6 mM IPTG and 25 g/ml carbenicillin. After overnight incubation, 100 eggs were placed per well and incubated at 20°C for 3 days. After 3 days of growth, 25 young adults per well were transferred into NGM wells containing 1mM aldicarb, and scored for paralysis after 100 minutes. For each 24 well plate assayed, empty vector pPD129.36, also referred to as L4440, (negative) and egl-3 (positive) controls wells were randomly included. All scoring was done blind, and an aldicarb resistant phenotype was assigned to a well if at least 25% of animals were not paralysed. The eri-1;lin-15B strain is slightly hypersensitive to aldicarb, compared to wild type controls, and has superficially normal locomotion behaviour. Two screens were performed in parallel using eri-1lin-15B double mutants or eri-1lin-15B dgk-1 triple mutants. Screens were done in quadruplicate, and phenotypes were confirmed by an additional round of quadruplicate screening. Genes were considered positive based on the following criteria: Ric or Dgk scores that are highly significant (p≤0.0001, Fisher’s Exact Test) (see Fig. 1b), an available loss-of-function mutation that is aldicarb resistant, protein localization to punctate structures in the DA motoneurons, or positive 2 generation RNAi aldicarb resistance. All positive clones were sequenced to confirm their identity.
For functional profiling, the following modifications were made: 200 l of dsRNA bacterial cultures were spotted onto RNAi NGM plates; 5-10 eggs were placed per well; and aldicarb assays were done after 6 days of growth (2 generations). All assays were done in quadruplicate and represented as a score out of 4. Only scores that were statistically significant (P<0.05, Fisher’s Exact Test) relative to empty vector controls were considered positive. Under these conditions, 40 (22%) of clones were lethal in 2 generations, and 84 (45%) scored below threshold, and were thus not included in the analysis. All 1 Gen and 2 Gen RNAi data are reported in Table S2. Clones scoring positively in the primary screens but negatively by 2 Gen RNAi were not enriched for false positives. 11/15 (73%) tested as homozygous mutants were aldicarb resistant. Levamisole (300uM, Sigma) assays were done 40 minutes after exposure. Suppression of aldicarb hypersensitivity under each condition was scored in quadruplicate and represented as a score out of 4.
The hierarchical clustering was performed using the hclust package in R language for statistical computing 101. Clusters were initially identified using Ward’s minimum variance method 102 with a Euclidian measure of distance. We then applied the following test to assess the robustness of these clusters: Data were re-clustered, a total of nine times, by varying the distance metric (manhattan, canberra and Pearson’s correlation) or agglomeration algorithm (average linkage, complete linkage, median linkage, single linkage and mcquitty). Clusters were judged to be robust if they were coherent in six or more of the nine methods. Only one of the eight clusters we initially identified failed this test. The position of the clusters relative to each other in the dendrograms produced by the nine different methods tended to change, even while the clusters themselves remained stable. Therefore, cluster membership is more consistent than positioning of clusters within the dendrogram. Clustering images were generated with custom scripts written in R.
Constructs and transgenes
Entry clones were obtained from the ORFeome project ( and cloned into the destination vector KP#1284 using the gateway strategy with LR clonase (Invitrogen) to make ORF::YFP expression clones. For this analysis, we used a YFP variant, Venus, which maintains its fluorescence in acidic compartments such as synaptic vesicles 103. KP#1284 contains an Sph I/BamH I unc-129 promoter fragment 104, cloned into MCSI of pPD49.26 ( andthe gateway selection marker ccdB flanked by the recombination sites arrR1 and attR2 cloned into the Nhe I/Kpn I sites of MCSII. The following substitutions in the coding region of yfp from pPD136.64 were created by PCR to generate Venus: F46L F64L M153T V163A S175G 103, and the resultant fragment was cloned in-frame into the 3’ end of the gateway insert using Age I/Kpn I. The ric-4 (short splice form), snn-1, and rab-3 were isolated by RT-PCR from total worm mRNA. rab-3 was N-terminally tagged with Venus. All other fusion proteins were C-terminal Venus tags. We found that the unc-129 promoter typically expressed in 9 cells in the ventral nerve cord that we identified as DA motoneurons based on their commissural morphology and posterior directed processes.
Wild type worms were injected with each ORF::Venus expression clone (25 ng/ul) with the co-injection marker KP# 708 (ttx-3::rfp, 50ug/ul) 105, as described 106. Clones that produced a punctate pattern in the dorsal cord were sequenced to confirm their identity. RAB-5 fusion protein expression was variably diffuse in some lines.
For the co-localization experiments, we used animals carrying the extra chromosomal array nuEx1065. nuEx1065 was made by co-injecting KP#1283 (10ng/ul) with the co-injection marker KP#704 (ttx-3::gfp, 25ng/ul). KP#1283 has the unc-129 promoter cloned into the Sph I/BamH I sites, and a 5’rfp-tagged genomic snb-1 fragment cloned into the Nhe I/Kpn I sites of pPD49.26. Animals carrying nuEx1065 were crossed to animals carrying each of the punctate ORF::Venus clone arrays and progeny expressing both injection markers were imaged. For unc-104 doubles, nuEx1065 animals were crossed into unc-104 mutants and homozygous F2 progeny were imaged.
For promoter expression studies, we sub-cloned promoter fragments generated by PCR from worm genomic DNA, into the AsiS I/Not I sites of KP#1285. KP#1285 is the NLS-GFP expression vector pPD96.04 ( AsiS I-Not I sites added in between the Sph I and BamH I sites in the polylinker. For nab-1 and ZK370.5/PDK rpomoters, the expression vector used was pPD95.75 (soluble GFP). Wild type worms were injected with a pool of each promoter construct (50ng/ul), the co-injection marker KP#708, and the cholinergic motoneuron marker KP#1281. KP#1281 has an Sph I/BamH I unc-17 promoter fragment 107, and a Kpn I/Sac I genomic rfp fragment cloned into pPD49.26.
The strain used for quantitative studies of GFP::SNB-1 puncta carried the integrant nuIs152. nuIs152 was generated by co-injecting KP#1282 (5ng/ul),the injection marker KP#708 ttx-3::rfp (40ng/ul), and a random 53mer oligo (1000ng/ul). KP#1282 is KP#1283 with Not I gfp replacing rfp. The nuIs152 integrant was outcrossed 10 times, before making double mutants. All double mutants made with deletion alleles from the knockout consortia were confirmed by PCR genotyping.
Fluorescence microscopy and quantitative analysis
All imaging experiments were done using a Zeiss Aviovert 100 microscope equipped with FITC/GFP or Texas red filters, and an ORCA CCD camera (Hamamatsu). Less than 2% bleed through between Venus and RFP using these filters was observed. Animals were immobilized with 30 mg/ml BDM (Sigma). A Zeiss Planapo 63× (NA 1.4) objective was used for imaging ORF::Venus localization and promoter::GFP expression, and an Olympus Planapo 100× (NA = 1.4) objective for GFP::SNB-1 quantitation. Worms were imaged in the region near the posterior gonad bend. Image stacks were captured and maximum intensity projections were obtained using Metamorph 4.5 software (Universal Imaging). Identical camera gain, exposure settings, and fluorescence filters were used for all the quantitative studies. Over the course of the quantitative studies, the fluorescent output of the bulb was determined to be essentially invariant (standard deviation <1%) as measured with 0.5µm fluorescent beads (FluoSpheres, Molecular Probes). Using these conditions, GFP fluorescence filled the 12-bit dynamic range, with minimal saturation. Line scans of ventral cord fluorescence were analysed in Igor Pro (WaveMetrics) using custom-written software 108. To calibrate our measurements of GFP fluorescence against the fluorescent output of the bulb, punctal fluorescence was expressed as a ratio of the absolute punctal fluorescence to the absolute mean fluorescence of the 0.5µm FluoSphere beads (Molecular Probes) measured during the experiment. Inter-punctal fluorescence was similarly normalized. All fluorescence values are normalized to wild type controls to facilitate comparison. No significant changes were seen in egl-8, nlp-12, sbt-1, pkc-1 and gska-3 mutants. Number of animals of each genotype imaged is as follows: 101 wild type, 21 unc-18, 25 unc-13, 26 fshr-1, 29 unc-31, 22 wwp-1, 21 tap-1, 24 arr-1, 28 unc-57, 29 egl-3, 38 cam-1, 27 osm-9, 28 sad-1, 29 nab-1, 24 let-60, 18 egl-8, 25 nlp-12, 22 sbt-1, 28 pkc-1, 36 gska-3, 5 prk-2.