WormBase Tree Display for Picture: WBPicture0000014086

WBPicture0000014086 Description: Figure S1. Characterization of ric-7, related to Figure 1. (A) The phylogenetic tree of RIC-7B orthologs shows rapid divergence among nematodes. The RIC-7Aisoform is present only in the Rhabditina clade. RIC-7 from C. elegans and B. malayi only share 23% identity; other mitochondrial proteins such as MIRO share58% identity in these species. Orthologs of the RIC-7 isoforms were identified by performing BLAST searches of other nematode sequence data and were hand assembled from multiple EST and genomic sequences. Sequence identity was determined by pairwise BLASTp alignment. (B) ric-7 mutants are defective foracetylcholine neurotransmission. Animals become paralyzed when exposed to the acetylcholinesterase inhibitor Aldicarb due to accumulation of acetylcholine inthe synaptic cleft. Paralysis in response to Aldicarb was assayed after 8 hrs. ric-7 mutants are resistant to Aldicarb, indicating impaired acetylcholine release (n=10-20 worms per data point). Similar results have been reported by Hao et al. [S1]. (C) In C. elegans GABA stimulates contraction of the enteric muscles [S2,S3]. ric-7 mutants display reduced enteric muscle contractions following posterior body muscle contractions, and this defect was rescued by expressing RIC-7(oxEx233) in the GABA neurons, 'RIC-7(+)'. n-values shown in each bar, p<0.01. (D) The active zone protein RIM (UNC-10) is normally distributed in the dorsalcord of ric-7(n2657) mutants. RFP is fused to the PDZ and C2A domains of RIM, which are sufficient for proper localization [S4]. The number of RIM puncta issimilar between the wild type and ric-7(n2657) mutants (avg # of RIM puncta per 50 um +/- SEM, 30 and 31 images, respectively, p=0.53 two-tailed t-test). (E) Asynaptic vesicle marker, the vesicular GABA transporter (vGAT/UNC-47), is normally localized in ric-7 mutants. vGAT was tagged with GFP and inserted as a single copy. The number of vGAT::GFP puncta is normal in ric-7 mutants (avg # of vGAT puncta per 50 um +/-SEM, 12 and 9 images, respectively, p=0.46 MannWhitneyTest). (F) Electron micrographs of neuromuscular junctions in the adult ventral nerve cord revealed that ric-7(n2657) has normal numbers of vesicles persynapse profile (wild type: 16 synapses, 75 profiles, ric-7(n2657): 15 synapses, 68 profiles). (G) The active zone protein RIM (UNC-10) is normally distributed in the dorsal cord of ric-7(n2657) mutants. (H) A synaptic vesicle marker, the vesicular GABA transporter (vGAT/UNC-47), is normally localized in ric-7mutants. (I) Dense core vesicles are distributed evenly throughout the axons of ric-7 mutants, shown here using the neuropeptide marker FLP-3::Venus. Scale bar = 10 um. (J) Electron micrographs of neuromuscular junctions show synaptic vesicles (arrowheads) surrounding the dense projection (*) for the wild type and ric-7(n2657). Scale bar = 250 nm. (K) ric-7 is expressed in neurons and head muscles. DNA 5 kb upstream of the ric-7a start codon through the second exon was fused to a nuclear localization signal (NLS) and GFP (left). Fluorescence was observed in most neurons in the head, as well as in the head muscles(middle), and motor neurons (right); examples of each are labeled. An asterisk marks autofluorescent gut granules. Anterior is to the left.
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  Crop: Crop_picture: WBPicture0000014087
  Acknowledgment: Template: Reprinted from <Journal_URL>, <Article_URL>, Copyright <Publication_year>, with permission from <Publisher_URL>.
    Publication_year: 2014
    Article_URL: DOI id 10.1016/j.cub.2014.02.025
    Journal_URL: CurrentBiology
    Publisher_URL: Elsevier
  Reference: WBPaper00045008