<i>cuti-1(RNAi)</i> worms display defects in seam cell function and epithelial integrity.

<p>A, B. Confocal micrographs of the seam cell boundaries of adult <i>control(RNAi)</i>;<i>dlg-1::gfp</i> and <i>cuti-1(RNAi)</i>;<i>dlg-1::gfp</i> worms respectively. <i>cuti-1(RNAi)</i>;<i>dlg-1::gfp</i> worms either failed to form a seam cell syncytium or failed to maintain the cell-cell contact between seam cells (B). C, D. Scanning electron micrographs of N2 worms fed control dsRNA and <i>cuti-1</i> dsRNA respectively. The lateral alae secreted by the underlying seam cells in <i>cuti-1(RNAi)</i> worms were bifurcated (arrow) (D). E, F. Confocal micrographs of adult <i>control(RNAi)</i>;<i>col-19::gfp</i> and <i>cuti-1(RNAi)</i>;<i>col-19::gfp</i> worms respectively. COL-19 is an adult-specific collagen that is expressed in the lateral alae (asterisks) and circumferential annuli (double-headed arrows) of the cuticle (E). Using COL-19 as a marker, the alae (asterisks) and annuli (double-headed arrow) appear highly disorganized in <i>cuti-1(RNAi)</i>;<i>col-19::gfp</i> adults (F). G. Nomarski micrograph of a <i>cuti-1(RNAi)</i> L4 worm showing vacuolation of the hypodermis (arrow). H. Scanning electron micrograph of an adult <i>cuti-1(RNAi)</i> worm showing a protruding vulva. I, J. Fluorescent micrographs of the anterior region of N2 adults fed control dsRNA and <i>cuti-1</i> dsRNA respectively. Worms were soaked in a 1 µg/ml solution of Hoechst 33258 (Sigma) for 15 mins. Hoechst 33258 is a membrane impermeable dye that fluoresces when bound to DNA. <i>Control(RNAi)</i> worms do not show fluorescence of nuclei when soaked in a solution of Hoechst 33258 (I). In contrast, nuclei of <i>cuti-1(RNAi)</i> worms fluoresce because of reduced barrier function of the cuticle and hypodermis (J). Scale bars 50 µm, except in C and D where scale bars 10 µm.</p

Multiple sequence alignment of CUTI-1 with known nematode homologues.

<p>A translated BLAST analysis was carried out through the European Bioinformatics Institute server. This identified homologues in <i>C. briggsae</i> (Cb; CBG12615), <i>Meloidogyne javanica</i> (Mj; BE578004), and <i>Heterodera glycines</i> (Hg; CB824616). In addition, a translated BLAST analysis was carried out against the <i>Haemonchus contortus</i> genome through the Wellcome Trust Sanger Institute and the <i>Brugia malayi</i> genome through The Institute for Genomic Research. This identified the partial <i>H. contortus</i> (Hc) homologue (haem-257m03.q1k) and the <i>B. malayi</i> homologue (Bm; Bm1_49050). Solid bars denote predicted transmembrane domains of <i>C. elegans</i> (Ce) CUTI-1. Roman numerals denote cytoplasmic regions I–V.</p

Postembryonic RNAi phenotypes observed following knockdown of expression of <i>cuti-1</i> in <i>C. elegans</i>.

<p>Nomarski micrographs of <i>C. elegans</i> fed on dsRNA-expressing bacteria for 3 days at 21°C. A. A gravid N2 hermaphrodite fed on control dsRNA. B–D. N2 <i>C. elegans</i> fed on <i>cuti-1</i> dsRNA displaying a variety of defects related to cuticle dysfunction that includes: dumpy body morphology and ruptured vulva (B), cuticle blistering (C) and cuticle constrictions caused by the previous cuticle wrapping around the body (D). E. A <i>control(RNAi)</i>;<i>him-8(e1489)</i> male showing fully elongated sensory rays (asterisk). F. A <i>cuti-1(RNAi)</i>;<i>him-8(e1489)</i> male showing sensory rays that have not fully elongated (asterisk) and blistering of the cuticle (arrow). Scale bars 50 µm.</p

The spatial and temporal expression of CUTI-1::GFP and <i>cuti-1</i> mRNA in <i>C. elegans</i>.

<p>A, B. Confocal micrographs of the expression pattern observed in CUTI-1::GFP transgenic <i>C. elegans</i>. A. A transgenic L4 worm. GFP expression is observed in the hypodermal (h) cells of the head, vulva (v) and the main body syncytia, hyp 7, which also surrounds the opening of the excretory canal (exc) and the anus (a). B. In addition, GFP is present in the seam cells and at the seam cell boundary (sc). The transcription of <i>cuti-1</i> cycles throughout development, peaking prior to each molt (C). This expression pattern is similar to that of the collagen, <i>sqt-1</i>, which is up-regulated two hours prior to each larval molt <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005117#pone.0005117-Johnstone4" target="_blank">[36]</a>. Scale bars 50 µm.</p

CUTI-1 interacts with the vesicle protein, VPS-39.

<p>A. Full-length CUTI-1 and each of the cytoplasmic regions, excluding region II, were fused to the Gal4 DNA binding domain (DB) and used to probe a cDNA library of proteins fused to the Gal4 activation domain (AD). Cotransformants were assayed for growth on selective media either lacking uracil (-URA) or histidine (-HIS), in addition to assaying the production of β-galactosidase (X-GAL). (A) shows an example of a colony containing CUTI-I and VPS-39 that tested positive for an interaction under all phenotypic conditions used. B. Schematic representation of <i>C. elegans</i> VPS-39 showing domain architecture. <i>C. elegans</i> VPS-39 is a 923 amino acid (aa) protein that contains an amino-terminal citron homology domain (CNH) and a carboxyl-terminal clathrin heavy chain repeat (CLH) motif. Underline denotes the region fused to AD in yeast two-hybrid experiments. C. The II and IV cytoplasmic regions of CUTI-1 were fused to GST and immobilised onto glutathione beads. The carboxyl terminus of VPS-39 fused to a FLAG epitope (VPS-39::FLAG) was then incubated with the GST protein for three hours to allow interactions to occur. Unbound VPS-39::FLAG was then removed before any bound interacting proteins were removed and detected by carrying out an anti-FLAG immunoblot. As a control, GST was used to determine if GST alone could interact with VPS-39::FLAG. TNT, 3′ VPS-39::FLAG product synthesized <i>in vitro</i> using the TNT® Coupled Reticulocyte Lysate System (Promega).</p

Ultrastructural analysis of <i>cuti-1(RNAi)</i> adults and dauer larvae.

<p>Transmission electron micrographs of N2 (A, B) and <i>daf-2(e1370)</i> worms (C, D). A, B. Adult N2 <i>C. elegans</i> fed control and <i>cuti-1</i> dsRNA respectively. In <i>cuti-1(RNAi)</i> worms electron dense material accumulated within the cuticle and vesicles or vesicle-like structures accumulated within the hypodermis (arrows) (B). <i>cuti-1(RNAi)</i> worms also tended to retain cuticle from the previous developmental stage. C, D. <i>daf-2(e1370)</i> dauers fed control and <i>cuti-1</i> dsRNA respectively. In <i>cuti-1(RNAi)</i> dauers, the epicuticle (ep) was present within the cortical layer (c) instead of at the apical surface. In addition, the cortical layer was expanded and had a granular appearance. The basal layer (b) was present, but was not striated compared with <i>control(RNAi)</i> worms. Vesicles or vesicle-like structures were also evident within the hypodermis (arrows). C, cuticle; H, hypodermis; UC, unshed cuticle. Scale bars 1 µm.</p

<i>vha-19(RNAi)</i>embryos are osmotically sensitive and appear to have a cytokinesis defect.

<p>A) Embryos cut out of the uterus of wild type (A-B,F) or pie-1::H2B::GFP [AZ212] (E) adult <i>C. elegans</i> fed control or <i>vha-19</i> dsRNA for 24 hours. Note the presence of an eggshell in all embryos in A (arrow heads), despite the random clusters of nuclei and compression of some <i>vha-19(RNAi)</i> embryos. Note also the presence of a polar body (pb) in a <i>vha-19(RNAi)</i> embryo in E, even though the nuclei are not cellularized (inset, bright field image), and that in F, non-cellularized, small clusters of nuclei (square bracket) are surrounded by cleavage furrows (arrows) in the <i>vha-19(RNAi)</i> embryo. C) The percentage of <i>control(RNAi)</i> and <i>vha-19(RNAi)</i> embryos that swelled or shrunk in deionized water (mQ H2O), 150 mM potassium chloride (KCl) and 300 mM KCl. Examples of embryos from this osmolarity experiment are shown in B. D) Embryos in the uterus of adults fed control or <i>vha-19</i> dsRNA for 48 hours and stained with Acridine Orange. Note that embryos in the uterus of <i>control(RNAi)</i> adult excluded Acridine Orange (arrows) whereas the embryos in the uterus of the <i>vha-19(RNAi)</i> adult were permeable to Acridine Orange (bracket). Scale bars represent 100 µm in D and 10 µm in A-B,E and F.</p

The progeny produced by mating <i>fog-2</i> male <i>C. elegans</i> with <i>fog-2</i> female <i>C. elegans</i> fed on either control or <i>vha-19</i> dsRNA.

<p>∧Significantly different to average brood size (p<0.01) or number of progeny that reached adulthood (p<0.0001) for <i>fog-2(q71) V</i>; <i>control(RNAi)</i>l x <i>fog-2(q71) V</i>; <i>control(RNAi)</i> mating.</p>*<p>Not significantly different to average brood size/number of progeny that reached adulthood for <i>fog-2(q71) V</i>; <i>vha-19(RNAi)</i> x <i>fog-2(q71) V</i>; <i>vha-19(RNAi)</i> mating (p>0.05).</p

VHA-19 is required for uptake of vitellogenin via trafficking of the RME-2 receptor.

<p>A) VIT-2::GFP <i>C. elegans</i> [DH1033] were fed control or <i>vha-19</i> dsRNA from the L4 stage for 48 hours and the oocytes examined. VIT-2::GFP did not reach the oocytes in adults fed <i>vha-19</i> dsRNA, instead it accumulated between the oocytes (thin arrows). Fat arrows indicate agglomerates of VIT-2::GFP. B) RME-2::GFP was not trafficked to the plasma membrane in RME-2::GFP; <i>vha-19(RNAi)</i> adults (arrows and dotted circle), whereas RME-2::GFP did reach the plasma membrane in worms fed control dsRNA (thin arrows between oocytes). Worms were fed <i>vha-19</i> or control dsRNA for 48 hours. Arrowheads in bright field panels indicate individual oocytes. Scale bars represent 10 µm.</p

<i>C. elegans</i> arrest as larvae when fed on <i>vha-19</i> dsRNA from the first larval stage.

<p><i>C. elegans</i> arrest as larvae when fed on <i>vha-19</i> dsRNA from the first larval stage.</p