The EH network in <i>C. elegans</i>. An interaction diagram is shown representing <i>C. elegans</i> EH proteins (red circles) together with their interactors (blue circles); the interactors are further grouped into functional categories that were derived from the Wormbase and the Gene Ontology databases, from the literature, or inferred from functions of the mammalian homologues.

<p>Interactions uncovered in this study by Y2H are shown by light blue lines. Interactions confirmed by <i>in vitro</i> binding assays are shown by dark blue lines. Interactions not fully depending on the EH domain are shown with dashed lines. Additional interactions, derived from the BioGRID database (<a href="http://thebiogrid.org/" target="_blank">http://thebiogrid.org/</a>) and from the literature, are shown by red lines. The picture was initially generated using the Osprey software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056383#pone.0056383-Breitkreutz1" target="_blank">[96]</a> and then edited with Adobe Illustrator.</p

Yeast Two Hybrid analysis of EH-proteins in <i>C. elegans</i>.

<p>(A) Schematic diagram of the five EH-containing proteins in <i>C. elegans</i>. Note that several isoforms are reported in wormbase. Here, we show the isoforms cloned, sequenced and used for the described experiments. Baits used for the Y2H are indicated by black lines. For EHS-1, two distinct baits were used in the screens, since a bait spanning the three EH domains showed self-activation. CC, coiled-coil region; SH3, region containing multiple SH3s in ITSN-1; PxxP, region containing multiple SH3-binding sites in EHS-1; DPFs, region containing multiple AP-2-binding sites in EHS-1; P-loop, nucleotide-binding domain in RME-1. (B) Results of the Y2H screen. The 26 identified EH-interactors are listed. Potential EH-binding motifs are indicated. Black, interactions detected in the initial screen; gray, interactions detected in the re-transformation assay (see text). The number of clones identified in the initial screen is also shown. No interactions were detected for R10E11.6. (C) The indicated genes were tested by quantitative PCR in the yeast library used for the Y2H screening. The number of EH-interacting motifs (NPF) and the frequency of identification in the Y2H (H, high; In, intermediate; L, low; No, no interaction) are shown at the bottom. The estimated number of copies present in the cDNA library is shown, by grey bars, in arbitrary units relative to the level of representation of <i>epn</i>-1 that was set to 100. As a comparison we show, using black bars, the frequency of isolation of the various clones in Y2H, again relative to the frequency of isolation of <i>epn</i>-1 that was set to 100 ( = 45 clones).</p

In vitro binding assays.

<p>Sixteen interactors, identified by Y2H (listed at the bottom), were expressed as GST-fusion proteins and used for <i>in vitro</i> binding assays with FLAG-EH proteins expressed in Phoenix cells. Results are the average of three independent experiments (examples are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056383#pone.0056383.s003" target="_blank">Figure S3</a>), and are expressed in arbitrary units on a scale 0–100, in which 100 represents the efficiency of the pull-down for the strongest interacting protein in each panel.</p

Effect of RNAi of EH interactors in various genetic backgrounds.

<p>Down-regulation of the EH-interactors was achieved by feeding RNA interference (RNAi), in the indicated strains, and animals were tested for aldicarb sensitivity. (A) In the column N2, the effect of RNAi on aldicarb sensitivity in wild type (N2) animals is reported (H, hypersensitive to aldicarb, R, resistant to aldicarb). In the other columns, the type of genetic interaction, detected in the various strains, is reported (S, suppressing; W, worsening; Rv, reverting; A, asynthetic; L, lethal; Ep, possibly RNAi epistatic; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056383#pone-0056383-t001" target="_blank">Table 1</a>). (B) Examples of the detected genetic interactions. Results are expressed as the change in the λ parameter in the best-fitting Weibull distribution with respect to WT. “KO strain”, null mutant for the EH-containing gene; “RNAi in WT”, N2 worms in which the EH-interactor was silenced by RNAi; “RNAi in KO strain”, null mutants for the EH-containing gene in which the EH-interactor was silenced by RNAi; Null hypothesis, mathematical sum of the observed phenotypes in the “KO strain” and “RNAi in WT conditions”. Details of the analysis are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056383#s2" target="_blank">Materials and Methods</a>.</p

A Snapshot of the Physical and Functional Wiring of the Eps15 Homology Domain Network in the Nematode

<div><p>Protein interaction modules coordinate the connections within and the activity of intracellular signaling networks. The Eps15 Homology (EH) module, a protein-protein interaction domain that is a key feature of the EH-network, was originally identified in a few proteins involved in endocytosis and vesicle trafficking, and has subsequently also been implicated in actin reorganization, nuclear shuttling, and DNA repair. Here we report an extensive characterization of the physical connections and of the functional wirings of the EH-network in the nematode. Our data show that one of the major physiological roles of the EH-network is in neurotransmission. In addition, we found that the proteins of the network intersect, and possibly coordinate, a number of “territories” of cellular activity including endocytosis/recycling/vesicle transport, actin dynamics, general metabolism and signal transduction, ubiquitination/degradation of proteins, DNA replication/repair, and miRNA biogenesis and processing.</p> </div

Description of genetic interactions.

<p>Aldicarb sensitivity was measured, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056383#s2" target="_blank">Materials and Methods</a>, at 0.5 mM aldicarb. A genetic interaction was scored when the aldicarb-response phenotype of the condition “RNAi in KO strain” was statistically different (p<0.05) from the sum of the individual phenotypes in the conditions “KO strain” and “RNAi in N2” (null hypothesis). The type of genetic interaction was further defined according to the effect that silencing of the EH-interactor gene had on the aldicarb response of the EH-mutant strain, by comparing the “RNAi in KO strain” to the “KO strain” conditions, as specified in the Table.</p>*<p>as defined by Drees et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056383#pone.0056383-Drees1" target="_blank">[97]</a>.</p

REPS-1 expression and function.

<p>(A) The <i>reps-1</i> locus. The deletion in the <i>tm2156</i> strain is also shown. (B) Protein lysates from wild-type and a transgenic line carrying a translational fusion of the <i>reps-1</i> gene with GFP (<i>reps-1p::REPS-1::GFP</i>) were probed with indicated antibodies. (C) Images (epifluorescence) of hermaphrodites carrying the <i>reps-1p::REPS-1::GFP</i> transgene. Anterior is to the left, ventral down. NR: nerve ring, INT: intestine, ESC: excretory system, V: vulva, NC: nerve cord, M: muscle cell. The asterisks indicate nerve commissures. Bars: 100 µm in a, 20 µm in b, 10 µm in c and d. (D) Aldicarb test on strains carrying mutations in EH proteins. Synchronized young adult animals were plated onto NGM plates containing 0.5 mM aldicarb and assayed after the indicated times for movement. The number of animals responding to a light touch with a platinum wire is reported. Results are the average of three independent experiments, each performed on 60 animals/strain. Note that the curves for N2 and <i>rme-</i>1 worms overlap almost completely. P values are indicated in the table.</p

Characteristics of EH interactors.

<p>Some characteristics of EH-interactors. Additional information is in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056383#pone.0056383.s006" target="_blank">Table S1</a>.</p>1<p>Human ortholog were identified through NCBI Homologene or by BLAST searches. (-) indicates that no human orthologue is immediately apparent; (*) indicates putative ortholog (best guess).</p>2<p>Previously known interactions between EH-containing proteins and EH-interactors were obtained from Wormbase or by literature search (indicated by the appropriate references). When the EH-containing protein is indicated (e.g. EHS-1), the physical interaction with the interactor has been described; when the gene is indicated (e.g. <i>ehs</i>-1), the genetic interaction between the genes has been described. (**); in the case of TFG-1, an interaction with the SH3 domains of ITSN-1 was described, by Y2H <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056383#pone.0056383-Wang1" target="_blank">[63]</a>, and TFG-1 was identified by mass-spec in anti-ITSN-1 immunoprecipitates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056383#pone.0056383-Wang1" target="_blank">[63]</a>.</p>3<p>The expression patterns in <i>C. elegans</i> were derived from Wormbase. NS, nervous system; INT, intestine; PHA, pharynx; BWM, body wall muscles; VPC, vulval precursor cells; VPC-de, VPC descendants; HYP, hypodermis; EMBR, expressed during embryogenesis; VM, vulval muscle; RS, reproductive system; EPI, epidermis; N/A, not annotated.</p>4<p>Functional categories were derived from Wormbase, from the Gene Ontology database, from literature data or inferred from functions of the human homologues. END, endocytosis; TRA, membrane and vesicular traffic; UB/DE, ubiquitin system and protein degradation; ACT, actin dynamics; miRNA, miRNA function; MET, metabolism; AP, apoptosis; SIGN, signaling; TRAN, transcription; REPL, DNA replication; REPA, DNA repair; UNKN, unknown. (?) indicates hypothetical function.</p