Byard EH et al. (1986) Stain Technol "A hot aldehyde-peroxide fixation method for electron microscopy of the free-living nematode Caenorhabditis elegans."

Sigurdson WJ, Woods RA, Byard EH

[Stain Technol, 1986]

An improved method for the fixation of the third and fourth larval stages and adults of Caenorhabditis elegans has been developed. Worms are placed in a mixture of 1.5% paraformaldehyde and 1.0% glutaraldehyde at pH 7.0 and 70 C and the suspension promptly cooled in a water bath at 20 C for 1 hr. The fixed worms are then immersed in a mixture of 5% glutaraldehyde and hydrogen peroxide at 4 C for 1 hr, stained en bloc in uranyl acetate, and embedded in resin for electron microscopy. The procedure results in superior fixation, particularly of microfilaments and microtubules. The high temperature of the initial fixation straightens the worms and thus facilitates serial sectioning.

Anna Elisabetta Salcini et al. (2002) European Worm Meeting "The EH network in C.elegans"

Pier Paolo Di Fiore, Lisa Trisciuoglio, Maria Grazia Malabarba, Anna Elisabetta Salcini

[European Worm Meeting, 2002]

The EH domain is an evolutionary conserved protein-protein interaction domain present in proteins from yeast to mammals. A number of its cellular ligands, together with its binding specificity, the NPF tripeptide, have been identified and demonstrated to define a complex network of protein interaction in eukaryotic cell. Many of the EH-containing and EH-binding proteins display characteristics of endocytic accessory proteins, suggesting a role for the EH network in regulation of various step in endocytic processes and, more in general, in protein sorting within the cell. Since endocytosis is a mechanism by which cells interact with the environment, interesting aspects of this process differ between unicellular and multicellular organisms. Our final goal is to define the functions of EH-containing proteins and their binding partners in a multicellular organism contest. In C.elegans, two EH-containing proteins have been identified and characterized: EHS-1 (Salcini et al. Nature Cell Biology, Vol.3, August, 2001) and RME-1 (Grant et al. Nature Cell Biology, Vol. 3, June, 2001) homologues of Eps15 and EHD-1, respectively. The first involved in synaptic-vesicle recycling, the second in yolk uptake in oocytes. Screening of C.elegans data bank revealed at least two other EH-containing proteins: Y39B6A.G, with no homology except in the EH domain, and Y116A8C.36, most likely the homologue of the Drosophila DAP160, a dynamin-binding protein localized in synapses. It is our interest to characterize the binding proprieties of EH-containing proteins, to create a map of the EH-NPF network and investigate the roles of these proteins in C.elegans. To this end, EH domains of the four C.elegans proteins will be used as baits in two-hybrid screen to clone interacting proteins. In silico interactors with NPF repeats, such Epsin protein, a known binding partner of Eps15 in mammals, or Amphiphysin protein, involved in mammals in endocytic processes, will be also analyzed.

Tsushima H et al. (2013) PLoS One "A snapshot of the physical and functional wiring of the Eps15 homology domain network in the nematode."

Cocito A, Di Fiore PP, D'Ario G, Verhoef LG, Tsushima H, Confalonieri S, Bartocci C, Senic-Matuglia F, Malabarba MG, Salcini AE

[PLoS One, 2013]

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.

Woods RA et al. (1989) Canadian Journal of Zoology "The genetics, ultrastructure, and tubulin polypeptides of mebendazole-resistant mutants of Caenorhabditis elegans."

Byard EH, Malone KMB, Spence AM, Sigurdson WJ, Woods RA

[Canadian Journal of Zoology, 1989]

Mebendazole-resistant mutants of Caenorhabditis elegans were isolated following mutagenesis of the wild-type strain, N2, with ethylmethane sulphonate. The mutants define a single autosomal gene and are allelic to ben-1. They grow, move, and reproduce normally in the presence of mebendazole and are cross-resistant to albendazole, cambendazole, fenbendazole, methylbenzimidazole carbamate, and thiabendazole. Accumulations of membrane-bound, electron-dense material associated with the nervous tissue and associated cells are seen in electron micrographs of L3, L4 and adult stages of N2 grown in the presence of mebendazole, but are not found in the mutants. The electron-dense material appears to be associated with the accessory cells of the neuropil rather than with neurons. The tubulins from wild type, N2, and the mutants were characterised by two-dimensional electrophoresis followed by silver staining and Western blotting with monoclonal antibodies to a- and B-tubulin. A single isotype of a-tubulin is apparent in both N2 and the mutants. One major and three minor B-tubulin isotypes, bt-1 to bt-3, can be discerned in N2; the minor isotype bt-2 is absent in all the mutants. These findings suggest that the isotype bt-2 is the primary site of action of benzimidazole-based drugs in C. elegans.

Grant B et al. (2001) Nat Cell Biol "Evidence that RME-1, a conserved C. elegans EH-domain protein, functions in endocytic recycling."

Paupard MC, Hirsh D, Lin SX, Hall DH, Grant B, Zhang Y

[Nat Cell Biol, 2001]

In genetic screens for new endocytosis genes in Caenorhabditis elegans we identified RME-1, a member of a conserved class of Epsl5-homology (EH)-domain proteins. Here we show that RME-1 is associated with the periphery of endocytic organelles, which is consistent with a direct role in endocytic transport. Endocytic defects in rme-l mutants indicate that the protein is likely to have a function in endocytic recycling. Evidence from studies of mammalian RME-1 also points to a function for RME-1 in recycling, specifically in the exit of membrane proteins from recycling endosomes. These studies show a conserved function in endocytic recycling for the RME-1 family of EH proteins.

Lin SX et al. (2001) Nat Cell Biol "Rme-1 regulates the distribution and function of the endocytic recycling compartment in mammalian cells."

Grant B, Maxfield FR, Hirsh D, Lin SX

[Nat Cell Biol, 2001]

RME-1 is an Eps15-homology (EH)-domain protein that was identified in a genetic screen for endocytosis gene in Caenorhabditis elegans. When expressed in a CHO cell line, the worm RME-1 protein and a mouse homologue are both associated with the endocytic recycling compartment. Here we show that expression of a dominant-negative construct with a point mutation near the EH domain results in redistribution of the endocytic recycling compartment and slowing down of transferrin receptor recycling. The delivery of a TGN38 chimaeric protein to the trans-Golgi network is also slowed down. The function of Rme-1 in endocytic recycling is evolutionarily conserved in metazoans as shown by the protein's properties in C. elegans.

Page LJ et al. (1999) J Cell Biol "Gamma-synergin: an EH domain-containing protein that interacts with gamma-adaptin."

Page LJ, Lui WW, Sowerby PJ, Robinson MS

[J Cell Biol, 1999]

The AP-1 adaptor complex is associated with the TGN, where it links selected membrane proteins to the clathrin lattice, enabling these proteins to be concentrated in clathrin-coated vesicles. To identify other proteins that participate in the clathrin-coated vesicle cycle at the TGN, we have carried out a yeast two- hybrid library screen using the gamma-adaptin subunit of the AP-1 complex as bait. Two novel, ubiquitously expressed proteins were found: p34, which interacts with both gamma-adaptin and alpha-adaptin, and gamma-synergin, an alternatively spliced protein with an apparent molecular mass of approximately 110-190 kD, which only interacts with gamma-adaptin. gamma-Synergin is associated with AP-1 both in the cytosol and on TGN membranes, and it is strongly enriched in clathrin-coated vesicles. It binds directly to the ear domain of gamma-adaptin and it contains an Eps15 homology (EH) domain, although the EH domain is not part of the gamma-adaptin binding site. In cells expressing alpha-adaptin with the gamma-adaptin ear, a construct that goes mainly to the plasma membrane, much of the gamma-synergin is also rerouted to the plasma membrane, indicating that it follows AP-1 onto membranes rather than leading it there. The presence of an EH domain suggests that gamma-synergin links the AP-1 complex to another protein or proteins.

Saumya Pant et al. (2007) International Worm Meeting "An RNAi Screen For Genes Affecting Endocytic Recycling."

Barth D. Grant, Saumya Pant, Zita Balklava

[International Worm Meeting, 2007]

Endocytosis is a vesicle mediated process for the internalization of receptor-bound ligands, fluid, solutes and other associated macromolecules. Receptors are often recycled to the plasma membrane to participate in further rounds of endocytosis (endocytic recycling). Endocytic recycling has an important role in maintaining cellular homeostasis of membrane ~ lipid components and in maintaining distinct apical vs. basolateral domains in polarized cells. We have previously shown that the RME-1 protein mediates endocytic recycling in several C.elegans tissues. RME-1 is a conserved protein homologous to four mammalian proteins called mRme-1/Ehd1, Ehd2, Ehd3 and Ehd4. All of these proteins contain an ATP binding N-terminal P-loop that is critical for membrane association and homo-oligomerization. The central coiled-coil region is also involved in oligomerization. The C-termini of RME-1 family proteins contain a single eps15-homology (EH) domain. Classically, EH domains are found in endocytosis proteins where they function as protein-protein interaction motifs, binding target proteins through Asparagine-Proline-Phenylalanine (NPF) sequences. Mammalian Ehds are known to bind target proteins such as Syndapin, Rabenosyn5, EHBP1 and Numb in this way. Curiously, the worm homologues of all these RME-1 interaction partners lack the NPF motif. We are investigating if worm syndapin (sdpn-1) can still interact with and affect RME-1 in C.elegans. If this is true we want to further investigate how the proteins interact in the absence of the classical EH-NPF interaction, whether syndapin is a regulator of endocytic recycling in polarized cells like the worm intestine and neurons, and whether the molecular machinery involved in this recycling complex are conserved from worms to mammals. Preliminary evidence supports a role for SDPN-1 in the RME-1-mediated recycling pathway of the worm intestine. We additionally utilized an RNAi based approach in a GFP-RME-1 expressing strain to directly screen for genes that affect recycling endosome morphology. We focused on worm homologs of known or suspected regulators of membrane trafficking, predicted worm proteins bearing repeated NPF motifs, and candidate endocytic regulators identified in a genome-wide RNAi screen performed in our lab (Balklava et al). We found several interesting genes affecting endocytic recycling. We followed up on some potentially interesting candidates using mutant analysis and worm expression studies. We will report our findings in the meeting.

Grant BD et al. (2008) Traffic "Mechanisms of EHD/RME-1 protein function in endocytic transport."

Grant BD, Caplan S

[Traffic, 2008]

The evolutionarily conserved EHD/RME-1 family of C-terminal EH-domain proteins has recently come under intense scrutiny because of its importance in intracellular membrane transport, especially with regard to the recycling of receptors from endosomes to the plasma membrane. Recent studies have shed new light on the mode by which these ATPases function on endosomal membranes in mammals and C. elegans. This review highlights our current understanding of the physiological roles of these proteins in vivo, discussing conserved features as well as emerging functional differences between individual mammalian paralogs. In addition, these findings are discussed in light of the identification of novel EHD/RME-1 protein and lipid interactions, and new structural data for proteins in this family, indicating intriguing similarities to the Dynamin superfamily of large GTPases.

George M et al. (2007) BMC Cell Biol "Shared as well as distinct roles of EHD proteins revealed by biochemical and functional comparisons in mammalian cells and C. elegans."

Parikh PT, Band H, Solomon A, Band V, George M, Rainey MA, Gao Q, Ying G

[BMC Cell Biol, 2007]

BACKGROUND: The four highly homologous human EHD proteins (EHD1-4) form a distinct subfamily of the Eps15 homology domain-containing protein family and are thought to regulate endocytic recycling. Certain members of this family have been studied in different cellular contexts; however, a lack of concurrent analyses of all four proteins has impeded an appreciation of their redundant versus distinct functions. RESULTS: Here, we analyzed the four EHD proteins both in mammalian cells and in a cross-species complementation assay using a C. elegans mutant lacking the EHD ortholog RME-1. We show that all human EHD proteins rescue the vacuolated intestinal phenotype of C. elegans rme-1 mutant, are simultaneously expressed in a panel of mammalian cell lines and tissues tested, and variably homo- and hetero-oligomerize and colocalize with each other and Rab11, a recycling endosome marker. Small interfering RNA (siRNA) knock-down of EHD1, 2 and 4, and expression of dominant-negative EH domain deletion mutants showed that loss of EHD1 and 3 (and to a lesser extent EHD4) but not EHD2 function retarded transferrin exit from the endocytic recycling compartment. EH domain deletion mutants of EHD1 and 3 but not 2 or 4, induced a striking perinuclear clustering of co-transfected Rab11. Knock-down analyses indicated that EHD1 and 2 regulate the exit of cargo from the recycling endosome while EHD4, similar to that reported for EHD3 (Naslavsky et al. (2006) Mol. Biol. Cell 17, 163), regulates transport from the early endosome to the recycling endosome. CONCLUSION: Altogether, our studies suggest that concurrently expressed human EHD proteins perform shared as well as discrete functions in the endocytic recycling pathway and lay a foundation for future studies to identify and characterize the molecular pathways involved.