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.

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.

Anbing Shi et al. (2006) Development & Evolution Meeting "Alx-1 Function in C. elegans Membrane Traffic"

Anbing Shi, Vanessa Figueroa, Saumya Pant, Carlos Chih-Hsiung Chen, Barth Grant

[Development & Evolution Meeting, 2006]

The endocytic pathway of eukaryotes is essential for the internalization and trafficking of macromolecules, fluid, membranes, and membrane proteins. We have previously shown that RME-1 functions as a critical regulator of endocytic recycling events in both clathrin medicated and clathrin independent endocytosis events. RME-1 is a conserved EHD family protein harboring a C-terminal EH domain. In a yeast 2-hybrid screen with the RME-1-EH domain we identified a novel binding partner called Alx-1. Alx-1 is the C. elegans homolog of mammalian Alix and yeast Bro1p. Published reports indicate that Alix/Bro1p functions in the multivesicular body (MVB) pathway. MVBs are endosomes containing lumenal vesicles formed by inward invagination of the limiting membrane of endosomes. MVBs are believed to mediate transport between early endosomes and late endosomes/lysosomes. Membrane proteins sorted into the lumenal vesicles of MVBs are degraded upon fusion of the MVBs with lysosomes. Oligomeric protein complexes containing ubiquitin binding protein Hrs and ESCRT (endosomal sorting complexes required for transport) complexes mediate sorting of cargo at the limiting membranes of the MVBs and invagination of the endosome membrane. Studies in yeast have shown that Bro1 functions in concert with components of the ESCRT machinery to regulate MVB formation (Odorizzi et al., 2003). Alix also associates with structural proteins of the cytoskeleton, including actin and tubulins (Schmidt et al., 2003). Recently, Alix has been reported to function in the actin-dependent intracellular positioning of endosomes in tissue culture cells (Cabezas et al., 2005). Although Alix has been suggested to function in multiple cellular processes, its precise functions still remain somewhat mysterious. The interaction with RME-1 suggests either that ALX-1 plays a role in recycling or that RME-1 plays a role in the MVB. We have taken a functional approach to determine ALX-1 function in vivo taking advantage of the consortium knockout allele alx-1 (gk275) and by colocalization analysis of ALX-1 with various endosome makers. Our progress in this analysis will be presented at the meeting.

Saumya Pant et al. (2006) Development & Evolution Meeting "An RNAi Screen For Genes Affecting Endocytic Recycling"

Zita Balklava, Barth Grant, Saumya Pant

[Development & Evolution Meeting, 2006]

Endocytosis is a vesicle mediated process for the internalization of ligand-bound receptors, fluid, solutes and other associated macromolecules. Once internalized, cargo-laden vesicles fuse with endosomes where receptors and ligands can be sorted. Ligands often dissociate from receptors in a pH-dependent manner and are degraded in the late endosomes or lysosomes. 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 and lipid components. In particular, polarized cells depend on accurate sorting during endocytic recycling to maintain distinct apical vs. basolateral domains. 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) motifs.Mammalian Ehds are known to bind target proteins syndapin, rabenosyn5, EHBP1 and Numb in this way. Curiously all of the worm homologues of these proteins lack the NPF motif. We have utilized an RNAi based approach in a GFP-RME-1 expressing strain to directly screen for genes that affect recycling endosome morphology. So far we have focused on 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 have found several interesting genes affecting endocytic recycling. We will present our progress in this screen at the meeting.

Barth D Grant et al. (2001) International C. elegans Meeting "RME-1, a novel EH domain protein required for endocytic recycling in C. elegans and mammalian cells"

David H Hall, Frederick R Maxfield, David Hirsh, Yinhua Zhang, Marie-Christine Paupard, Sharron X Lin, Barth D Grant

[International C. elegans Meeting, 2001]

The endocytic pathway is essential for the internalization and trafficking of macromolecules, fluid, membranes, and membrane proteins. The steps involved in uptake and endocytic trafficking within the endosomal system have been described, but many of the components mediating these steps at the molecular level remain to be identified. In particular, little is known about the proteins required for endocytic recycling, a critical membrane trafficking event in all cells. Functional characterization of a new endocytosis gene, rme-1 , by C. elegans genetics, suggests that it functions in endocytic recycling. rme-1 mutants display endocytosis defects in several cell types. These defects include strongly reduced uptake of yolk proteins by oocytes, a receptor-mediated process. In rme-1 mutants, yolk receptors accumulate in an abnormally expanded cortical endosomal compartment, implicated in recycling. rme-1 mutant intestinal cells display an abnormal accumulation of basolateral, but not apical, fluid-phase endocytosis markers in large vacuolated structures. Uptake and trafficking of the membrane dye FM 4-64 from the basolateral membranes of the intestine is unaffected by rme-1 mutations, suggesting that membrane internalization and late endosomal trafficking are independent of rme-1 . RME-1 is a member of a new class of proteins, evolutionarily conserved among multicellular organisms, bearing an amino-terminal P-loop domain, a central coiled-coil domain, and a carboxy-terminal EH domain. Yeast two-hybrid analysis indicates that the P-loop and coiled-coil regions are required for multimerization. RME-1 is a cytoplasmic protein associated with the periphery of endocytic organelles, consistent with a direct role for RME-1 in endocytic trafficking. We find that Ce-RME-1 and mammalian Rme-1 (mRme-1) expressed in CHO cells localizes primarily to the endocytic-recycling compartment (ERC). In cells expressing dominant negative mRme-1 G429R, a mutation near the EH domain equivalent to a worm dominant negative rme-1 mutation, we identified a kinetic slowing of transferrin (Tf) recycling with no apparent alteration of the rate constant for Tf internalization. In addition, TGN38 delivery to the TGN from the ERC was slowed in mRme-1 G429R expressing cells. Together, these data suggest that RME-1 functions specifically in the export of molecules from the ERC.

Hall DH et al. (2000) East Coast Worm Meeting "rme-1, a new endocytosis gene implicated in recycling"

Hirsh DI, Hall DH, Paupard M-C, Grant BD, Zhang Y

[East Coast Worm Meeting, 2000]

Endocytosis is essential to many aspects of metazoan life, including nutrient uptake, synaptic vesicle recycling, and growth factor receptor down-regulation. Much remains to be learned about this important cellular pathway. We have isolated a collection of endocytosis defective mutants, the rme genes. Recently, we focused on the analysis of rme-1, a gene that may regulate endocytic recycling. rme-1 mutants are defective in endocytosis in multiple cell types including oocytes and coelomocytes. rme-1 mutants also develop large vacuoles in the intestine and hypodermis. The intestinal vacuoles accumulate endocytosis markers, but only from their basolateral (pseudocoelomic) surfaces. Specifically, we found that fluorescent dextran microinjected into the pseudocoelom rapidly accumulates within rme-1 mutant vacuoles. Another endocytosis marker microinjected into the pseudocoelom, the membrane dye FM4-64, is excluded from rme-1 mutant vacuoles and instead accumulates in autofluorescent gut granules (lysosomes). Because fluid-phase markers like dextran can enter the recycling pathway, and FM4-64 does not, rme-1 mutant vacuoles may represent aberrant recycling endosomes. We found that rme-1 encodes a member of a new family of proteins bearing a C-terminal EH, or eps15 homology, domain. The EH domain has been found at the N-terminus of a small number of proteins, each of which has been implicated in endocytosis. RME-1 also contains a predicted P-loop sequence associated with ATP or GTP binding in many proteins, as well as a predicted coiled-coil domain. Homologs of RME-1 are found in the human and fly genome databases, but have not been studied functionally. No homologs of RME-1 are found in the complete yeast genome sequence. Sequencing of rme-1 cDNAs and 5 RACE analysis identified four different rme-1 mRNAs differing at their 5 ends, encoding four predicted RME-1 isoforms. Sequencing of our rme-1 mutant alleles revealed mutations in highly conserved residues of the RME-1 region shared by all isoforms. Immunofluorescence with anti-RME-1 Abs shows that RME-1 is a cytoplasmic protein expressed in all cells, and that it is associated with the periphery of basolateral endosomes in the wild-type intestine.

Pant, S. et al. (2009) International Worm Meeting "AMPH-1/Amphiphysin/BIN1: A Novel Regulator of Endocytic Recycling."

Patel, K., Caplan, S., Carr, C., Grant, B., Pant, S., Sharma, M.

[International Worm Meeting, 2009]

Receptor-mediated endocytosis is crucial for the internalization of receptor-bound ligands of many types. Cargo-laden internalized vesicles fuse with early endosomes. Endosome maturation allows sorting with degradation in the late endosomes and lysosomes or recycling to the plasma membrane to participate in further rounds of endocytosis. Endocytic recycling maintains cellular homeostasis of membrane and lipid components and in polarized cells allows accurate sorting to maintain distinct apical and basolateral domains. The Grant Lab utilizes Caenorhabditis elegans as a model system to uncover the molecular players involved in endocytic recycling. We have previously established that the RME-1 protein is found on the endocytic recycling compartment and mediates endocytic recycling in several C. elegans tissues. RME-1 is a conserved protein homologous to four mammalian proteins EHD1-EHD4. The protein is an ATPase with an ATP binding N-terminal P-loop that is critical for membrane association and homo-oligomerization. The recent EHD2 crystal structure reveals that, while being ATPases, EHD/RME-1 family proteins bear structural similarity to the clathrin-coated pit GTPase Dynamin. The C-termini of RME-1 family proteins contain a single Eps15-homology (EH) domain. Classically, EH domains function as protein interaction motifs in endocytosis proteins, binding target proteins through Asparagine-Proline-Phenylalanine (NPF) motifs. Mammalian EHD proteins bind to Syndapin, Rabenosyn5, EHBP1, and Numb in this manner.Utilizing an RNAi based approach in a GFP-RME-1 expressing worm strain, we screened for proteins causing alteration in recycling endosome morphology. We focused on predicted worm proteins bearing multiple NPF motifs. Through this screen we identified C. elegansAMPH-1/Amphiphysin as a novel regulator of endocytic recycling. Utilizing genetic analysis we establish a novel role for AMPH-1 in the regulation of recycling endosome morphology and recycling of transmembrane cargo. We demonstrate that endogenous RME-1 colocalizes with and associates with endogenous AMPH-1, and this association has a role in recycling. We show that in vitro recombinant purified RME-1 and AMPH-1 bind and tubulate phosphatidylserine liposomes independently, but their properties are distinct when in complex. We propose that the AMPH-1/RME-1 complex functions at the recycling endosome, promoting tubulation and/or scission of cargo carriers destined for the plasma membrane. We show phylogenetic conservation of the role of Amphiphysin family proteins in endosome function using siRNA analysis in HeLa cells.

Saumya Pant et al. (2010) East Asia Worm Meeting "AMPH-1/Amphiphysin/Bin1 functions with RME-1/Ehd1 in endocytic recycling"

Kruti Patel, Steve Caplan, Chavela Carr, Saumya Pant, Mahak Sharma, Barth D Grant

[East Asia Worm Meeting, 2010]

RME-1/EHD1 family proteins are key residents of the recycling endosome required for endosome-to-plasma membrane transport in C. elegans and mammals. Recent studies suggest similarities between the RME-1/EHD proteins and the Dynamin GTPase superfamily of mechanochemical pinchases, which promote membrane fission. Here we show that endogenous C. elegans AMPH-1, the only C. elegans member of the Amphiphysin/BIN1 family of BAR-domain-containing proteins, colocalizes with RME-1 on recycling endosomes in vivo, that amph-1-deletion mutants are defective in recycling endosome morphology and function, and that binding of AMPH-1 Asn-Pro-Phe(Asp/Glu) sequences to the RME-1 EH-domain promotes the recycling of transmembrane cargo. We also show a requirement for human BIN1 (also known as Amphiphysin 2) in EHD1-regulated endocytic recycling. In vitro, we find that purified recombinant AMPH-1/RME-1 complexes produce short, coated membrane tubules that are qualitatively distinct from those produced by either protein alone. Our results indicate that AMPH-1 and RME-1 cooperatively regulate endocytic recycling, probably through functions required for the production of cargo carriers that exit the recycling endosome for the cell surface.

Morten K. Larsen et al. (2006) European Worm Meeting "MAA-1, A Novel Acyl-CoA Binding Protein Involved in Endosomal Vesicle Transport in C. elegans"

Morten K. Larsen, Simon Tuck, Jens K. Knudsen, Nils J. Faergeman

[European Worm Meeting, 2006]

Morten K. Larsen1, Simon Tuck1, Nils J. Faergeman2 and Jens K. Knudsen2. The formation of transport vesicles during membrane trafficking requires an intricate interplay between many proteins including those coating the vesicles, adaptor proteins that recruit components of the coat, and small GTPases involved in initiation of vesicle formation. In addition to the protein factors, membrane bilayer lipids are increasingly being recognised as molecules participating actively during vesicle formation. For example, formation of transport carriers is promoted by the hydrolysis of acyl-CoA lipid esters in vitro. The mechanisms by which these lipid esters are directed to the appropriate membranes during vesicle biogenesis is not yet understood. Here we present the first study of MAA-1, a novel member of the acyl-CoA-binding protein family. We show that in C. elegans, MAA-1 is membrane associated and localizes to intracellular membrane organelles in several polarised tissues in C. elegans. MAA-1 binds fatty acyl-CoA in vitro and the ability to form high affinity ligand-protein complexes is necessary for function. Lack of maa-1 reduces the rate of endosomal recycling and enhances phenotypes caused by rme-1, an EH domain protein involved in endosomal recycling. Our results are consistent with a role for acyl-CoA and MAA-1 during endosomal recycling in vivo.

Anbing Shi et al. (2007) International Worm Meeting "A novel function for ALX-1/Alix in endocytic recycling."

Barth D Grant, Anbing Shi, Vanessa Figueroa, Carlos Chih-Hsiung Chen, Saumya Pant

[International Worm Meeting, 2007]

The endocytic pathway of eukaryotes is essential for the internalization and trafficking of macromolecules, fluid, membranes, and membrane proteins. We have previously shown that RME-1 functions as a critical regulator of endocytic recycling of cargo proteins that are internalized by clathrin-mediated and clathrin-independent endocytosis events. RME-1 is a conserved EHD family protein harboring a C-terminal EH domain. In a yeast 2-hybrid screen using the RME-1-EH domain as bait we identified a novel binding partner called ALX-1. ALX-1 is the C.elegans homolog of mammalian Alix and yeast Bro1p. Published reports indicate that Alix/Bro1p family proteins function in the multivesicular body(MVB) pathway. Oligomeric protein complexes containing ubiquitin binding protein Hrs and ESCRT complexes mediate sorting of cargo at the MVBs limiting membranes and membrane invagination. Studies in yeast have shown that Bro1 functions in concert with components of the ESCRT to regulate MVB formation. Alix is also required for the budding of retroviruses like HIV from the plasma membrane, and the actin-dependent intracellular positioning of endosomes in tissue culture cells. Although Alix has been suggested to function in multiple cellular processes, its precise functions still remain somewhat mysterious. The interaction with RME-1 suggested that ALX-1 might play a role in recycling, or that RME-1 plays a role in the MVB. Our studies did not find any role for RME-1 in membrane protein degradation, but revealed a requirement for ALX-1 in the recycling of clathrin-independent membrane proteins. In this study we define protein domains required for RME-1 and ALX-1 interaction, and have taken a functional approach to determine ALX-1 function in vivo taking advantage of the consortium knockout allele alx-1(gk275). We show a distinct functional involvement for ALX-1 in the RME-1-associated, clathrin-independent, endocytic recycling pathway of polarized epithelial cells of the C.elegans intestine. Loss of ALX-1 results in intracellular accumulation of both RME-1 and a clathrin independent endocytic recycling cargo protein, which suggests that the recycling of clathrin-independent cargo requires ALX-1. No changes were observed in the localization of the clathrin-dependent cargo protein hTfR, indicating that clathrin dependent cargo recycles normally in the absence of ALX-1. We also show that ALX-1 localizes to RME-1-positive recycling endosomes, and that mutant forms of ALX-1 that cannot bind RME-1 fail to rescue recycling phenotypes. Finally we also show functional association of ALX-1 with the membrane-protein degradation pathway of the C.elegans embryo, a function that is independent of RME-1.