Grant BD et al. (1996) East Coast Worm Meeting "sel-1, Negative Regulator of lin-12 and glp-1."

Grant BD, Greenwald IS

[East Coast Worm Meeting, 1996]

The lin-12 gene of C. elegans encodes a founding member of the lin-12/Notch family of putative receptor proteins. Two members of this family in C. elegans, the lin-12 and glp-1 genes, mediate many cell fate decisions throughout development. Suppressor analysis has yielded a number of candidate genes, including the sel-1 gene, which may function with lin-12 and glp-1 in determining cell fates (Sundaram and Greenwald, 1993) . We will present here further genetic and molecular characterization of the sel-1 gene (see Grant and Greenwald, 1996). We have shown that the null phenotype of sel-1 is wild-type, except for an elevation in lin-12 and glp-1 activities when assayed in sensitized genetic backgrounds. We also found that by genetic criteria, sel-1 is a specific modifier of lin-12 and glp-1 activities. Cloning and sequencing of the sel-1 locus revealed that SEL-1 is a predicted extracellular protein, with a putative secretory signal sequence, potential GPI linkage signal, and a region of homology to predicted proteins from humans and yeast. Sequence analysis of sel-1 mutants combined with structure / function studies indicate that the homologous region is very important to sel-1 function. Immuno-localization experiments indicate that SEL-1 is a widely distributed protein found at the cell-surface and within the cell body (possibly within membrane bound vesicles). SEL-1 is found in cells undergoing lin-12 mediated cell fate decisions. Experiments addressing local versus diffusable models for SEL-1 activity will be discussed. Sundaram, M. and I. Greenwald 1993 Suppressors of a lin-12 hypomorph define genes that interact with both lin-12 and glp-1 in Caenorhabditis elegans. Genetics 135: 765-783. Grant, B. and I. Greenwald 1996 The Caenorhabditis elegans sel-1 gene, a negative regulator of lin-12 and glp-1, encodes a predicted extracellular protein. Genetics in press.

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.

Grant BD et al. (1993) Worm Breeder's Gazette "Genetic and Molecular Analysis of sel-1, a Suppressor of lin-12 and glp-1"

Greenwald IS, Grant BD

[Worm Breeder's Gazette, 1993]

The sel-1 gene may participate in several different cell fate decisions mediated by lin-12 or glp-1 (Sundaram & Greenwald, Genetics, in press). Mutations in the sel-1 gene were found to suppress multiple lin-12 (n676n930)associated defects, enhance lin-12 gain of function defects, and suppress the maternal effect lethal glp-1 allele e2142 .None of the alleles originally studied display phenotypes in a wild type background. We are continuing the characterization of the sel-1 gene by determining its null phenotype, molecular nature, and cellular focus. Gene dosage studies indicated that existing sel-1 mutations are reduction or loss of function alleles (Sundaram & Greenwald, Genetics, in press). Since sel-1 alleles were originally identified by reverting the egg laying defect of a lin-12 hypomorph, sel-1 (0)alleles would not have been obtained if they caused an Egl, sterile, or lethal phenotype. We therefore performed a noncomplementation screen for sel-1 (0) alleles based on the observation that sel-1 /Dfsuppresses glp-1 (e2142)but sel-1 /+does not. Our preliminary analysis of 10,500 chromosomes screened after EMS mutagenesis indicates that we have isolated two new sel-1 alleles. Both new alleles suppress lin-12 and glp-1 ,and display no other obvious phenotype. This information combined with the gene dosage studies indicates that the sel-1 null phenotype is probably wild type. We mapped sel-1 to a position between the cloned polymorphism arP5 and the cloned gene lin-25 (Tuck & Greenwald, WBG 12(5):49). This interval of about 200kb is completely covered by overlapping cosmid clones. We tested clones from this interval for sel-1 gene activity by two assays. First, we found that cosmids C06C11 and T17C2 complement the suppression of glp-1 (e2142)when coinjected with a dpy-20 (+)marker. Curiously, no complementation occurred with arrays formed with the rol-6 (d)marker. Second, we found that a 10kb HindIII subclone from C06C11 complements suppression of lin-12 (n676n930)as well as suppression of glp-1 (e2142).We are currently sequencing our smallest rescuing clone of 6.8kb, and isolating cDNAs derived from this region.

Grant BD et al. (1997) International C. elegans Meeting "Analysis of Receptor-Mediated Endocytosis in C. elegans"

Hirsh DI, Grant BD, Zhang YH

[International C. elegans Meeting, 1997]

Clathrin-mediated endocytosis of molecules bound to cell surface receptors mediates many aspects of cellular function, including nutrient uptake from the circulatory system (e.g. LDL and yolk), antigen uptake by the immune system, synaptic vesicle recycling, membrane turnover, and growth factor receptor down-regulation. We have developed a new system to study endocytosis in worm oocytes utilizing a GFP tagged version of the yolk protein YP170. YP170-GFP can be directly observed in vivo during synthesis, transport, and endocytosis. We have begun to examine the function of potential C. elegans endocytosis genes by injecting their cognate antisense RNAs into the adult germline, and examining any subsequent effects on YP170-GFP uptake and distribution. Each gene we examined encodes a homologue of a known or suspected vertebrate endocytosis component. We have injected antisense RNAs for over 30 candidate endocytosis genes, identifying eight genes which seem to be specifically required for efficient uptake of YP170-GFP by oocytes. We have also identified one gene which alters YP170-GFP localization within the oocyte and embryo when injected as an antisense RNA. Recently we also performed antisense experiments designed to block germline secretory function, and identified several specific phenotypes which should help us to distinguish between genes important for secretory rather than endocytic function in this assay system. Recently we began analyzing the expression patterns of the nine endocytic pathway genes we identified in our antisense screen. We are also beginning a large scale genetic screen for mutants defective in YP170-GFP endocytosis. We believe that C. elegans offers a special opportunity to use forward and reverse genetics to identify many new components essential for endocytosis and to decipher their functions at the molecular level.

Grant BD et al. (1998) East Coast Worm Meeting "Endocytosis Mutants and The Yolk Receptor in C. elegans"

Zhang YH, Pedraza LG, Wallenstein E, Hirsh DI, Grant BD

[East Coast Worm Meeting, 1998]

We are using C. elegans genetics to look for new genes involved in receptor-mediated endocytosis. We have isolated 23 mutants, in 11 complementation groups, defective in the endocytosis of YP170-GFP by oocytes. Some of these mutants are temperature sensitive lethals. We anticipate that our mutants will identify key components of the general endocytic machinery as well as gene products that specifically mediate yolk uptake. The most severe YP170-GFP uptake defective mutants identify a gene encoding a putative receptor for vitellogenins (yolk proteins) in the C. elegans oocyte. During the normal maturation of oocytes within the C. elegans germline yolk proteins (YPs), encoded by the vit genes and secreted by the intestine, are actively endocytosed from the psuedocoelomic space and stored within membrane bound compartments sometimes called yolk platelets. A vit-2::gfp transgene, encoding YP170-GFP, has allowed us to visualize this process in vivo and study its properties by fluorescence microscopy. RNAi analysis in this genetic background allowed us to test the function of potential components of the C. elegans endocytic machinery identified by the genome project. This analysis indicates that YP170 endocytosis requires clathrin coated pit components (clathrin heavy chain, adaptins, dynamin) and regulators of endosomal function (rab5, rab7, rab11), as expected from studies of lipoprotein uptake in other organisms. 	One complementation group, comprised of three alleles, appears to mutate T11F8.c, a partially sequenced member of the LRP family of lipoprotein and proteinase receptors. We are currently confirming this inference by transformation rescue and sequencing of mutant alleles. RNAi data by Bossinger et al. (1997 IWM Abstract 61) had previously indicated a possible role for T11F8.c in yolk uptake. Our results show that T11F8.c RNAi blocks YP170-GFP uptake. We are tentatively naming this gene lrp-2, in anticipation of the complete genomic sequence which is in progress at the sequencing consortium (Many thanks to Bob Waterston and Stephanie Chissoe). Immunofluorescence experiments, using polyclonal antisera we produced against intracellular or extracellular LRP-2 fusion proteins, indicate that LRP-2 is an abundant plasma membrane protein expressed specifically in oocytes. Our putative lrp-2 mutants all show an absence or mislocalization of LRP-2 in oocytes. 	We are currently engaged in a more detailed phenotypic analysis of endocytic defects in the other ten complementation groups identified in our genetic screen utilizing anti-LRP-2 antibodies and other specific antisera we have developed against general endocytic pathway components identified in our RNAi screen (see abstract by Y. Zhang et al.).

Grant BD et al. (1997) Development "Structure, function, and expression of SEL-1, a negative regulator of LIN-12 and GLP-1 in C. elegans."

Grant BD, Greenwald IS

[Development, 1997]

Previous work indicated that sel-1 functions as a negative regulator of lin-12 activity, and predicted that SEL-1 is a secreted or membrane associated protein. In this study, we describe cell ablation experiments that suggest sel-1 mutations elevate lin-12 activity cell autonomously. We also use transgenic approaches to demonstrate that the predicted signal sequence of SEL-1 can direct secretion and is important for function, while a C-terminal hydrophobic region is not required for SEL-1 function. In addition, by analyzing SEL-1 localization using specific antisera we find that SEL-1 is localized intracellularly, with a punctate staining pattern suggestive of membrane bound vesicles. We incorporate these observations, and new information about a related yeast gene, into a proposal for a possible mechanism for SEL-1 function in LIN-12 turnover.

Grant BD et al. (1999) Mol Biol Cell "Receptor-mediated endocytosis in the Caenorhabditis elegans oocyte."

Hirsh DI, Grant BD

[Mol Biol Cell, 1999]

The Caenorhabditis elegans oocyte is a highly amenable system for forward and reverse genetic analysis of receptor-mediated endocytosis. We describe the use of transgenic strains expressing a vitellogenin::green fluorescent protein (YP170::GFP) fusion to monitor yolk endocytosis by the C. elegans oocyte in vivo. This YP170::GFP reporter was used to assay the functions of C. elegans predicted proteins homologous to vertebrate endocytosis factors using RNA-mediated interference. We show that the basic components and pathways of endocytic trafficking are conserved between C. elegans and vertebrates, and that this system can be used to test the endocytic functions of any new gene. We also used the YP170::GFP assay to identify rme (receptor-mediated endocytosis) mutants. We describe a new member of the low-density lipoprotein receptor superfamily, RME-2, identified in our screens for endocytosis defective mutants. We show that RME-2 is the C. elegans yolk receptor.

Grant BD et al. (2003) Curr Opin Cell Biol "Functional genomic maps in Caenorhabditis elegans."

Wilkinson HA, Grant BD

[Curr Opin Cell Biol, 2003]

The completion of the Caenorhabditis elegans genome sequence was the initial step toward the use of whole-genome analysis in this model organism. Advances in C. elegans genomics include transcript profiling, gene-function screens using RNA-mediated interference, and protein-interaction mapping using the yeast two-hybrid system. Recent reports have employed these methods to gain new insights into diverse biological problems such as tissue-specific gene expression, cell-fate specification, genome organization, the DNA damage response, and early embryonic development. These studies combined genomic approaches to probe complex biological pathways on an

Grant BD et al. (1994) Worm Breeder's Gazette "sel-1, a Negative Regulator of lin-12 and glp-1 Activity, Encodes a Novel Extracellular Protein."

Greenwald IS, Grant BD

[Worm Breeder's Gazette, 1994]

sel-l, a negative regulator of lin-12 and glp-l activity, encodes a novel extracellular protein. Barth Grant and Iva Greenwald HHMI, Columbia University Dept. of Biochemistry, New York, NY 10032

Grant BD et al. (1996) Genetics "The Caenorhabditis elegans sel-1 gene, a negative regulator of lin-12 and glp-1, encodes a predicted extracellular protein."

Grant BD, Greenwald IS

[Genetics, 1996]

The Caenorhabditis elegans lin-12 and glp-1 genes encode members of the LIN-12/NOTCH family of receptors. The sel-1 gene was identified as an extragenic suppressor of a lin-12 hypomorphic mutant. We show in this report that the sel-1 null phenotype is wild type, except for an apparent elevation in lin-12 and glp-1 activity in sensitized genetic backgrounds, and that this genetic interaction seems to be lin-12 and glp-1 specific. We also find that sel-1 encodes a predicted extracellular protein, with a domain sharing sequence similarity to predicted proteins from humans and yeast. SEL-1 may interact with the LIN-12 and GLP-1 receptors and/or their respective ligands to down-regulate signaling.