Cook, Daniel et al. (2017) International Worm Meeting "The C. elegans natural diversity resource."

Andersen, Erik, Zdraljevic, Stefan, Roberts, Joshua, Cook, Daniel

[International Worm Meeting, 2017]

Caenorhabditis elegans is used as a model in over 1,100 labs around the world and has enabled biological discoveries in many diverse fields. These advances have been fueled by an extensive genetic toolkit, including strains, reagents, and databases. However, the majority of C. elegans research focuses on the laboratory-domesticated N2 strain, neglecting potential insights gleaned from natural populations. Studies of C. elegans natural variation can identify the genetic factors underlying biomedically relevant traits and genome evolution. To address the need for resources to study natural variation, we developed the Caenorhabditis elegans Natural Diversity Resource (CeNDR) - available at www.elegansvariation.org. The web-based CeNDR platform includes three areas: (1) a central repository for the deposition, organization, and dissemination of wild C. elegans strains, including detailed information for each strain (e.g. collection date, GPS location, substrate, and elevation); (2) a data portal for dissemination of whole-genome sequence data in BAM or CRAM formats and variant data in VCF format for each of the 383 wild isolates, including a powerful interactive genome browser that can be used to interrogate genetic variation across the population for genes or regions of interest; (3) a genome-wide association mapping portal to enable mappings of quantitative traits measured using wild C. elegans strains, including a comprehensive report of significance, variation, measures of selection, etc. We believe that CeNDR will become an indispensable tool within the C. elegans genetic toolkit to enable researchers to examine natural populations and identify interesting new biological phenomena.

Morrison, Kayleigh N. et al. (2021) MicroPubl Biol

Shakes, Diane C., Ragle, James Matthew, Uyehara, Christopher M., Ward, Jordan D., Morrison, Kayleigh N.

[MicroPubl Biol, 2021]

During the process of cell differentiation, specific cytoskeletal proteins can sequentially assemble into a wide variety of diverse molecular superstructures. Nematode spermatogenesis provides a powerful system for studying these transitions since sperm-specific transcription ceases prior to the meiotic divisions and translation ceases shortly thereafter (Chu and Shakes, 2013). Therefore, structural transitions that follow the meiotic divisions must be carried out by the remodeling of already synthesized proteins. The Major Sperm Protein (MSP) is a nematode-specific cytoskeletal element whose polymerization dynamics drive the pseudopod-based motility of the activated sperm (Roberts, 2005). In C. elegans, MSP additionally functions as the extracellular signaling molecule for triggering both ovulation and oocyte maturation (Miller et al., 2003). MSP is highly abundant in sperm, where it reaches 10-15% of total and 40% of soluble cellular protein (Roberts 2005). Within developing spermatocytes, MSP is packaged into fibrous bodymembranous organelle (FB-MO) complexes (Fig. 1A, Roberts et al., 1986). By assembling into paracrystalline FBs, MSP is both sequestered away from the critical meiotic processes of chromosome segregation and cytokinesis while also being packaged for efficient segregation into spermatids during the post-meiotic partitioning process (Chu and Shakes 2013, Nishimura and LHernault, 2010, Price et al., 2021). Following the meiotic divisions and sperm individualization, FBs disassemble, and MSP disperses as dimers throughout the spermatid cytoplasm (Fig. 1A). When sperm activate to form motile spermatozoa, MSP polymerization within the pseudopod drives the motility of the crawling sperm (Chu and Shakes, 2013). Thus, MSP exists in at least three distinct molecular states: 1) in highly organized paracrystalline FBs within developing spermatocytes 2) as unpolymerized dimers within spermatids, and 3) in dynamically polymerizing filaments and fibers within crawling spermatozoa.

Carta L (1998) East Coast Worm Meeting "TOXICITY OF 4 SERRATIA MARCESCENS BACTERIAL STRAINS TOWARD C. ELGANS N2, PGP-3 AND OTHER NEMATODES IS INVERSELY RELATED TO PIGMENT"

Carta L

[East Coast Worm Meeting, 1998]

We are testing a number of biocontrol bacteria (eg. Burkholderia, Pseudomonas and Stenotrophomonas) for toxicity to bacterial feeding nematodes including C. elegans N2 and pgp-3. Serratia marcescens in particular had a measurable effect on number of eggs over 24 hours and relative motility in microtiter wells. Serratia marcescens strains with increasing amounts of red pigmentation (BF1-5@ (albino), D1, N4-5, NIMA) were tested for relative motility to C. elegans N2, pgp3, Oscheius myriophila DF5020, Panagrellus redivivus PS 1163, Mesorhabditis sp. PS1170 and Zeldia punctata PS 1153. Toxicity directly increases with decreasing amount of pigment in Caenorhabditis, Oscheius and Panagrellus. The order of relative toxicity is reversed for the 2 highly pigmented strains and the 2 least pigmented strains (2143 vs. 1234) in Mesorhabditis and Zeldia. These nematodes are phylogenetically distant from C. elegans. Bacterial strains were provided by Phyllis Martin and Dan Roberts, USDA.

Nguyen TT et al. (2014) Curr Protoc Toxicol "F3-Isoprostanes as a Measure of in vivo Oxidative Damage in Caenorhabditis elegans."

Aschner M, Nguyen TT

[Curr Protoc Toxicol, 2014]

Oxidative stress has been implicated in the development of a wide variety of disease processes, including cardiovascular disease, cancer, and neurodegenerative diseases, as well as progressive and normal aging processes. Isoprostanes (IsoPs) are prostaglandin-like compounds that are generated in vivo from lipid peroxidation of arachidonic acid (AA, C20:4, -6) and other polyunsaturated fatty acids (PUFA). Since the discovery of IsoPs by Morrow and Roberts in 1990, quantification of IsoPs has been shown to be an excellent source of biomarkers of in vivo oxidative damage. Eicosapentaenoic acid (EPA, C20:5, -3) is the most abundant PUFA in Caenorhabditis elegans and gives rise to F3-IsoPs upon nonenzymatic free-radical-catalyzed lipid peroxidation. The protocol presented is the current methodology that our laboratory uses to quantify F3-IsoPs in C. elegans using gas chromatography/mass spectrometry (GC/MS). The methods described herein have been optimized and validated to provide the best sensitivity and selectivity for quantification of F3-IsoPs from C. elegans lysates.

Tan JH et al. (2020) Elife "Alternative splicing of coq-2 controls the level of rhodoquinone in animals."

Davis RE, Lautens M, Reinl SR, Tan JH, Shepherd JN, Schertzberg MR, Romanelli-Cedrez L, Salinas G, Wang J, Fraser AG

[Elife, 2020]

Parasitic helminths use two benzoquinones as electron carriers in the electron transport chain. In normoxia they use ubiquinone (UQ), but in the anaerobic conditions inside the host, they require rhodoquinone (RQ) and greatly increase RQ levels. We previously showed the switch from UQ to RQ synthesis is driven by a change in substrates by the polyprenyltransferase COQ-2 (Del Borrello et al., 2019; Roberts Buceta et al., 2019) - how this substrate choice is made is unknown. Here, we show helminths make two <i>coq-2</i> splice forms, <i>coq-2a</i> and <i>coq-2e</i>, and the <i>coq-2e-</i>specific exon is only found in species that make RQ. We show that in <i>C. elegans</i> COQ-2e is required for efficient RQ synthesis and for survival in cyanide. Crucially, parasites switch from COQ-2a to COQ-2e as they transition into anaerobic environments. We conclude helminths switch from UQ to RQ synthesis principally via changes in the alternative splicing of <i>coq-2.</i>

Ward S et al. (1983) Worm Breeder's Gazette "Monoclonal and Polyclonal Antibodies that Bind to Sperm Antigens"

Hogan E, Ward S

[Worm Breeder's Gazette, 1983]

We have examined a number of monoclonal and polyclonal antisera generously provided to us by other labs in order to assess their binding to sperm antigens. The antisera include the following: two rabbit sera and eight mouse monoclonals raised against muscle proteins by Ross Francis: five anti-sperm mouse monoclonals prepared by Susi Strome and obtained initially from Michael Klass; eight anti-sperm mouse monoclonals prepared by Fred Pavalko and Tom Roberts; and a rabbit anti-glyceraldehyde phosphate dehydrogenase (GAPDH) serum obtained from Patrice Yarborough and Ralph Hecht. Our interest in these sera is to use them to identify new sperm antigens whose cellular localization and developmental history can be compared with the sperm specific antigens we have alrealy examined. All the antisera were first assayed for tissue specificity by a solid phase immunoassay with proteins from sperm, eggs, larvae or fem- 1( hc17) hemaphrodites which have no sperm. Those antisera that showed significant binding to sperm proteins were then characterized further by immunostaining of sperm or fem-1 proteins transferred from 1-D or 2- D gels to nitrocellulose ('Western transfers'). In addition the cellular localization of antigens was determined by immunofluorescence.

Murthy PK et al. (1997) Folia Parasitol (Praha) "Fate of infective larvae of Brugia malayi in the peritoneal cavity of Mastomys natalensis and Meriones unguiculatus."

Tyagi K, Chatterjee RK, Murthy PK, Murthy PS

[Folia Parasitol (Praha), 1997]

The fate of intraperitoneally inoculated infective third-stage larvae (L3) of the nematode Brugia malayi Lichtenstein and the status of the peritoneal macrophage function were investigated in the susceptible rodent hosts Mastomys natalensis Roberts and Meriones unguiculatus Milne-Edwards (jird). Jirds and M. natalensis were inoculated intraperitoneally with 125 and 250 L3 and the worm burden and peritoneal macrophage function in the two species were compared at different days post-inoculation (DPI). None of the infected M. natalensis had adult worms in the peritoneal cavity; very few degenerating L3 surrounded by peritoneal cells were recovered 7 and 15 DPI. In contrast, all the infected jirds showed the parasite in different stages of development and the worm burden at different days PI was more in 250 L3 dose group than in 125 L3 dose group. The phagocytic function of peritoneal macrophages of normal M. natalensis was twice higher than that of jirds. This function was found significantly suppressed in both host species at 15 DPI; at 35 DPI, the activity was still at this low level in the jird, while that in M. natalensis reverted to uninfected age- and sex-matched control levels. These findings demonstrate that the peritoneal environment of M. natalensis is not conducive to the development of B. malayi and this is probably related to high macrophage activity in the peritoneum of this host compared to that found in the jird.

Pavalko FM et al. (1983) Worm Breeder's Gazette "amonoclonal and polyclonal antibodies that bind to sperm antigens."

Roberts TM, Pavalko FM

[Worm Breeder's Gazette, 1983]

We have been applying hybridoma technology to our research by generating monoclonal antibodies (MAb's) against C. elegans sperm. We have produced 10 mAb's in our lab and have obtained 2 others from Susie Strome at Boulder. Seven of these mAb's target antigens present on the cell surface. We have been purifying monoclonal antibodies from ascites fluid by DEAE Affi-Gel blue chromatography (supplied by Bio-Rad). Although yields have been lower than ideal (40-60%), these antibodies are free of all contaminants except mouse transferrin. Membrane lipid flow and directed movement of pseudopod surface components on sperm was previously reported by Roberts and Ward (J. Cell Bio. 92: 113-120). We have evidence that the movement of antigen-antibody complexes may be due to a continual flow of membrane lipid (see Bretscher, 1976, Nature 260: 21-23) from the tip of the pseudopod back toward the cell body. Bretscher's lipid flow model of membrane movement predicts that membrane components which diffuse slowly are displaced by the lipid stream whereas those which diffuse rapidly effectively escape lipid flow. Because the rate of diffusion is related to molecular weight, large complexes (e.g. antibody- crosslinked membrane proteins) would be swept along more rapidly than individual molecules (e.g.- unbound membrane proteins). We have tested this prediction by comparing clearance rates of various sized complexes form the cell pseudopod. On sperm, relatively small antigen- mAb complexes clear from the pseudopod surface slowly (2-3 min) while larger, cross-linked antigen-mAb-2 antibody complexes are cleared more rapidly (30-45 seconds). Using mAb's conjugated directly to colloidal gold particles (CGP), we have obtained evidence that new surface antigens are preferentially inserted at the tips of pseudopodial projections and then move rearward toward the cell body pseudopod junction (Pavalko and Roberts, C. elegans Meeting Abstracts, 1983). This observation predicts that there must be a pool of antigen in the cytoplasm which would be available for insertion into the membrane. Labelling thin sections of spermatozoa with CGP conjugates of mAb's 11, 63, and 56 (each of which bind to the cell surface in indirect immunofluorescence assays) reveals antigen on the surface (plasma membrane, exposed face of fused membranous organelles and MO contents). In addition, these antibodies label the cytoplasmic laminar membranes and the pseudopod cytoplasm. Many of the gold particles in the cytoplasm lie just beneath the plasma membrane suggesting that they are bound to antigens that were destined for insertion onto the surface. Because there are no vesicles in the cytoplasm, the mechanism which transports these antigens is unknown. We will be trying to characterize the antigens targeted by the mAb's and to solve the question of how they are shuttled around the cell.

Michael Dybbs et al. (2001) International C. elegans Meeting "A screen for new Hic mutants"

Josh Kaplan, Michael Dybbs

[International C. elegans Meeting, 2001]

ABSTRACT International Worm Meeting 2001 A screen for new Hic mutants Michael Dybbs and Joshua Kaplan 361 LSA, Dept. of Mol. and Cell Biology, University of California, Berkeley, CA 94720 Previous studies by our lab and others have shown that release of acetylcholine at the C. elegans neuromuscular junction (NMJ) is regulated by a G-protein signaling network. These studies have shown that serotoninergic signaling through goa-1 G a o and dgk-1 DAG kinase inhibits acetylcholine release. An opposing muscarinic signaling pathway through egl-30 G a q and egl-8 PLC b enhances acetylcholine release via the production of diacylglycerol ( DAG). Two RGS proteins, egl-10 RGS and eat-16 RGS, further regulate this pathway by stimulating the GTPase activity of goa- 1 G a o ( eat-16 RGS) and egl-30 G a q ( egl-10 RGS). Mutations in the inhibitory pathway ( goa-1 , dgk-1 ) make animals hypersensitive to paralysis by the acetylcholine esterase inhibitor, aldicarb (Hic) Mutations in the muscarinic pathway ( egl-30 , egl-8 ) make animals resistant to aldicarb (Ric). In order to identify additional molecules which negatively regulate release, we are conducting a broad screen for Hic mutants. Aldicarb sensitivity could be a result of either mutations that effect presynaptic neurotransmitter release or postsynaptic response to neurotransmitter in the muscle. We are distinguishing these effects using levamisole, a cholinergic agonist that acts directly on the acetylcholine receptors in the muscle. We are focusing on mutants that display a wild-type muscle sensitivity and are thus acting presynaptically. Mutants isolated in our pilots screens will be described.

Brett Roberts et al. (2006) European Worm Meeting "Investigation of Interactions between Mutant Collagens Using C. elegans as a Model Organism"

Genevieve Stapleton, Brett Roberts, Iain Johnstone

[European Worm Meeting, 2006]

Brett Roberts, Genevieve Stapleton and Iain Johnstone. Extracellular matrix (ECM) provides the structural basis of tissues and organs in animals. Collagen proteins form a diverse range of supramolecular components in ECM and mutations in various human collagen genes which result either in a reduction or loss of a collagen species from the ECM, or in some cases insertion of mutant collagen, are responsible for a wide variety of inherited diseases, including osteogenesis imperfecta, Ehlers-Danlos Syndrome and some forms of osteoporosis, osteoarthritis and invertebral disc disease. Excessive collagen synthesis is also detrimental resulting in fibrosis.. In C. elegans the cuticle is an ECM comprised primarily of collagens, encoded by a large gene family, which interact to form distinct substructures within the cuticle. Previously we have demonstrated specific requirements for collagen partners during assembly of cuticle collagen sub-structures. We observe distinct differences between the behaviour of certain classes of mutant collagen. The most common class, Gly substitution mutants within the repetitive Gly-X-Y domains cause retention of the mutant collagen in the ER. However null mutations in some cuticle collagen genes result in neither assembly nor ER retention of obligate partner collagens. We are interested in studying the fate of this partnerless collagen; various approaches will be presented.. We are also interested in the retention of Gly sub collagen in the ER. We have performed a mutant screen in a dpy-7(e88) glycine substitution mutant background to identify modifiers of the dpy-7(e88) phenotype. Two classes of mutants have been isolated: those that significantly enhance the severity of the dpy-7(e88) phenotype, resulting in shorter, fatter animals; and those that display a distinct Roller modifier phenotype. Our preliminary analyses of these new modifier alleles will be described.