Scott Alper et al. (2005) International Worm Meeting "Development of two high-throughput RNA-interference assays to investigate innate immunity"

David Schwartz, Jonathan H Freedman, Paul Dunlap, Scott Alper

[International Worm Meeting, 2005]

In response to infection, humans mount an immediate innate immune response as well as a slower but more specific adaptive immune response. The immediate response involves secretion of antimicrobial compounds and infiltration of cytotoxic and phagocytic cells at the site of infection. While innate immunity plays a vital role in pathogen defense, misregulation of innate immunity also plays a role in the onset of many human diseases including sepsis, asthma, atherosclerosis, and organ rejection. To investigate the genetics of innate immunity, we are using both C. elegans and mammalian tissue culture as model systems. Our lab has identified many candidate mouse innate immunity genes using DNA microarrays and QTL analysis. We are now developing two high throughput assays to determine the function of these genes. Both assays use RNA-interference to inhibit gene function. One assay uses the nematode C. elegans as a model system. In response to infection with a pathogen, C. elegans produces antimicrobial compounds as part of its innate immune response. We have engineered gfp fusions to antimicrobial genes induced by infection with Serratia marcescens or Staphylococcus aureus. Using RNAi, we are currently inhibiting C. elegans orthologs of candidate innate immunity genes to determine their effect on antimicrobial gene expression. Genes identified in this primary screen are then rescreened in assays to monitor C. elegans pathogen load and pathogen-mediated toxicity. In the other assay, mouse macrophages are treated with lipopolysaccharide (LPS) and cytokine production is measured. Using siRNAs to inhibit gene function, we are beginning to test the effect of candidate genes on cytokine production. In preliminary experiments, we have identified several novel genes that are required for production of TNF-alpha and IL-6. Using these two assays, we hope to rapidly identify and determine the function of candidate innate immunity genes.

Scott Alper et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Novel Innate Immunity Gene and Pathway Discovery Using Comparative Genomics"

Paul Dunlap, Jonathan Freedman, Windy Boyd, Brad Lackford, Scott Alper, David Schwartz, Rebecca Laws

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

In response to infection, humans mount an immediate innate immune response and a slower but more specific adaptive immune response. The innate response involves the action of phagocytic and cytotoxic cells that fight the infection. Therefore, the innate immune system plays a vital role in host defense. However mis-regulation of innate immunity can also contribute to a variety of immunological diseases, including asthma and sepsis. Thus, the identification of genes that regulate innate immunity is critical to understanding host defense and to identifying potential targets for treatment of infectious and immunological diseases. While many genes that regulate the response to Gram negative lipopolysaccharide (LPS) have been discovered, it is unclear how many other genes regulate this response. To identify novel regulators of the innate immune response to LPS (and other microbial toxins), we developed assays in two model systems to inhibit candidate genes by RNA-interference and monitor the subsequent immune response. Both models utilize a Gram negative bacterial stimulus. In one in vivo assay, the nematode C. elegans was stimulated with E. coli and production of antimicrobial proteins was monitored. In the second in vitro assay, mouse macrophages were stimulated with LPS and cytokine production was monitored. Genes that altered innate immune responsiveness in these systems were validated using mutant nematode models and are currently being tested in mutant murine models. These assays have led to the discovery of 11 genes that regulate the innate immune response in both systems and the identification of a novel protein interaction network with a conserved role in innate immunity regulation. These genes represent potential therapeutic targets for infectious and immunological diseases.

Consortium Wormbase (2000) Worm Breeder's Gazette "Update on the C. elegans database WormBase"

Consortium Wormbase

[Worm Breeder's Gazette, 2000]

WormBase (www.wormbase.org) is an international consortium of biologists and computer scientists dedicated to providing the research community with accurate, current, accessible information concerning the genetics, genomics and biology of C. elegans and some related nematodes. WormBase builds upon the existing ACeDB database of the C. elegans genome by providing curation from the literature, an expanded range of content and a user friendly web interface. The team that developed and maintained ACeDB (Richard Durbin, Jean Thierry-Mieg) remains an important part of WormBase. Lincoln Stein and colleagues at Cold Spring Harbor are leading the effort to develop the user interface, including visualization tools for the genome and genetic map. Teams at Sanger Centre (led by Richard Durbin) and the Genome Sequencing Center at Washington University, St. Louis (led by John Spieth) continue to curate the genomic sequence. Jean and Danielle Thierry-Mieg at NCBI spearhead importation of large-scale data sets from other projects. Paul Sternberg and colleagues at Caltech will curate new data including cell function in development, behavior and physiology, gene expression at a cellular level; and gene interactions. Paul Sternberg assumes overall responsibility for WormBase, and is delighted to hear feedback of any sort. WormBase has recently received major funding from the National Human Genome Research Institute at the US National Institutes of Health, and also receives support from the National Library of Medicine/NCBI and the British Medical Research Council. WormBase is an expansion of existing efforts, and as such continues to need you help and feedback. Even with the increased scope and funding, all past contributors to ACeDB remain involved. The Caenorhabditis Genetics Center (Jonathan Hodgkin and Sylvia Martinelli) collaborate with WormBase to curate the genetic map and related topics. Ian Hope and colleagues continue to supply expression data to WormBase. Leon Avery will continue his superb website and serves as one advisor to WormBase. While the major means of access to WormBase is via the world wide web, downloadable versions of WormBase as well as the acedb software engine will continue to be available.

Rachel McMullan et al. (2006) European Worm Meeting "Rho is a Pre-Synaptic Activator of Neurotransmitter Release at Pre-Existing Synapses in C.elegans"

Rachel McMullan, Emma Hiley, Paul Morrison, Stephen J. Nurrish

[European Worm Meeting, 2006]

Rachel McMullan, Emma Hiley, Paul Morrison and Stephen J. Nurrish. Rho GTPases have important roles in neuronal development but their function in adult neurons is less well understood. We demonstrate that pre-synaptic changes in Rho activity at C.elegans neuromuscular junctions can radically change animal behaviour via modulation of two separate pathways. In one, pre-synaptic Rho increases acetylcholine (ACh) release by stimulating the accumulation of diacylglycerol (DAG) and the DAG-binding protein UNC-13 at sites of neurotransmitter release; this pathway requires binding of Rho to the DAG kinase DGK-1. A second DGK-1-independent mechanism is revealed by the ability of a Rho inhibitor (C3 transferase) to decrease levels of release even in the absence of DGK-1; this pathway is independent of UNC-13 accumulation at release sites. We do not detect any Rho induced changes in neuronal morphology or synapse number, thus Rho facilitates synaptic transmission by a novel mechanism. Surprisingly, many commonly available human RhoA constructs contain an uncharacterised mutation that severely reduces binding of RhoA to DAG kinase. Thus a role for RhoA in controlling DAG levels has not been previously appreciated.

Minniti AN et al. (1995) International C. elegans Meeting "THE HERMAPHRODITE-INITIATED SPERMIOGENESIS PATHWAY."

Minniti AN, Nance JF, Sadler C, Ward S

[International C. elegans Meeting, 1995]

During spermiogenesis, round nonmotile spermatids are rapidly transformed into asymmetrical crawling spermatozoa. In addition to spe-8 and spe-12, we found two new genes, spe-27 and spe-29, that when mutated, disrupt spermiogenesis in an identical manner: mutant hermaphrodites are self-sterile, while mutant males are fertile. Sperm from both sexes activate abnormally in vitro, suggesting that these gene products are expressed in both sperm, but only hermaphrodites require them for activation. The hermaphrodite's spermatids, however, can be activated by mating. This phenotype has been explained by a model that has two distinct pathways of activation, one for males and one for hermaphrodites (Shakes and Ward, 1989). The four genes are needed only for the hermaphrodite pathway. spe-27 and spe-12 have been cloned. Their mRNA is found exclusively in the germline during spermatogenesis. Their predicted protein sequences show no similarities to known proteins. We are currently cloning spe-29. To further understand how these genes act during spermiogenesis we are determining the subcellular localization of their gene products. In addition, suppressor analysis is under way (see abstract by Paul Muhlrad and Samuel Ward) to look for other genes in this pathway and for interactions between these genes.

Stiernagle TL et al. (1995) International C. elegans Meeting "CAENORHABDITIS GENETICS CENTER."

Martinelli SD, Hodgkin JA, Herman RK, Stiernagle TL

[International C. elegans Meeting, 1995]

The Caenorhabditis Genetics Center (CGC), supported by NIH NCRR, supplies Caenorhabditis strains and information to researchers. The St. Paul team is responsible for acquiring, maintaining and distributing worm stocks, generating and maintaining a C. elegans bibliography, and publishing the Worm Breeder's Gazette (WBG). The Cambridge team acts as a clearing house for C. elegans genetic nomenclature and maintains the genetic map. The CGC now has over 2150 different strains. We strive to have at least one allele of every published gene and all chromosome rearrangements, duplications and deficiencies. In addition, we have several strains of species closely related to C. elegans. Strains are available upon written request, which should include a brief statement of the intended use of the strains. Email requests (to stier@molbio.cbs.umn.edu) are satisfactory. The CGC bibliography currently includes over 2025 research articles and book chapters. The WBG is published 3 times each year and currently has over 675 subscribers. Various types of information from the CGC are available electronically. By gopher you can get current strain lists, the WBG subscriber directory, WBG Tables of Contents and the CGC bibliography. We can also email files to you. We like to be acknowledged in papers for providing strains. We also like to receive reprints of worm papers.

Balaji, Vishnu et al. (2021) International Worm Meeting "Ubiquitin-Dependent Dimer-Monomer Switch Defines Substrate Specificity and Processivity of the E3 Ligase CHIP"

Loewe, Thomas, Pokryzwa, Wojciech, Hoppe, Thorsten, Lorenz, Robin, Balaji, Vishnu, Mueller, Leonie

[International Worm Meeting, 2021]

Proteostasis is achieved by quality control pathways that support the generation of correctly folded proteins, prevent protein misfolding and remove toxic proteins. The quality control E3 ligase CHIP ubiquitylates damaged proteins consigned by chaperone partners for disposal through the endo-lysosomal pathway, proteasomal degradation, or autophagy. Additionally, CHIP has been reported to modulate essential signaling pathways by specifically delivering a myriad of native proteins to destined fates homeostasis (Paul I and Ghosh M.K., 2015). We aimed at understanding the substrate specificity and processivity through a "structure to function" approach, by examining the modeled 3D structure of the C. elegans ortholog of CHIP, CHN-1, based on the reported structure of murine CHIP (Zhang M et al., 2005). Using C. elegans and mammalian cells as model system and with various genetic and biochemical analyses, we demonstrate that monomeric CHN-1/CHIP has preserved ubiquitylation activity and promotes longevity via the DAF-2 Insulin-like signaling pathway (Tawo R et al., 2017). Our data reveal that the CHN-1/CHIP autoubiquitylation and its chaperone binding interplay modulates the alteration between monomer and dimer (Balaji V and Hoppe T., 2020). Together, the conserved dimer-monomer transition provides a molecular switch regulating CHN-1/CHIP activity in response to proteotoxic stress and aging.

Caldicott IM et al. (1991) International C. elegans Meeting "A GENETIC AND PHENOTYPIC ANALYSIS OF unc-120."

Waterston RH, Caldicott IM

[International C. elegans Meeting, 1991]

The temperature sensitive mutant unc-120 (st364ts) displays a novel muscle phenotype when examined by polarized light and when stained with phalloidin and myosin antibodies. The number of thick and thin filament bands per muscle cell is reduced in a non uniform manner throughout st364 animals. In animals raised at 20 C a gradual deterioration in the animal's ability to move occurs, until as adults they are almost completely paralyzed. As adults these animals display from 5 to 9 staining bands compared to the wild type number of 9 to 10. In animals raised at 15 C a distinction between movements of mutant and wild type animals cannot be made until well into adulthood at which point the unc-120 animals are sluggish . unc-120 adults raised at 15 oc have between 7 and 10 staining bands. The interesting phenotype of unc-120 (st364tsJ prompted us to conduct genetic and physical mapping studies of the gene. We have placed unc-120 in the cluster on linkage group I between mei-l and lin- 28 and we have obtained cosmid complementation with the cosmid C48Bl. The cosmid complementation was performed by testing strains putatively containing cosmids spanning the region of interest, provided to us by Paul Mains, against unc-120. We are now repeating the rescue experiments by direct injection of C48Bl and the overlapping cosmids C26C7 and C05D8 which did not rescue in the complementation experiments.

Murtaugh J et al. (1991) International C. elegans Meeting "GUANINE NUCLEOTIDE-DEPENDENT SIGNALING PATHWAYS IN C. Elegans: ISOLATION OF TWO GENES ENCODING 20 kDa ADP-RIBOSYLATION FACTORS."

Takada T, Lee C, Murtaugh J, Fleming JT, Vaughan M, Moss J, Mishima K, Fire A

[International C. elegans Meeting, 1991]

ADP-ribosylation factors (ARFs) comprise a highly conserved family of guanine nucleotidebinding proteins initially identified by their ability to stimulate, in a GTP-dependent process, the ADP- ribosyltransferase activity of cholera toxin, and are believed to function in golgimediated vesicular transport and secretion. We sought to determine whether one or multiple ARF genes are present in C. elegans. Strategies using either polymerase chain reaction (PCR), or hybridization of a mixed degenerate oligonucleotide encoding a conserved ARF sequence to lambda DNA libraries identified two genes, designated ARF-a and ARF-b. These genes were found to have 543 and 552 bp open reading frames, coding for proteins of 181 and 184 amino acids, respectively. Conservation of sequence between C. elegans ARFs and those of other eukaryotes was noted throughout the coding region and, in particular, in putative GTP binding and hydrolyzing domains. Nothern analysis revealed three ARF mRNA species. Southern analysis of C. elegans genomic DNA using both oligonucleotide and PCR-derived probes was consistent with the presence of these two ARF genes. ARF-a was physically mapped by Paul Coulson to a central contig on chromosome III. A partially purified low molecular weight ( 20 kDa) factor from C. elegans stimulated, in the presence of GTP, cholera toxin-catalyzed ADP-ribosylation of proteins and auto-ADP-ribosylation of the toxin catalytic unit, consistent with the expression of a GTP- dependent ARF- like protein in C. elegans.

Anthony A Ferrante et al. (2001) International C. elegans Meeting "Automated Sorting of C. elegans Muv Mutants According to Pseudovulva Number"

Russell Gershman, Anthony A Ferrante, W Peter Hansen, Steve Alam, Peter Kalutkiewicz

[International C. elegans Meeting, 2001]

The UBI COPAS BIOSORT system disperses and maintains nematodes in liquid suspension, straightens them, and flows them one-by-one through a laser beam. The system analyzes and sorts each animal according to both size and fluorescence without harm. For this study the system was modified such that positional information was retained as the nematodes were scanned along their longitudinal axis. Rather than averaging the fluorescence signal over the length of the animal, the signal was measured at approximately 100 points along the length of adult C. elegans . Profiling algorithms were developed that determined the location and the number of fluorescent features of the animals. The prototype was used to characterize and sort multivulva (Muv) C. elegans according to the number of vulvae. The Muv strains MT309 lin-15 and MT388 lin-12 (both obtained from the C. elegans Genetics Center, University of Minnesota, St. Paul), as well as wild-type strain N2, were stained using NEMASELECT Male/Herma stain (Union Biometrica, Inc.) to mark the location of their vulvae. Electronic profiles were collected with the new signal processing electronics and single nematodes were deposited in individual wells of a 96-well plate. Nematodes and their associated profiles were visually compared. Close correlation between the number of stained vulva and the number of fluorescence peaks in the profiles was observed. The prototype software accurately scored and sorted the animals according to vulva number.