Richardson, Claire E. et al. (2011) International Worm Meeting "Physiological IRE-1-XBP-1 and PERK/PEK-1 Signaling in C. elegans Intestinal Homeostasis and Immunity."

Kim, Dennis H., Kinkel, Stephenie, Richardson, Claire E.

[International Worm Meeting, 2011]

Endoplasmic reticulum (ER) stress activates the Unfolded Protein Response (UPR), a compensatory signaling response mediated by the IRE-1, PERK/PEK-1, and ATF-6 transmembrane proteins. Genetic studies have implicated roles for UPR signaling in animal development, but the function of the UPR under physiological conditions, in the absence of chemical agents administered to induce ER stress, is not well understood. Here, we show that in Caenorhabditis elegans, XBP-1 deficiency results in dramatically increased activities of IRE-1 and PEK-1, reflecting constitutive ER stress under basal physiological conditions. Innate immune activation and elevated physiological temperatures exacerbate this basal ER stress and necessitate IRE-1- XBP-1 and PEK-1 signaling for development and survival. Our data suggest that the negative feedback loops involving the activation of the IRE-1-XBP-1 and PERK/PEK-1 pathways serve essential roles not only at the extremes of exogenously imposed ER stress, but also in the maintenance of ER homeostasis under physiological conditions.

Richardson, Claire E. et al. (2009) International Worm Meeting "The IRE-1 Branch of the Unfolded Protein Response Functions in C. elegans Pathogen Resistance During Larval Development."

Kim, Dennis H., Kooistra, Tristan, Richardson, Claire E.

[International Worm Meeting, 2009]

The Unfolded Protein Response (UPR), a mechanism to sense and respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER), has an important role in development and stress resistance from C. elegans to mammals. We asked if the UPR functions in C. elegans immune defense against the bacterial pathogen Pseudomonas aeruginosa. We found that the conserved IRE-1-XBP-1 branch of the UPR is required specifically for immunity during larval development. IRE-1 is an endoribonuclease that activates the transcription factor XBP-1 by splicing a short exon from its mRNA. Using quantitative PCR, we show that the relative amount of IRE-1-spliced xbp-1 transcript increases after pathogen exposure. Furthermore, a fluorescent reporter for IRE-1 activation is induced after exposure to Pseudomonas specifically at the site of infection - the intestine. We analyzed the relationship between the IRE-1 branch of the UPR and the PMK-1 pathway, which is known to promote C. elegans immunity, and found that IRE-1 activation in response to Pseudomonas is downstream of the PMK-1 pathway. The IRE-1-XBP-1 branch of the UPR has been recently shown to be required for cellular defense against pore-forming toxins, also functioning downstream of PMK-1 (Bischoff et al, 2008). Our data point to a more general role for the UPR in innate immunity against infection. Since PMK-1 pathway activates transcription of putative pathogen resistance proteins, we suggest that the IRE-1 branch of the UPR promotes pathogen resistance by accommodating a PMK-1-driven influx of pathogen resistance proteins to the secretory pathway. Bischof, L.J., Kao, C.-Y., Los, F.C.O., Gonzalez, M.R., Shen, Z., Briggs, S.P., van der Goot, F.G., Aroian, R.V. (2008) Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo. PLOS pathogens, 4, 1-11.

Tillman, Erik et al. (2015) International Worm Meeting "Genetic Analysis of Endoplasmic Reticulum Homeostasis and Tolerance to Infection in C. elegans."

Tillman, Erik, Reddy, Kirthi, Kim, Dennis, Richardson, Claire, Cattie, Douglas

[International Worm Meeting, 2015]

We have previously identified an essential role for the conserved XBP-1 transcription factor in the maintenance of endoplasmic reticulum (ER) homeostasis during infection of Caenorhabditis elegans with pathogenic bacteria (Richardson, CE et al., Nature 2010). We conducted a genetic screen for mutations that suppress the lethality of xbp-1 loss during infection with Pseudomonas aeruginosa. We are currently carrying out the molecular characterization of suppressors that point to novel mechanisms that promote ER homeostasis independent of previously described pathways including the unfolded protein response (UPR).

Cattie, Douglas et al. (2013) International Worm Meeting "Mutations in the Translation Initiation Factor Subunit eIF-3.k Suppress the Stress Sensitivity of xbp-1 Mutants."

Reddy, Kirthi, Kim, Dennis, Richardson, Claire, Cattie, Douglas

[International Worm Meeting, 2013]

The Unfolded Protein Response (UPR) is a conserved homeostatic mechanism that functions to balance the folding capacity of the endoplasmic reticulum (ER) with the flux of unfolded protein into the compartment. We are interested in the physiological challenges that necessitate UPR function in C. elegans. Previously, we demonstrated a specific requirement for the ire-1/xbp-1 branch of the UPR in mediating the secretory load that results from innate immune activation, as mutants deficient in xbp-1 die during larval development when infected with the human opportunistic pathogen Pseudomonas aeruginosa. In order to better understand the physiological and pathological role of the UPR we conducted a forward genetic screen to identify mutations that would facilitate larval development of xbp-1 mutant worms developing on P. aeruginosa. From a screen of approximately 10,000 haploid genomes, we isolated 24 mutants and have further characterized three complementation groups. One suppressor mutation of xbp-1 larval lethality on P. aeruginosa was identified in the translation initiation factor subunit eIF-3.k. Mutants deficient in eIF-3.k also exhibited enhanced resistance to tunicamycin treatment and elevated temperatures, indicating that the resilience of these mutants is not limited to pathogen-induced proteotoxic stress. RNAi knockdown of several other eIF-3 subunits in the xbp-1 mutant background similarly restored successful larval development when infected with pathogenic bacteria. Mutations that result in attenuated translation likely contribute to survival in response to ER proteotoxic stress much in the way PEK-1 phosphorylation of eIF2a functions in the UPR by limiting the influx of unfolded protein into the ER. We are currently investigating whether ER stress tolerance is restricted to knockdown of eIF-3 complex components or whether attenuating translation by other genetic mechanisms also confers these phenotypes.

Cattie, Douglas et al. (2015) International Worm Meeting "Regulation of Stress Physiology and Longevity by the eIF3 Translation Initiation Complex in C. elegans."

Cattie, Douglas, Richardson, Claire E., Reddy, Kirthi C., Kim, Dennis H.

[International Worm Meeting, 2015]

The translation initiation factor EIF-3 is a 13-subunit protein complex that plays a critical role in coordinating the assembly of the 43S pre-initiation complex. Prior studies have defined a core complex of subunits required for translation initiation in vitro for both yeast and mammalian cells, though still more subunits are required for viability in vivo. We have probed the requirement for these subunits in an organismal context using C. elegans and identified two subunits that are entirely dispensable for normal development, EIF-3.K and EIF-3.L. Animals lacking either of these highly conserved subunits exhibit wild-type growth rates and harbor no measurable defect in bulk protein translation. Furthermore, each of these mutant strains extend lifespan 40% beyond that of wild-type animals. Lifespan extension caused by EIF-3.K deletion is dependent on the FOXO transcription factor DAF-16. In addition, mutations in eif-3.K and eif-3.L confer enhanced resistance to endoplasmic reticulum (ER) stress, independent of DAF-16 and XBP-1. While many studies have established a reciprocal relationship between metabolism and lifespan, these mutants represent a perturbation to the core translational machinery that affects organismal physiology without a concomitant decrease in bulk translation. Given the role of EIF-3 in translation initiation, it is possible that these subunits regulate the efficiency by which certain transcripts are translated over others, and that in their absence the ribosome is tuned towards translation of proteins that promote detoxification or cellular maintenance. We are conducting ribosomal profiling and genetic studies aimed at defining EIF-3.K site of action in modulating longevity and stress physiology.

Claire Lecroisey et al. (2006) European Worm Meeting "Implication of the Dense Body Protein Dyc-1 in the Dystrophin Dependent Muscle Degeneration"

Kathrin Gieseler, Laurent Segalat, Claire Lecroisey

[European Worm Meeting, 2006]

Claire Lecroisey, Kathrin Gieseler and Laurent Sgalat. The molecular mechanism underlying muscle necrosis in dystrophinopathies remains elusive. Our group addresses this question by using the genetically amenable animal model Caenorhabditis elegans, which has the same overall sarcomere composition and architecture as those of vertebrates.. In C. elegans, mutation of the dystrophin homologue, dys-1(cx18), produces a peculiar behavioural phenotype (hyperactivity, a tendency to hypercontract). In a sensitized hlh-1(cc561ts) background, which is a mild mutation of the myogenic factor MyoD, the dys-1(cx18) mutation also leads to a progressive muscle necrosis. The dyc-1 gene was previously identified in a genetic screen because its mutation leads to a phenotype similar to that of dys-1(cx18) mutation, which suggests that the two genes are functionally linked. Like dys-1(cx18); hlh-1(cc561ts), the double mutant dyc-1(cx32); hlh-1(cc561ts) also shows a progressive muscle degeneration. Moreover, Dyc-1 overexpression partially suppresses the dys-1(cx18); hlh-1(cc561ts) phenotype.. In the sarcomere, Dyc-1 is localized at the edge of the dense body, the nematode muscle adhesion structure functionally equivalent to vertebrate Z disc, where actin filaments are anchored. Two hybrid and immunocytochemistry experiments indicate that Dyc-1, Deb-1 (the vinculin homologue and main component of dense bodies), Zyx-1 (a focal adhesion protein), and Atn-1 (alpha-actinin homologue) may form a complex at the dense body. Our results suggest that the dense body might be the site of early pathological events occurring in the absence of dystrophin.. We are currently trying to demonstrate that dystrophin impairs the function of dense bodies proteins.

Grooms, N. et al. (2019) International Worm Meeting "Fluorescent screen for identifying axon regeneration genes."

Fitzgerald, M., Chung, S., Grooms, N., Urena, S.

[International Worm Meeting, 2019]

Statement of purpose: Central nervous system (CNS) injuries are prevalent and currently lack effective therapies. A conditioning lesion of the peripheral sensory axon triggers robust central axon regeneration in mammals (Richardson and Issa. Nature. 1984). Lesion conditioning could drive powerful therapies for CNS injuries. We established a model for lesion-conditioned regeneration in the invertebrate roundworm C. elegans. This approach allows for a much more comprehensive and faster search for neuronal regeneration genes compared to mammalian models. Methods: The C. elegans model permits unbiased screening. Ethyl methanesulfonate was used to stochastically introduce mutations and disrupt the regeneration pathway (Brenner. Genetics. 1974). In our screen, green fluorescence protein intensity is used as a proxy for neuronal regeneration. We selected candidate worms based on reduced intensity. Disruptions to sensory pathways in C. elegans has been shown to produce ectopic outgrowths under physiological stress. Therefore, studying ectopic outgrowth could identify genes involved in regeneration pathways (Chung, et al. PNAS. 2016). Ectopic outgrowth of dim strains is assessed via fluorescent microscopy by counting axonal outgrowths after culturing at 25 deg C. We are also quantifying the fluorescent intensity. The fluorescence of each strain is quantified using InSpeck Image Intensity Calibration Kits as a standard. Maximum intensity projections of 3D images were taken of worms next to beads for quantification. Laser surgery will be performed to assess regeneration in strains with reduced ectopic outgrowth. One-step whole genome sequencing will identify the affected gene in strains with reduced regeneration (Doitsidou, et al. PLOS ONE. 2010). Summary of results: Following mutagenesis, we isolated twelve strains, originating from six distinct F1s. These mutations have varying penetrance. In each selected strain, around 30-50% of the worms have reduced fluorescence. Ectopic outgrowth studies are expected to show decreased axonal outgrowths. The quantification of fluorescent intensities allows us to correlate intensity with regeneration on an individual and on an overall population basis. The identified genes will provide a roadmap for accelerating vertebrate lesion conditioning research. Affiliations: Funding provided by Morton Cure Paralysis Fund.

Jagan Srinivasan et al. (2007) International Worm Meeting "Evolution of a sensory response network."

Jagan Srinivasan, Paul W. Sternberg

[International Worm Meeting, 2007]

We are trying to understand how neural circuits evolve to mediate ethologically relevant behaviors. While model systems help elucidate how sensory information from diverse environmental conditions is represented and processed in the neural, remarkably little is known about the evolution of neural circuits, and how selective forces shape the final architecture of neural circuits and processing circuits (Dumont & Richardson, 1986, Science). Nematodes are an ancient phylum comprising millions of species from diverse habitats. They have molecularly diverse nervous systems which in principle can help them sense several types of environmental stimuli. To trace the evolutionary history of such a sensory repertoire, we tested three different avoidance behaviors; osmotic avoidance, response to nose touch, and volatile chemical repellence in six diverse species of free-living nematodes, a) Caenorhabditis elegans (N2), b) Caenorhabditis briggsae (AF16), c) Caenorhabditis sp. 3 (PS1010), d) Pristionchus pacificus (PS312) e) Cruznema tripartitum (PS1351) and f) Panagrellus redivivus (PS2298). We found that all species tested exhibit the three avoidance behaviors. However, we also find that sensory sensitivity to the different stimuli differs among the tested nematode species. In C. elegans , response to these stimuli are mediated by the ‘polymodal ASH neurons (Kaplan and Horvitz PNAS, 1993, Hart et al. Nature, 1995, Hilliard et al Curr Biol. 2002). We identified the pairs of putative ASH neurons in different nematode species by their anatomical positions and ablated them. Ablation of the ASH neurons in these species resulted in an inability to avoid these stimuli. By further ablation experiments, we find that there is increase or decrease in the set of sensory neurons mediating osmosensation and mechanosensation. In P. pacificus , osmosensation involves the ADL neuron. In Caenorhabditis sp. 3 , mechanosensation is solely mediated by the ASH neuron as compared to 3 neurons in C. elegans . The overall conservation of ASH mediated behaviors suggests that polymodality is an ancestral feature and is evolutionarily stable, but can evolve by alterations in the level of sensitivity and the relative contributions of sensory neurons.

Tillman, E.J. et al. (2017) International Worm Meeting "FKH-9 modulates stress physiology and endoplasmic reticulum homeostasis during pathogen infection in C. elegans."

Kim, D.H., Reddy, K.C., Richardson, C.E., Tillman, E.J., Cattie, D.J.

[International Worm Meeting, 2017]

Animals maintain cellular homeostasis in response to environmental challenges, including microbial pathogens. Distinct stress response signaling pathways promote protein folding homeostasis in the cytoplasm, endoplasmic reticulum (ER), and the mitochondria, and a role for each has been implicated during infection of C. elegans with pathogenic bacteria. We previously demonstrated that the ER Unfolded Protein Response (UPR) is induced during infection of C. elegans with Pseudomonas aeruginosa, and that the UPR regulator XBP-1 has an essential role during infection and immune activation (1). To identify signaling mechanisms that can compensate for XBP-1 deficiency during pathogen infection, we conducted a forward genetic screen for suppressors of xbp-1 mutant lethality on the pathogenic bacteria Pesuodomonas aeruginosa. We recently reported that mutations in the eIF3k and eIF3l translation initiation factor subunits can suppress the lethality of the xbp-1 mutant in the presence of P. aeruginosa (2). Here, we show that mutations in the Forkhead family transcription factor FKH-9 also suppress the lethality of the xbp-1 mutant on P. aeruginosa. We observe that mutations in fkh-9 confer improved intestinal ER morphology and organismal tunicamycin resistance, independent of previously defined UPR signaling pathways. To define how FKH-9 activity modulates ER homeostasis, we have conducted ChIP-seq analysis to define direct transcriptional targets of FKH-9, along with functional genetic analysis, which suggest a role for FKH-9 in integrative stress signaling. Our data suggest that XBP-1-independent pathways contribute to maintenance of ER homeostasis and survival during infection and innate immune activation in C. elegans. References: 1. Richardson, C. E., Kooistra, T. & Kim, D. H. An essential role for XBP-1 in host protection against immune activation in C. elegans. Nature 463, 1092-1095 (2010). 2. Cattie, D. J. et al. Mutations in Nonessential eIF3k and eIF3l Genes Confer Lifespan Extension and Enhanced Resistance to ER Stress in Caenorhabditis elegans. PLoS Genet 12, e1006326 (2016).

Edgley ML et al. (1991) International C. elegans Meeting "Caenorhabditis genetics center."

Riddle DL, Edgley ML, Estevez AOZ

[International C. elegans Meeting, 1991]

The CGC has been in operation for 12 years, supplying Caenorhabditis strains and information to researchers. It is responsible for coordination of C. elegans genetic nomenclature, and acts as the central clearing house for naming strains, genes and alleles. Information distributed includes the genetic map, genetic map data, strain information, a bibliography of 1300 research articles and book chapters, and the C. elegans research newsletter, the Worm Breeder's Gazette, which is distributed to 450 subscribers worldwide. A collection of 1550 strains has been established, representing 30 wildtype isolates of C. elegans, wild-type strains of C. briggsae, C. remanei and C. vulgariensis, as well as alleles of 773 mutant genes and 254 chromosome rearrangements. Stocks are stored permanently in liquid nitrogen. Approximately 1350 different strains have been distributed to research laboratories in 7800 separate mailings. Investigators in 210 laboratories in 21 countries have either received strains from the CGC or contributed strains to the CGC collection. The current version of the C. elegans genetic map includes 26 pages, and contains 920 genes and 320 rearrangements. It is produced using the computer drawing program Designer (Micrografx, Inc., Richardson TX), which runs under Microsoft Windows on IBM-compatible microcomputers. The map is published biannually in Genetic Maps (S.J. O'Brien, editor; Cold Spring Harbor Laboratory) and semi-annually in the Worm Breeder's Gazette. Caenorhabditis strains and information are available upon request. All strain, bibliographic and genetic information is maintained on a microcomputer database system, and the various files are available as printouts or data on diskette. Operation of the CGC will be transferred to other investigators at the end of the current CGC contract (September 29,1992). One possible plan emerged at the last C. elegans meeting, in which Bob Herman will assume responsibility for the strain collection, and Jonathan Hodgkin will assume responsibility for the genetic map. Plans for continuation of other CGC services remain to be made. New developments in the use of computers to integrate all information on the worm will enhance the usefulness of data collected by the CGC (see Thierry-Mieg, Durbin, Schatz and Ward, this meeting). The CGC is supported by the NIH National Center for Research Resources.