Wang, Juan et al. (2017) International Worm Meeting "Biogenesis and Function of Social Extracellular Vesicles (EVs)."

Barr, Maureen, Wang, Juan, Silva, Malan

[International Worm Meeting, 2017]

Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.

Kyung Won Kim et al. (2005) International Worm Meeting "RNP-8 interacts with the GLD-2 cytoplasmic poly(A) polymerase and regulates its polyadenylation activity"

Kyung Won Kim, Judith Kimble, Liaoteng Wang

[International Worm Meeting, 2005]

Polyadenylation is critical for mRNA stability and translational activation. During oocyte maturation and early embryonic development, cytoplasmic polyadenylation of preexisting mRNAs promotes their translation. The C. elegans gld-2 gene is required for multiple steps in germline development, including the mitosis/meiosis decision. GLD-2 is a cytoplasmic poly(A) polymerase (PAP) that lacks an RNA recognition motif (1); similar PAPs have been identified in virtually all eukaryotic organisms (2). A yeast two-hybrid screen using GLD-2 as bait isolated GLD-3, RNP-8 and LARP-1 (1; L. Wang and J. Kimble, unpublished). GLD-2 has little PAP activity on its own, but it is stimulated in vitro by GLD-3 (1). We have now focused on RNP-8 to ask whether this GLD-2 interactor also stimulates GLD-2 activity and to determine whether RNP-8 has a major role in germline development. Preliminary data reveal that rnp-8(RNAi) and gld-2 mutants have similar defects during oogenesis. Both gld-2 and rnp-8 mRNAs are abundant in embryos, L4 larvae, and adults, and rnp-8 mRNA is enriched in the germ line. To test whether RNP-8 stimulates the GLD-2 PAP activity, we performed assays for PAP activity in vitro. Preliminary data suggest that RNP-8 can indeed stimulate GLD-2 PAP activity. Taken together, we suggest that RNP-8 stimulates GLD-2 activity to control germline development.1. Wang, L., Eckmann, C.R., Kadyk, L.C., Wickens, M., and Kimble J. (2002), A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans. Nature 419: 312-316 2. Kwak, J.E., Wang, L., Ballantyne, S., Kimble J. and Wickens, M. (2004), Mammalian GLD-2 homologs are poly(A) polymerases. PNAS 101: 4407-4412

Kendall Wu et al. (2004) West Coast Worm Meeting "Identification of Genetic Biomarkers and Regulators of Aging in C. elegans"

James Lund, Min Jiang, John Wang, Yueyi Liu, Stuart Kim, Kendall Wu

[West Coast Worm Meeting, 2004]

Despite the wealth of knowledge about conditions and mutations that cause worms to live longer, we still have an incomplete understanding of what varies from young to old worms -- especially at the molecular level. A long-term goal of our laboratory is to determine the molecular changes that occur as an organism ages in order to better understand the aging process and its regulation. Using the nematode Caenorhabditis elegans ( C. elegans ) as a genetic model for aging, we have used DNA microarrays to identify a common set of genes regulated throughout several age-related conditions: genes that change in old age (Lund et al. , 2002), genes that change in the exit from the dauer state (Wang and Kim, 2003), and genes that change expression in long-lived mutants of the insulin-like pathway such as the age-1 and daf-16 mutants (unpublished data). Analysis of the upstream regions of these age-regulated genes showed that they are heavily enriched for a GATA DNA consensus sequence suggesting that a GATA transcription factor may be involved in regulating the expression of these genes. This GATA motif may represent a novel regulatory pathway of the aging process that might act either together or separately from the daf-2 /insulin-like pathway. Using GFP reporters of these genes, we have begun to determine how these genes are age-regulated and to identify the key tissues regulating lifespan. We have also used RNAi to abrogate the expression of these genes to determine their role in longevity. The results of these studies will define a set of molecular biomarkers that will allow a more accurate description of the aging process. In addition, these biomarkers will allow identification of new aging mutants perhaps leading to identification of mutants with accelerated aging. Lund, J., Tedesco, P., Duke, K., Wang, J., Kim, S. K., and Johnson, T. E. (2002). Transcriptional profile of aging in C. elegans. Curr Biol 12, 1566-1573. Wang, J., and Kim, S. K. (2003). Global analysis of dauer gene expression in Caenorhabditis elegans. Development 130, 1621-1634.

Singh, Ramya et al. (2019) International Worm Meeting "The Unfolded Protein Response inhibits GLP-1/Notch signalling associated germline stem cell proliferation."

Racher, Hilary, B Smit, Ryan, Hansen, Dave, Singh, Ramya

[International Worm Meeting, 2019]

Stem cell proliferation in the C. elegans germline is primarily supported by the GLP-1/Notch signalling pathway. We have found that induction of the Unfolded Protein Response inhibits GLP-1/Notch signalling levels thereby resulting in suppression of stem cell proliferation. We first focussed on a Derlin (Degradation in the Endoplasmic Reticulum) protein, called cup-2, known to be involved in the ER-Associated degradation pathway of protein quality control. Loss of cup-2 has previously been shown to lead to ER stress and activate the Unfolded Protein Response, a pathway that helps protect cells against ER stress (Ye et al. 2004 & Schaheen et al. 2009). We found that loss of cup-2 suppressed several Notch-dependent germline tumours but could not suppress Notch-independent germline tumours. Loss of the closely related Derlin gene, der-2, was also able to suppress a Notch-dependent tumour and loss of both cup-2 and der-2 additively suppressed a Notch-dependent tumour. Using a readout for Notch signalling, we found that the level of Notch signalling in cup-2(0); glp-1(gf) germlines was reduced to nearly wild-type levels in contrast to the higher levels seen in the glp-1(gf) background. Since Derlin proteins are involved in protein quality control, and the loss of their activity induces ER stress, we asked whether chemical induction of ER stress could also suppress Notch-dependent tumours. We found that induction of ER stress using Dithiothreitol (DTT) or Thapsigargin was able to suppress Notch-dependent tumours but could not suppress a Notch-independent tumour. We further found that cup-2 suppression is dependent on XBP-1, a key player in the Unfolded Protein Response pathway. Therefore, we conclude that activation of the Unfolded Protein Response by inducing ER stress, reduces GLP-1/Notch signalling levels in the germline. This work highlights the importance of protein quality control mechanisms in promoting GLP-1/Notch signalling in the germline. References Ye Y, Shibata Y, Yun C, et al (2004) A membrane protein complex mediates retro- translocation from the ER lumen into the cytosol. Schaheen B, Dang H, Fares H (2009) Derlin-dependent accumulation of integral membrane proteins at cell surfaces. J Cell Sci 122:2228-2239. doi: 10.1242/jcs.048892

Bamps, Sophie et al. (2009) International Worm Meeting "Caloric restriction induces elevated expression of sir-2.1 in C. elegans."

Wirtz, Julia, Lake, Duncan, Savory, Fiona, Hope, Ian A., Bamps, Sophie

[International Worm Meeting, 2009]

Caloric restriction results in lifespan extension in yeast, worms, Drosophila and mammals. Yeast studies showed extension of lifespan in response to caloric restriction involves sir-2 the founding member of a widely conserved gene family encoding the sirtuins, enzymes with histone deacetylase activity. Like in yeast, Drosophila and mammals, over-expression of the C. elegans sirtuin gene sir-2.1 leads to extension of life span and deletion of the gene shortens life span (Tissenbaum & Guarente, 2001; Wang & Tissenbaum, 2006). Previously, a C-terminal translational gfp reporter fusion with sir-2.1 under the control of a 300 nucleotide intergenic promoter fragment (in strains HT809, HT810) was reported to reveal expression of sir-2.1 in most of the nerve cells in the head and the tail, and in the hypodermis (Wang & Tissenbaum, 2006). Recombineering allowed us to insert both a mCherry reporter gene just before the stop codon of sir-2.1 and a gfp reporter just before the stop codon of R11A8.6, within a 29.3kb fosmid genomic DNA clone. R11A8.6 is the first gene and sir-2.1 is the downstream gene in a two gene, 4.4kb operon. The mCherry expression pattern under normal growth conditions, with abundant food, indicated that sir-2.1 is indeed expressed, as previously described, in many nerves in the head. However, GFP was also seen, along with mCherry, in the gut and in head muscles. This reveals that expression of sir-2.1 is broader than previously described, fitting the expectation that the SIR2.1 function in influencing lifespan would be required in most cell types. Furthermore, the broader expression arises from transcription starting with the upstream gene of the sir-2.1 operon. SIR-2.1::mCherry is nuclear, consistent with the proposed function in controlling gene expression, while R11A8.6::GFP is cytoplasmic, consistent with the enzymatic role of the glutathione-S-transferase encoded by R11A8.6. Finally, and importantly, the expression of sir-2.1::mCherry and R11A8.6::gfp rises dramatically upon starvation. No such increase is seen with sir-2.1::gfp under the control of the intergenic promoter fragment suggesting that elevated sir-2.1 expression upon starvation depends on transcription across the operon from the upstream gene. Curiously, under starvation conditions, SIR-2.1::mCherry is not nuclear-localized, but is localized in puncta in the cytoplasm. Elevated transcription of sir-2.1 also corresponds with elevation of expression of sir-2.1 orthologues in other organisms upon starvation. We thank Heidi Tissenbaum for strains HT809, HT810. Tissenbaum & Guarente, Nature, 2001. 410, 227 Wang & Tissenbaum, Mech. Ageing Dev. 2006. 127, 48.

Alcantara, Alfredo Jr. et al. (2021) International Worm Meeting "Simulated Microgravity Impairs C. elegans Gut Immunity"

Higashibata, Akira, Higashitani, Atsushi, Lee, Jin Il, Alcantara, Alfredo Jr., Hashizume, Toko

[International Worm Meeting, 2021]

Humanity had been fascinated with space exploration and habitation. However, weak gravity force in outer space, termed as microgravity, has been known to alter normal human biology and induce health risks, such as muscle atrophy and compromised immunity. The underlying processes on how gravity is sensed by the body and how it affects immunity are still unclear. The nematode Caenorhabditis elegans, is a well-known model organism in the field of innate immunity and space research. Similar to humans, worm immune response signaling is regulating by MAPK and TGFbeta pathways. Previous microgravity experiments in C. elegans have shown an increase in p38 MAPK pmk-1 expression in simulated microgravity (Li, Wang, & Wang, 2018) and decreased expression of TGFbeta ligand dbl-1 in space (Harada et al., 2016). In this study, we have utilized Enterobacter cloacae carrying tdTomato fluorescence reporter to observe gut colonization in C. elegans under simulated microgravity using a 3D clinostat. E. cloacae is a known commensal in C. elegans' gut but is invasive in immunocompromised dbl-1 mutant worms (Berg et al., 2019). Our preliminary results have shown that simulated microgravity can induce E. cloacae gut colonization in wild type C. elegans and aggravated colonization in pmk-1 mutants, while there is no difference observed in dbl-1 mutants which have robust colonization in both normal earth gravity and simulated microgravity, which demonstrates potential involvement of these pathways. Our study aims to discover the underlying mechanism of gravity sensing and how it affects the immune signaling pathways. In addition, spaceflights are already scheduled to send C. elegans aboard the International Space Station (ISS) for actual space gravity experiments.

Bayer, Emily et al. (2015) International Worm Meeting "Investigating the genetic basis of neuronal dimorphisms in C. elegans."

Oren-Suissa, Meital, Hobert, Oliver, Bayer, Emily

[International Worm Meeting, 2015]

Sexual dimorphism is a widely acknowledged biological phenomenon, yet the mechanisms underlying specific developmental dimorphisms are largely unknown. As a result of the defined connectome available for both C. elegans males and hermaphrodites, it is clear that there are dimorphic wiring differences in shared neurons between the adult animals of the two sexes (1). Several of these dimorphisms are found in the phasmid sensory neurons, whose synapses onto the command interneurons are sexually dimorphic, suggesting that they have sex-specific functions. I am confirming the predicted sex-specific functions of the phasmid neurons using behavioral assays. To study how these dimorphic patterns of connectivity (and function) are established, I am using transsynaptic labeling (GRASP technology; 2) to visualize both male-specific and hermaphrodite-specific synaptic connections of phasmid neurons. Sexual determination is regulated across many invertebrate and vertebrate species by the highly-conserved doublesex/mab (DM) domain genes. In C. elegans, the DM domain class contains 11 paralogs, the majority of which have no known function. I analyzed expression patterns for 8 of the dmd genes in both larval and adult stages in the two sexes to identify dimorphisms, and identified dmd-4 as a candidate for dimorphic regulation in the PHA and PHB phasmid neurons. dmd-4 is an embryonic lethal gene, so I am generating a conditional knockout allele and perform mosaic rescue analysis to determine if dmd-4 is part of the genetic regulatory system for dimorphism in the phasmid neurons, and if this function is cell-autonomous. Ultimately, we will seek to elucidate the genetic regulatory logic of dimorphisms in shared neurons.1. Jarrell TA, Wang Y, Bloniarz AE, Brittin CA, Xu M, Thomson JN, Albertson DG, Hall DH, Emmons SW. 2012. Science 337, 437-444.2. Feinberg EH, VanHoven MK, Bendesky A, Wang G, Fetter RD, Shen K, Bargmann CI. 2008. Neuron 57(3), 353-363.

Kyung Won Kim et al. (2007) International Worm Meeting "RNP-8, a newly identified co-activator of GLD-2 cytoplasmic poly(A) polymerase, and its control of germline development."

Kyung Won Kim, Liaoteng Wang, Judith Kimble

[International Worm Meeting, 2007]

Polyadenylation is critical for mRNA stability and translation. In general, poly(A) tail elongation can stabilize and translationally activate mRNAs, while poly(A) tail removal can trigger mRNA degradation and repress translation. One key regulator of polyadenylation is GLD-2, a widely-conserved cytoplasmic poly(A) polymerase (PAP) (1, 2). GLD-2 proteins are catalytic subunits but have no recognizable RNA-binding domain (1). Our work focuses on GLD-2 in the nematode C. elegans and its control of germline development. Our current model is that a nematode GLD-2 is recruited to specific mRNAs by specific RNA-binding partners. GLD-2 has low PAP activity on its own, but its activity is stimulated by interactions with GLD-3/Bicaudal-C, a conserved RNA-binding protein (1). To identify other GLD-2 partners, we conducted a yeast two-hybrid screen and identified RNP-8, a novel RRM protein that is predicted to bind RNA. Consistent with our model, we find that RNP-8 stimulates GLD-2 PAP activity in vitro. Both rnp-8 mRNA and RNP-8 protein are germline-enriched and abundant in the cytoplasm of germ cells in the pachytene stage of meiotic prophase and in maturing oocytes. An rnp-8 deletion mutant exhibits low percentage of the Mog (for masculinization of germline) phenotype, suggesting that RNP-8 promotes oogenesis. Importantly, its minor phenotypic defects are enhanced by removal of other RNA regulators controlling germline development. 1. Wang, L., Eckmann, C.R., Kadyk, L.C., Wickens, M. and Kimble, J. (2002) A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans. Nature 419: 312-316. 2. Kwak, J.E., Wang, L., Ballantyne, S., Kimble, J., and Wickens, M. (2004) Mammalian GLD-2 homologs are poly(A) polymerases. Proc Natl Acad Sci USA 101, 4407-4412.

Nayoung Suh et al. (2003) International Worm Meeting "Identifying GLD-2 target mRNAs in C. elegans"

Nayoung Suh, Judith Kimble

[International Worm Meeting, 2003]

We are interested in understanding the molecular controls of how germ cells are regulated to leave the mitotic cell cycle and differentiate. The gld-2 gene is a central player in this control in the nematode Caenorhabditis elegans (Kadyk and Kimble, 1998). The C. elegans GLD-2 protein belongs to the DNA nucleotidyltransferase superfamily; its closest homologs are S. pombe Cid1 and Cid 13, S. cerevisiae TRF4/TRF5, and classical eukaryotic poly(A) polymerase (PAP). Unlike conventional PAP, GLD-2 does not contain any apparent RNA binding motif. In the simplest model, GLD-2 acts as a heterodimer with the RNA binding partner (Wang et al., 2002). By interacting with different RNA binding proteins, GLD-2 PAP activity might target diverse specific mRNAs. Therefore, identifying target mRNAs of GLD-2 is necessary to understand how an essential regulator of the germ line governs its development and early embryogenesis. To identify target mRNAs of GLD-2, we will employ two different approaches. For the biochemical approach, we will co-immunoprecipitate GLD-2 associated mRNAs. This strategy has been used successfully to identify GLD-1 targets (Lee and Schedl, 2001). To date, we have made the DNA construct for an epitope-tagged GLD-2. We will establish the transgenic animal expressing this construct and test if it is functional. The other approach is to measure the poly(A) tail length of candidate target mRNAs to test for GLD-2-dependent polyadenylation. We will use a "Poly(A) test (PAT)" assay, a PCR-based technique that amplifies poly(A) tails. We are currently establishing the experimental techniques for examining the length of specific mRNA tails. Kadyk and Kimble (1998) Development 125, 1803-13; Lee and Schedl (2001) Genes Dev. 15, 2408-20; Wang et al. (2002) Nature 419, 312-16.

Pei-ken Hsu et al. (2004) West Coast Worm Meeting "Structural and Functional Analysis of PSR-1 in C. elegans"

Ming-chia Lee, Yi-Chun Wu, Pei-ken Hsu

[West Coast Worm Meeting, 2004]

In the development of multicellular organisms, the proper removal of cell corpses generated by programmed cell death prevents the possible dispersal of harmful cellular contents from dying cells. A recent study has shown that psr-1 , the C. elegans homolog of human PSR (phosphatidylserine receptor), is important for cell-corpse engulfment (Wang et al., 2003). PSR-1 is proposed to function as a receptor and act upstream of the pathway mediated by CED-2, CED-5, CED-10, and CED-12 signaling molecules, possibly through physical interactions with CED-5 and CED-12 to control the engulfment process. In order to understand how PSR-1 interacts with CED-5 and CED-12, we mapped the binding region in PSR-1 for CED-5 and CED-12 interactions. We found that the amino acids 135-257 are required but not sufficient for CED-5 binding and the amino acids 212-257 of PSR-1 are required and sufficient for CED-12 binding. In addition, PSR-1 is predicted to have one tyrosine phosphorylation site. To investigate its function in cell-corpse engulfment we changed the tyrosine to phenylalanine in PSR-1 and found that the mutant PSR-1 failed to rescue the cell-corpse engulfment defect in psr-1 mutants. Therefore, the tyrosine residue and possibly its phosphorylation is important for PSR-1 function. To explore the expression pattern of PSR-1, we generated polyclonal antibodies against GST-PSR-1 fusion protein. We are currently investigating the subcellular localization of PSR-1. Reference: Wang, X., Wu, Y., Fadok, V.A, Lee, M., Gengyo-Ando. K., Cheng, L., Ledwich, D., Hsu, P., Chen, J., Chou, B., Henson, P., Mitani, S., Xue, D. Science 302, 1563-1566 (2003).