Chen, Wen et al. (2015) International Worm Meeting "Excretory-secretory products (ESP) profiling of Caenorhabditis elegans provide clues for environmental and developmental regulation."

Chen, Wen, Sternberg, Paul

[International Worm Meeting, 2015]

During the life cycle of parasitic and free-living nematodes, a wide range of biomolecules especially proteinases are continuously excreted or secreted into the environment, contributing to their survival, reproduction, habitat establishment, regulation of host immune system and various other functions. ESPs are active directly at the parasite-host or worm-environment interface and many ESP proteinases are enzymatically active, making them promising drug targets and anti-parasitic vaccine candidates. However, the mechanisms and related pathways are not clear given the complex processes occurring throughout different life stages. Also, whether the parasitic proteinases inherited and evolved whole or partly from free-living ancestral worms in response to parasitism has not been addressed. Thus, the investigation of ESP using C. elegans would provide rich information concerning ESP composition and the activity and substrate specificity of proteinases. Comparison of ESPs with parasitic nematodes might reveal novel functional pathways. We used liquid chromatography (LC)/MS-based proteomics to profile proteins released during in vitro maintenance of C. elegans asynchronous cultures and identified about 235 C. elegans proteins so far. General substrate and class specific substrates were used to test ESP general and cathepsin B-, D-like and collagenase activities. In addition, different culture conditions, for example, temperature, different E. coli diet, dauer-forming condition, were explored to show the adjustments in ESP profile according to environment. Annotation of identified proteins showed that many are metalloproteases, cysteine proteases and other catalytic proteases. Some of these proteases are implicated in the molting cycle, carbohydrate metabolism, growth rate regulation among other biological processes. We plan to use genetic methods to further explore specific functional roles of these ESP proteins. .

Chen, Wen et al. (2019) International Worm Meeting "Proteomic study of Caenorhabditis elegans Excretory-secretory products (ESP) shows conserved genes groups responding to environmental signals."

Chen, Wen, Sternberg, Paul

[International Worm Meeting, 2019]

Excretory-secretory products (ESP) are first characterized and defined in parasitic nematode proteomics studies as the combination of various biomolecules that are continuously excreted or secreted into the environment throughout the whole life cycle. ESP is particularly interesting to many scientists as anti-parasitic vaccine candidates and promising drug target since large portions of ESP are active enzymes that potentially function directly at the parasite-host or worm-environment interfaces. ESPs are also reported to play pivotal roles in many critical pathways, regulating nematode survival, reproduction, food processing and innate immune response. However, proteomics study using parasitic nematodes as model are limited due to lack of whole genome sequence knowledge and lack of genome-editing tools. Thus the number of ESP identified is limited and many functions of ESP proteins are elucidated. Therefore, we use the most studied nematode, Caenorhabditis elegans, as the model to characterize the composition of excreted/secreted proteins with the help of liquid chromatography (LC)/MS. In summary, we characterized more than 500 proteins with mix-staged worms, including many metalloproteases, cysteine proteases and lysozymes. We first confirmed the representative proteins are secreted/excreted from intestine and hypodermis using GFP tagged expression experiments and signal peptide prediction. With gene ontology analysis, many proteins are indicated to play roles in defending bacteria infection and regulating pathogen susceptibility. We compared the reported Caenorhabditis elegans transcriptomes culturing on different bacteria and pathogens with our ESP data, leading to the identification of a specific group of proteins that are up regulated in response to pathogen instead of bacteria. On the other hand, we compare the published ESPs of parasitic nematodes to C. elegans data. Through protein family analysis, we confirmed the conservation of pathways between species that having drastically different life styles, suggesting C. elegans ESP study can serve as the platform to facilitate nematode studies.

Chen, Shaw-Wen et al. (2013) International Worm Meeting "Using Stable Isotope Tracers to Understand the Role of Membrane Maintenance in Aging."

Chen, Shaw-Wen, Olsen, Carissa Perez

[International Worm Meeting, 2013]

The fatty acid composition of an animal's phospholipids has a great impact on the properties of the membrane including its permeability, fluidity, and susceptibility to damage. In fact, altered membrane makeup has been associated with a number of diseases as well as with natural aging. The membrane's integrity is also heavily influenced by its capacity to remove or repair damage; however, little is known about how membrane maintenance mechanisms impact membrane quality over time. Using dietary stable isotope tracers in C. elegans, we can quantify fatty acid replacement in membranes by following the dietary carbon into phospholipids via gas chromatography/mass spectrometry. These studies have found that in young sterile adults about a third of the membrane fatty acids are replaced or remodeled within a 6 hour period. This significant amount of new fatty acid indicates that the membrane needs a continual infusion of new lipids for replacement of consumed and damaged molecules. Additionally, we have found that, in these young adults, the majority of the new fatty acid tails are directly derived from dietary fat. Currently, we are defining how other metabolic pathways such as lipid synthesis influence the maintenance of membranes by conducting a targeted RNAi screen in conjunction with stable isotope labeling. Over aging, the composition of the membrane changes with a specific loss of polyunsaturated fatty acid species. This loss is predicted to be a result of damage as this group of fatty acids is particularly susceptible to reactive oxygen species attack. We hypothesized that reduced membrane maintenance may contribute to the altered membrane structure observed with natural aging. Indeed, we have found that as nematodes age, the amount of dietary resources funneled into the membrane is dramatically reduced. We are currently examining the relationship between membrane maintenance and longevity in different genetic backgrounds. This effort combined with our studies in young adult populations will allow us to identify important regulatory pathways for the maintenance of appropriate membrane composition and to further understand the role of membrane maintenance in aging.

Wang, Xiaodong et al. (2013) International Worm Meeting "Exploring Gene Expression and Transcriptional Regulation Data in WormBase."

Wang, Xiaodong, Chen, Wen, Raciti, Daneila

[International Worm Meeting, 2013]

Gene expression studies not only inform us on potential gene function but also serve as a measure of cellular and organismal phenotype and provide the foundation for building transcriptional regulatory networks. WormBase offers extensive coverage of C. elegans gene expression from small-scale as well as genome-scale studies such as modENCODE. WormBase includes data from expression results based on reporter gene analysis, immunostaining, in situ hybridization, single molecule FISH (smFISH), RNAseq, and microarrays, among others. Recently, we have started to curate gene expression data from other species. Over the past four years WormBase has adapted SPELL as a tool to access large-scale gene expression data. These now include microarray, tiling array, and RNAseq data. The WormBase implementations of SPELL include the following functions: 1. A display of expression levels of individual genes in each experimental dataset. 2. A search capability for genes with similar expression profiles and biological processes. 3. Access to whole experiment datasets, which are available for download. All SPELL microarray, tiling array and RNAseq data are mapped to the current WormBase release, keeping these data current with the latest gene models. Transcriptional regulation information is captured from reported changes in gene expression due to gene mutation, small molecule/chemical, or heat shock/physical treatment. These data can be viewed as part of gene interaction networks using Cytoscape as an interactive browser. In addition, WormBase accommodates a number of canonical transcriptional factor binding sites using Position Weight Matrix (PWM)/Position Frequency Matrix (PFM) data obtained through literature curation. These data can be accessed with the MotifFinder plugin in GBrowse. Large-scale transcriptional regulation data from modEncode, such as ChIp-seq, transcription factor binding sites (enhancers, silencers, promoters), histone modification sites, and DNAseI hypersensitivity sites, etc., are also integrated into WormBase. These data can also be viewed with GBrowse in their own track. In this poster we will give an overview of the gene expression and regulation data and the ways to access and mine these data.

Kato, Mihoko et al. (2013) International Worm Meeting "A novel protein complex required for the collective migration of the male somatic gonad."

Chou, Tsui-Fen, Chen, Wen, Yu, Collin Z., Sternberg, Paul W., Kato, Mihoko

[International Worm Meeting, 2013]

The morphogenesis of the C. elegans male gonad occurs by the migration of the individual linker cell and the adhesion of the proliferating follower cells behind this leader. We identified a conserved, single-pass transmembrane protein, TAG-256, that is required for the gonadal cells to stay connected during their migration; in mutants with a partial tag-256 deletion, dissociated gonadal cells are left behind during the migration, and this mutant can be rescued with a wild-type genomic copy of tag-256. TAG-256::YFP fusion protein expresses in the entire somatic gonad and localizes to the plasma membrane. Antibodies generated to different domains of TAG-256 show additional localization of the extracellular domain to large cytoplasmic vesicles and the intracellular domain to the nucleus, suggesting proteolytic cleavage. Since TAG-256 has no known interactors, we used SILAC mass spectrometry on human 293T cells to identify protein interactors of its human ortholog, ITFG1. Sixty-six proteins were ³ 5-fold enriched in immunoprecipitates with ITFG1, and of these, 46 proteins (70%) were conserved in C. elegans. To identify the genes that function together with tag-256 in C. elegans, we performed RNAi of the 46 interactors having worm orthologs in wild-type animals, and looked for a gonad detachment phenotype. ruvb-1, ruvb-2 and tba-2 yielded the same phenotype as tag-256, and we confirmed the physical interaction of their human orthologs by Western blot. We are currently investigating the subcellular localization of this new complex and the role of the AAA+ ATPases, RUVB-1 and RUVB-2, in this process. Based on the membrane localization of TAG-256 and gonad detachment phenotype of the mutant, we propose that TAG-256 has a physical role in cell-cell adhesion.

Chai, Cynthia et al. (2021) International Worm Meeting "Discovery of a Highly-conserved Behavioral Role for an Interneuron Neuropeptide Receptor"

Cohen, Sarah, Chai, Cynthia, Park, Heenam, Wong, Wan-Rong, WAN, Xuan, Chen, Wen, Sternberg, Paul

[International Worm Meeting, 2021]

Throughout the animal kingdom, neuropeptide signaling mediates many survival-promoting behaviors such as foraging and the escape response. In C. elegans, members of two of the largest neuropeptide gene families, flp and nlp, bind to their cognate frpr and npr-encoding receptors to regulate many aspects of nematode behavior and physiology. However, the expression and function of several neuropeptide G-protein coupled receptors (GPCRs) in C. elegans still remain unknown. Here, we characterize 21 frpr and npr neuropeptide GPCRs with no pre-existing reagents and discover a conserved behavioral role for the interneuron neuropeptide receptor FRPR-14. We systematically screened 21 CRISPR-generated GPCR null mutants using two behavioral paradigms, the posterior light touch-induced response and freely-moving locomotion tracking assays. Although half of the receptor mutants screened had at least one phenotype, we found that only the frpr-14 mutant was defective in both. A frpr-14 GFP reporter is expressed in the AVJ and AIB interneurons, both of which are presynaptic to several command interneurons. However, only AVJ-specific frpr-14 cDNA expression was sufficient to rescue the full repertoire of mutant phenotypes suggesting a broader behavioral role for AVJ compared to AIB. To identify FRPR-14's ligand in these behavioral contexts, we first screened all available flp mutants for visible motility defects. Following up with genetic epistasis analysis, we find FLP-3 to be a potential ligand for FRPR-14. We next applied a comparative approach by studying the orthologous Cbr-frpr-14 in Caenorhabditis briggsae. A Cbr-frpr-14 GFP reporter is expressed in a single pair of C. brigggsae interneurons with strikingly similar morphology and relative anatomical position as the C. elegans AVJ neurons. While cross-species C. elegans AVJ-specific expression of Cbr-frpr-14 cDNA rescued all mutant phenotypes, a Cbr-frpr-14 null mutant was only defective in the posterior light touch-induced response. Although FRPR-14's function at the cellular level is evolutionarily-conserved, the less evident conservation at the organismal level might be due to differing ligand expression patterns and/or neural architecture between species. Our results provide a framework for further studies on the evolution and function of neuropeptide signaling networks between two closely-related, genetically tractable species.

Frederick J Tan et al. (2004) West Coast Worm Meeting "Functions of the Outer Mitochondrial Membrane Protein CED-9"

R Blake Hill, Andrew Z Fire, Frederick J Tan

[West Coast Worm Meeting, 2004]

CED-9 (CEll Death abnormal) negatively regulates the programmed cell death pathway in C. elegans . This membrane associated pro-survival protein prevents the activation of the CED-3 caspase. Loss of function ced-9 animals arrest at early embryogenesis, presumably dying from ectopic cell deaths. Conversely, gain of function animals appear healthy, albeit with extra cells (Hengartner, Ellis and Horvitz, 1992). These effects have been ascribed to the ability of CED-9 to sequester the CED-4/CED-3 caspase complex at the mitochondria (Chen, et al. , 2000). We hope to understand the structural features of CED-9 that participate in its function. To this end, we are assaying the activity of various CED-9 mutants and derivatives for ability to prevent cell death and respond to appropriate modulating signals. A question that we consider to be central is whether the C-terminal transmembrane domain of CED-9 serves only as a targeting sequence to the mitochondrion, or plays a more active role. We have demonstrated that this domain is essential for mitochondrial localization and plays some role in the function of CED-9. Distinction between localization and other structural and functional roles is in progress. Hengartner MO, Ellis RE and HR Horvitz. 1992. Caenorhabditis elegans gene ced-9 protects cells from programmed cell death. Nature 356 , 494-499. Chen F, Hersh BM, Conradt B, Zhou Z, Riemer D, Gruenbaum Y, and HR Horvitz. 2000. Translocation of C. elegans CED-4 to Nuclear Membranes During Programmed Cell Death. Science 287 , 1485-1489.

S Chen et al. (1999) International C. elegans Meeting "Are Complex N-glycans Essential for the Development of the Nematode C. elegans?"

AM Spence, S Zhou, S Chen, M Sarkar, H Schachter

[International C. elegans Meeting, 1999]

UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I) is a key enzyme in the synthesis of hybrid and complex N-glycans. Disruption of the GnT I gene in the mouse results in arrest of embryogenesis. Defects in the synthesis of complex N-glycans have been shown to be associated with several human congenital diseases. We are investigating the role of complex N-glycans in C. elegans . Three C. elegans genes homologous to mammalian GnT I have been identified by searching the genome databases. They are designated gly-12, gly-13 and gly-14 . cDNAs encoding these predicted genes have been cloned and their expression patterns determined. Both gly-12 and gly-14 encode active enzymes while gly-13 does not, towards the substrate tested [Chen et al ., 1999]. To better understand its biological function, we wanted to isolate a GnT I null mutant. We screened worms that were UV irradiated in the presence of trimethylpsoralen(TMP) and isolated a mutant with a 1.6kb deletion of gly-12 . The deletion covers from intron 6 to exon 12, a DNA fragment encoding most of the GnT I C-terminal catalytic domain. The mutants have no obvious phenotypic defects. A Tc1 insertion has been detected within intron 9 of gly-14 . Screening for Tc1 derived deletions is under way. We will create double mutants that lack both gly-12 and gly-14 to test the redundancy of these two GnT I genes. Chen, S., Zhou, S., Sarkar, M., Spence, A. M., and Schachter, H. (1999) J. Biol. Chem. 274 , 288-297.

Ni, J. et al. (2017) International Worm Meeting "The triggering mechanism of C. elegans germline nuclear RNAi at the native targets."

Ni, J., Gu, S., Kalinava, N.

[International Worm Meeting, 2017]

We wish to understand the molecular mechanisms that distinguish "self" and "non-self" DNA in the germline genome of C. elegans. Nuclear RNAi refers to a set of small RNA-guided chromatin-based gene-silencing pathways (e.g., heterochromatin formation and transcriptional silencing), and plays an essential role in genome surveillance in yeast, plants, and animals. We previously identified a large set of genomic loci that are targeted by germline nuclear RNAi in C. elegans [1,2]. The triggering mechanisms at these native targets appear to be highly complex and are poorly understood. We are using two different approaches to resolve this gap. (1) We are identifying aberrant features associated with germline nuclear RNAi-targeted transcripts. To this end, we are using an unbiased RNA-seq approach to characterize both polyA and non-polyA transcripts in both wild type and mutant animals. In addition, subcellular localization of the native target transcripts are being examined using single-molecule FISH and RNA-seq combined with biochemical fractionation. (2) We are using CRISPR-mediated genome editing to test whether the silencing responses at native targets are indeed triggered by the aberrant features that are identified in (1). To date, we have found that "self" and "non-self" transcripts differ in RNA-pocessing, as well as subcellular localization. By using CRISPR, we have identified cis-regulatory elements that are required for the silencing response. Reference: 1. Ni JZ, Chen E, Gu SG. BMC Genomics. 2014. PMID: 25534009 2. Ni JZ, Kalinava N, Chen E, Huang A, Trinh T, Gu SG. Epigenetics Chromatin. 2016. PMID: 26779286

Karla Opperman et al. (2007) International Worm Meeting "A structure-function analysis of the C. elegans L1CAMs."

Lihsia Chen, Xuelin Wang, Karla Opperman, Shan Zhou

[International Worm Meeting, 2007]

Cell adhesion molecules play important roles during development and in maintaining tissue integrity. L1CAMs are a family of immunoglobulin (Ig)-like cell adhesion molecules that are important in vertebrate nervous system development. L1CAMs are conserved in C. elegans, and are encoded by the lad-1/sax-7 and lad-2 genes. SAX-7 and LAD-2 have distinct functions in the nervous system despite overlapping neuronal expression. SAX-7 is required to maintain neuronal positioning while LAD-2 participates in axon pathfinding. We are performing a structure-function analysis to determine how both proteins mediate their functions. Because the cytoplasmic tails between both proteins are distinct, we are focusing our analysis on how the cytoplasmic tail regulates L1CAM functions. Indeed, while the extracellular domains of SAX-7 and LAD-2 are conserved, the LAD-2 cytoplasmic tail is completely divergent and does not contain any of motifs conserved in SAX-7 and vertebrate L1CAMs. These motifs include the ankyrin-binding motif, which links L1CAMs to the spectrin-actin cytoskeleton, the PDZ-binding motif, and conserved tyrosine residues that are phosphorylated. Our preliminary results reveal that both conserved motifs, as well as phosphorylation one of the tyrosine residues, which regulates ankyrin binding, all contribute to the function of SAX-7 in neuronal position maintenance. (1) Chen L, Ong B, Bennett V. J Cell Biol. 2001 Aug 20;154(4):841-55. (2) Wang X, Kweon J, Larson S, Chen L. Dev Biol. 2005 Aug 15;284(2):273-91. (3) Sasakura H, Inada H, Kuhara A, Fusaoka E, Takemoto D, Takeuchi K, Mori I. EMBO J. 2005 Apr 6;24(7):1477-88. Epub 2005 Mar 17.