Dietrich, N. et al. (2017) International Worm Meeting "Homeostatic response to zinc deficiency in C. elegans is mediated by the LZA enhancer and the zinc transporter ZIPT-2.3."

Tan, C., Kornfeld, K., Dietrich, N., Schneider, D.

[International Worm Meeting, 2017]

The essential element zinc plays an important structural and functional role in proteins in all living organisms. Zinc homeostasis is critical, because both zinc deficiency and excess are deleterious; in humans, defective zinc homeostasis leads to several disease states. The mechanisms utilized by animals to respond to excess zinc have been extensively characterized, but much less is known about how animals sense and respond to zinc deficiency. A primary method animals use to increase zinc content is through the zinc transport family known as the Zrt, Irt-like proteins (ZIPs). Caenorhabditis elegans is a powerful experimental system to study the mechanisms of zinc deficiency based on sophisticated genetic and cell biological approaches, and studies of C. elegans are likely to be relevant to humans, since both worms and humans have 14 ZIP family members. To characterize the response to zinc deficiency, we measured the transcriptional response of all 14 C. elegans ZIP genes and identified three that are activated in response to low levels of dietary zinc. By analyzing the promoters of these genes, we identified an evolutionarily conserved cis-regulatory element, which we named the low zinc activating (LZA) element. The LZA is both necessary and sufficient for the induction of mRNA in response to low dietary zinc. To determine factors that function in trans to mediate the activity of the LZA, we used RNAi of candidate genes and showed the LZA requires the function of the GATA transcription factor ELT-2 and the mediator complex member MDT-15 to cause an increase in mRNA expression in low zinc conditions. The function of this element is also conserved in human cells, as cells transfected with the LZA in a vertebrate promoter displayed a sequence dependent induction of a reporter gene. These results identify a novel and conserved enhancer that responds to low zinc in animals. One of the genes induced by low zinc is zipt-2.3. To analyze the function of this gene, we identified a deletion allele and showed that mutant animals display hypersensitivity to zinc deficiency. Thus, zipt-2.3 is necessary to survive a low zinc challenge. To analyze the biochemical activity of ZIPT-2.3, we expressed the protein in vertebrate cells and measured zinc uptake. ZIPT-2.3 mediated zinc uptake, demonstrating that it functions as a zinc transporter. These results elucidate low zinc homeostasis by identifying a zinc transporter that is induced by low zinc and is necessary to respond to zinc deficient conditions.

Dietrich, N et al. (2019) International Worm Meeting "Zinc homeostasis is mediated by reciprocal regulation of CDF-2 and ZIPT-2.3, transporters that store and release zinc from lysosome-related organelles in intestinal cells."

Dietrich, N, Tan, C.H., Schneider, D.L., Mendoza, A.D.

[International Worm Meeting, 2019]

Zinc is required for a large number of cellular processes because it promotes protein structure and enzymatic activity, and it can function as a signaling molecule. Because both zinc deficiency and excess are deleterious. Zinc homeostasis is critical, and organisms have evolved mechanisms to obtain, store, and mobilize zinc. To investigate zinc homeostasis in C. elegans, we used reverse genetics by analyzing the conserved family of zinc importers SLC39A, Zrt, Irt-Like Proteins (ZIP) that transports zinc into the cytosol. C. elegans has 14 predicted family members, called zipt genes. We focused on zipt-2.3 because it is robustly activated by zinc deficient conditions through the LZA enhancer element (Dietrich et al 2017). ZIPT-2.3 protein expressed in mammalian cells promoted zinc uptake, indicating it functions biochemically as a zinc transporter. In transgenic animals, ZIPT-2.3::mCherry was expressed specifically in lysosome-related organelles in intestinal cells (gut granules). Gut granules express the zinc transporter CDF-2, which promotes zinc storage. In zinc replete conditions CDF-2 and ZIPT-2.3 fully colocalize in the spherical organelle. Remarkably, in zinc deficient conditions lysosome-related organelles form an interior pocket, which contains ZIPT-2.3, while CDF-2 remains on the exterior. In excess zinc conditions, the organelle is remodeled to form a bilobed structure; ZIPT-2.3 localizes to the smaller lobe, while CDF-2 is present on both lobes. Furthermore, cdf-2 and zipt-2.3 displayed reciprocal regulation: in zinc excess conditions, cdf-2 is highly expressed and zipt-2.3 is repressed, whereas in zinc deficient conditions, zipt-2.3 is highly expressed and cdf-2 is repressed. Based on these observations, we hypothesize that CDF-2 and ZIPT-2.3 mediate storage and release of zinc from gut granules, respectively, to mediate zinc homeostasis, which is facilitated by remodeling of the organelle. Consistent with this model, the zipt-2.3(lf) mutation caused hypersensitivity to zinc deficient conditions, whereas overexpression of zipt-2.3 was sufficient to cause hypersensitivity to zinc excess conditions. zipt-2.3(lf) mutants displayed retention of zinc in the gut granules when challenged with zinc deficient conditions, suggesting they are defective in mobilization of zinc. These findings elucidate a mechanism of zinc homeostasis based on reciprocal regulation of CDF-2 and ZIPT-2.3 that store and release zinc from gut granules.

Martin Gutternigg et al. (2006) European Worm Meeting "Glycosidases of Caenorhabditis elegans involved in N-glycan processing"

Martin Gutternigg, Matthias Hackl, Ute Stemmer, Iain B. H. Wilson, Petra Geier, Gunter Lochnit, Ramona Ranftl, Katharina Paschinger, Verena Jantsch, Dorothea Lubich

[European Worm Meeting, 2006]

Martin Gutternigg, Dorothea Lubich, Matthias Hackl, Katharina Paschinger, Ute Stemmer, Verena Jantsch1, Gnter Lochnit2, Ramona Ranftl, Petra Geier and Iain B. H. Wilson. Recent data indicates that in addition to the Golgi ?-mannosidases, the model nematode Caenorhabditis elegans also possesses, like insects, an N-acetylhexosaminidase activity putatively involved in N-glycan processing in the Golgi. The presence of such an activity is invoked, not just on the basis of the detected enzyme activity, but also to explain the absence of terminal N-acetylglucosamine residues on structures which require the prior action of N-acetylglucosaminyltransferase I during their biosynthesis. In order to understand the genetic basis for these activities, we have cloned cDNAs encoding members of both glycohydrolase families 20 and 38 from the worm. The encoded glycosidases were expressed in the yeast Pichia pastoris as soluble forms lacking putative cytoplasmic and transmembrane domains. Four glycohydrolase family 20 members were shown to cleave p-nitrophenyl-?-N-acetylglucosaminide and/or p-nitrophenyl-?-N-acetylgalactosaminide, but showed contrasting specificities with regard to N-glycan substrates. On the other hand, one glycohydrolase family 38 member was shown to be active using p-nitrophenyl-?-mannoside as a substrate and, in addition, had mannosidase II activity. Analysis of the glycans of the relevant mutant showed large-scale changes in the N-glycosylation spectrum. These, therefore, are the first data on the activity of Caenorhabditis glycosidases towards N-glycan substrates and should aid the further elucidation of N-glycan processing in this organism.

ZHANG, Gaotian et al. (2015) International Worm Meeting "Characterization of nematode-infecting microsporidia and natural variation in sensitivity of Caenorhabditis nematodes to microsporidia."

Zhang, Gaotian, Felix, Marie-Anne

[International Worm Meeting, 2015]

The microsporidian Nematocida parisii is the first characterized intracellular pathogen of Caenorhabditis elegans; and a second Nematocida species, called N. sp. 1, was also reported from an Indian C. briggsae isolate (Troemel et al. 2008). In our lab we further established a collection of wild rhabditid nematodes suspected with microsporidian infection. Here, we characterize molecularly these wild microsporidia and study their specificity of infection.First, the potentially infected rhabditid isolates were characterized using newly designed PCR primers specific for 18S and beta-tubulin genes of microsporidia. In addition to C. elegans, we found wild C. briggsae and Oscheius tipulae as natural hosts for N. parisii; in addition to C. briggsae, we found C. elegans and C. remanei as natural hosts of N. sp. 1. We further found three new microsporidian species. Two of them are close to the Nematocida species in microsporidian clade II and preliminarily named N.-like sp. 2 and N.-like sp. 3. The third species (or group of species), infecting Oscheius, is very distant, and found in microsporidian clade IV, that includes most human pathogens. They are closest to Orthosomella operophterae (with 18S), and thus preliminarily named Orthosomella-like spp.. Phylogenetic analyses with the 18S and beta-tubulin genes provide the same relationships of Nematocida spp. and the three putative new microsporidia species. Co-infections of two microsporidian species occurr in two wild O. tipulae strains, of which one infected with N. parisii and O.-like spp., the other infected with N. sp. 1 and O.-like spp. (confirmed by specific FISH probes).Secondly, two transgenic C. elegans strains ERT54 jyIs8[C17H1.6p::gfp; myo-2p::mCherry] and ERT72 jyIs15[F26F2.1p::gfp; myo-2::mCherry], which express a constitutive fluorescent Cherry marker and will induce GFP upon infection with N. parisii (Bakowski et al. 2014), were used in infection tests. Our results show that N. parisii, N. sp. 1, N.-like sp. 2 and N.-like sp. 3 can infect ERT54 and ERT72, forming meronts and spores; N. sp. 1 does not induce the GFP reporter (or only rarely), while the other three induce it consistently, suggesting that C. elegans has a different transcriptional response to the infection of N. sp. 1 than to other Nematocida spp. (see also abstract of Luallen et al.). The results also showed that O.-like spp. could not infect the two transgenic C. elegans strains (nor induce the GFP signals).

Jeon, JiHae et al. (2021) International Worm Meeting "N. parisii compensates for genomic loss of dihydroceramide desaturase through reliance on C. elegans sphingolipid biosynthesis"

Jeon, JiHae, Burton, Nick, Reinke, Aaron, Zhao, Winnie

[International Worm Meeting, 2021]

Microsporidia are obligate intracellular parasites that have lost many metabolic enzymes, resulting in the smallest known eukaryotic genomes. Microsporidia are highly dependant upon host nutrients, but it is poorly understood the effect that these parasites have on host metabolism. Using N. parisii, a natural microsporidian parasite of C. elegans, we describe how infection alters host lipid metabolism. We show that the C. elegans lipase ATGL-1 is upregulated in response to N. parisii infection and that host fat stores are depleted. Metabolic profiling of infected animals reveals large changes in lipid composition including changes consistent with nutrient starvation. Additionally, we identify novel ceramides only generated in animals infected with N. parisii. Genomic analysis reveled that N. parisii has lost the enzymes necessary for the de novo synthesis of these sphingolipids. Mutations in C. elegans dihydroceramide desaturases F33D4.4 and ttm-5 or acid ceramidase asah-2, reduce N. parisii growth. Supplementation with sphingosine, a substrate for ceramide synthesis, enhances N. parisii proliferation. Together, our data demonstrate that N. parisii exploits sphingolipid biosynthesis in C. elegans and reveals an evolutionary strategy used by microsporidian parasites to cope with extreme genomic reduction.

di Luccio, Eric et al. (2021) International Worm Meeting "N-NOSE is an innovative, non-invasive and highly sensitive cancer screening method based on the chemotaxis of C. elegans"

di Luccio, Eric, Hirotsu, Takaaki

[International Worm Meeting, 2021]

A simple, affordable, non-invasive, and highly sensitive cancer detection method is the Holy Grail in health screening. Especially early diagnosis of cancer is critical to increase the likelihood of recovery. N-NOSE (Nematode-NOSE) is a pioneering cancer screening method based on the chemotaxis of C. elegans which shows evasive behavior from the urine of healthy individuals while being attracted to the urine of cancer patients. N-NOSE has an average cancer-detection sensitivity of 86.3% in a clinical setting and detect the presence of cancer for 15 types at all stages: stomach, colorectal, lung, breast, uterine, liver, prostate, esophageal, ovarian, bile duct, gallbladder, bladder, kidney, pharyngeal, and pancreatic cancer. N-NOSE relies on a single drop of urine, is non-invasive and cheap, and is notably more sensitive than the blood tumor markers CEA, CA19-9 and CA15-3. In this presentation, we detail the C. elegans olfaction and chemotaxis method behind N-NOSE. Next, we review and summarize our recently published clinical studies on N-NOSE performed in several hospitals in Japan and compare N-NOSE performance to other cancer screening methods. Then, we briefly comment on the next generation of N-NOSE methods currently under development. Lastly, we outline our "World Consortium" R&D grant initiative to anyone in the world with the goal of contributing to the development of next generations of N-NOSE cancer-specific using nematodes. N-NOSE by Hirotsu Bio Science has been launched commercially in 2020 in Japan and is quickly seeking to expand overseas.

Pierce-Shimomura JT et al. (2000) West Coast Worm Meeting "Mutation in the LIM homeobox gene lim-6 disrupts asymmetric function of the ASE chemosensory neurons"

Gaston MR, Lockery SR, Pierce-Shimomura JT, Pearson BJ

[West Coast Worm Meeting, 2000]

C. elegans detects chemicals with 11 pairs of bilaterally symmetric sensory neurons. All of the right-left members of these pairs share similarity in lineage, morphology, and synaptic connectivity. However, one of these pairs (ASE) expresses genes asymmetrically1. ASEL expresses two putative chemoreceptor guanylyl cyclases gcy-6&7 and the homeobox gene lim-6, while ASER expresses a different guanylyl cyclase gcy-5. The ASE neurons are important for chemotaxis to soluble attractants including salts3. We tested whether the asymmetry in expression pattern correlated with an asymmetry in function by ablating ASER, ASEL, or both neurons in individual worms and tracked each animal during chemotaxis in gradients of ammonium chloride, sodium acetate, potassium acetate, and ammonium acetate. To measure chance performance, we tracked worms (n=74) in the absence of a gradient, but analyzed them as if they were in a gradient. Most ASER- (n=29) and ASE- (n=27) worms failed to reach the peak of the NH4Cl gradient, while most ASEL- worms (n=22) reached the peak similar to sham worms (anaesthetized and recovered but not ablated; n=35). Likewise, most ASER- (n=17) and ASE- (n=15) worms failed to reach the peak of the K-acetate gradient, while most ASEL- worms (n=20) reached the peak similar to sham worms. Surprisingly, most ASEL- (n=26) and ASE- (n=12) worms failed to reach the peak of the Na-acetate gradient, while most ASER- worms (n=25) reached the peak similar to sham worms. Worms did not perform better than chance in the ammonium-acetate gradient (n=37). Thus, ASER controls chemotaxis to Cl- and K+, while ASEL controls chemotaxis to Na+. In lim-6 deletion mutants gcy-5 is ectopically expressed in ASEL, while gcy-6&7 expression is correctly restricted to ASEL2. To examine if LIM-6 specifies asymmetry in ASE function, we tested whether ASE neurons retain their asymmetric function in lim-6 mutants. In contrast to wildtype worms, many ASER- lim-6 worms (n=28) were able to reach the peak of the ammonium chloride gradient. Thus, a homeobox gene is involved in breaking symmetry in neuronal function. 1. Yu et al (1997) PNAS 95:3384-7 2. Hobert et al (1999) Dev 126:1547-62 3. Bargmann & Horvitz (1991) Neuron 7:729-42

Muimo R et al. (1995) International C. elegans Meeting "NEMATODE ARYLALKYLAMINE N-ACETYL- TRANSFERASES: PURIFICATION AND CHARACTERISATION FROM ASCARIDIA GALLI"

Muimo R, Isaac RE

[International C. elegans Meeting, 1995]

We have previously shown that nematodes have high levels of arylalkylamine N-acetyltransferase activity and that N-acetylation could be a primary route for regulating biogenic amine levels in nematodes. Three arylalkylamine N-acetyltransferase enzymes have recently been identified and purified to electrophoretic homogeneity from a whole worm homogenate preparation of Ascaridia galli using ammonium sulphate precipitation followed by hydrophobic interaction, metal chelate and ion exchange chromatography. A major peak of activity was associated with a 40 kDa protein band on SDS-PAGE and we now report the characterisation of this enzyme. The enzyme activity was very labile and showed a strong dependency on the presence of dithiothreitol for activity during purification. The enzyme had ability to N-acetylate arylalkyl-, phenyl-, and catechol-amines and ,furthermore, the N-acetylation of 5-HT was inhibited in a dose-dependent manner by amines from these groups. Enzyme activity was maximal at pH 6.5 when measured at 37 oC with tyramine and acetyl CoA as substrates. The enzyme had a high affinity for tyramine (Km 6.6 *M) and was inhibited by Cu2+, Zn2+ and Hg2+ions (1mM). The Km for acetyl CoA was 18.8 *M. Arylamines and hydrazines, substrates for the mammalian liver acetyltransferase enzymes, did not inhibit or markedly affect the enzyme activity towards tyramine indicating that the A. galli arylalkylamine N-acetyltransferase enzyme (40 kDa) is not similar to the mammalian liver arylamine N-acetyltransferase enzymes. The other two arylalkylamine N-acetyltransferase enzyme activities are being characterised and their properties will also be reported.

Rahman, Maisha et al. (2021) International Worm Meeting "Specific N-glycans fine-tune somatosensory dendrite patterning"

Diaz-Balzac, Carlos, Rahman, Maisha, Bulow, Hannes

[International Worm Meeting, 2021]

Dendrites are essential for the transmission and processing of stimuli through the nervous system, and their development requires the precise orchestration of many proteins. For example, the C. elegans PVD somatosensory neurons require a conserved cell-adhesion 'menorin' complex - comprised of skin-derived MNR-1/Menorin and SAX-7/L1CAM, muscle secreted LECT-2/Chondromodulin II, and the transmembrane receptor, DMA-1/LRR-TM, in PVD - for their stereotyped arborization. We found that a key enzyme in the N-glycosylation pathway, AMAN-2/Golgi alpha-mannosidase II, plays a role in fine-tuning PVD dendrite patterning. aman-2/GM-II encodes an evolutionarily conserved enzyme required for the formation of complex and paucimannose N-glycans. Mutations in aman-2/GM-II result in dendritic trees with reduced complexity, and enhance the severity of dendrite defects in hypomorphic alleles of members of the menorin complex. AMAN-2/GM-II requires enzymatic activity in PVD to form higher order branches, suggesting that N-glycosylation of a menorin complex component in PVD itself may be significant. Consistent with this hypothesis, we find that DMA-1/LRR-TM is glycosylated in vivo with primarily high-mannose/hybrid N-glycans, and that DMA-1/LRR-TM carries larger, abnormal N-glycans in aman-2/GM-II mutants. Importantly, we determined that the presence of abnormal N-glycans, rather than the absence of wildtype N-glycans, is the root cause of the observed defects, and likely leads to altered protein-protein interactions. Lastly, we found that specific N-glycosylation sites in DMA-1/LRR-TM are important for PVD dendrite morphogenesis. Collectively, our findings suggest that specific N-glycan structures fine-tune dendrite patterning, possibly by regulating complex formation of the DMA-1/LRR receptor with other components of the menorin complex.

Shinji Ihara et al. (2004) East Asia Worm Meeting "Glycosylation of MIG-17 ADAM protease required for cell migration in C. elgans"

Kiyoji Nishiwaki, Shinji Ihara

[East Asia Worm Meeting, 2004]

Protein glycosylation is widely found in cell surface and secreted proteins. The pattern of glycosylation can be structurally modified or altered during development, carcinogenesis and malignant transformation. The C. elegans MIG-17, a glycoprotein belonging to the ADAM (A Disintegrin And Metalloprotease) family, is secreted from the body wall muscle cells and localize on the gonad surface where it is required for correct migration of the gonadal distal tip cells (DTCs). To investigate the functional importance of N -glycosylation of the ADAM family proteins, we carried out site-directed mutagenesis of N -glycosylation sites of the MIG-17 protein. MIG-17 has nine potential N -glycosylation sites (i.e. NXS/T): six in the prodomain (Asn 52 , Asn 65 , Asn 123 , Asn 172 , Asn 183 , Asn 189 ), and three in the metalloproteinase domain (Asn 218 , Asn 219 , Asn 350 ). The asparagine residues in these sites were changed to glutamines in various combinations to eliminate N -glycosylation in this study. The MIG-17 protein failed to localize on the gonad and cannot rescue the DTC migration defects of mig-17 mutants in the total absence N -glycans. The MIG-17 protein lacking N -glycosylation of the prodomain also did not localize or rescue, but that lacking those of the metalloproteinase domain successfully did. The analysis of the MIG-17 proteins lacking individual N -glycosylation sites revealed that the substitution of either one of Asn 52 , Asn 65 , Asn 172 or Asn 189 in the prodomain partially perturb the rescuing activity of MIG-17. Furthermore, when MIG-17 lacking N -glycosylation of the prodomain was ectopically expressed as a transmembrane form in the DTCs, it successfully directed DTC migration in mig-17 mutants. These results suggest that N -glycosylation of the prodomain is essential to recruit the MIG-17 protein to the gonad surface, but that it is dispensable to control DTC migration after localization of MIG-17.Our findings shed light on an novel function of prodomain glycosylation that is required for targeting secreted ADAM proteins to specific tissues or cells.