Marsili, J. et al. (2017) International Worm Meeting "The nematode Caenorhabditis elegans displays a chemotaxis behavior to tuberculosis-specific odorants."

Nguyen , Q.H., Voisine, C., McFall , S.M., Marsili, J., Neto, M.F., Aldakeel , S.

[International Worm Meeting, 2017]

The World Health Organization's Millennium Development Goal of halting and reversing the tuberculosis (TB) epidemic by 2015 has been met; on average, incidence of TB has fallen 1.5% per year and is now 18% lower than in 2000. Despite these gains, TB remains one of the leading causes of morbidity and mortality globally-in 2014, 9.6 million people fell ill with TB and 1.5 million died from the disease. Each year, approximately one third of the TB infected population remain undiagnosed, equating to roughly 3 million people. A simple, affordable diagnostic test for pulmonary tuberculosis (TB) is urgently needed to improve detection of active Mycobacterium tuberculosis. Recently, it has been suggested that animal behavior can be used as a biosensor to signal the presence of human disease. For example, the giant African pouched rats can detect tuberculosis by sniffing sputum specimens while honeybees respond to three of the four tuberculosis-specific volatile organic compounds (TB-VOCs) detected in the breath of TB positive patients by proboscis extension. However, both rats and honeybees require animal housing facilities and professional trainers, where few laboratories in low-income, high-burden TB settings possess the needed infrastructure. Rather than using trained animals, we propose exploiting the innate sensing capabilities of the nematode Caenorhabditis elegans, which has demonstrated reproducible, behavioral responses to over 100 VOCs. Our data shows that the innate olfactory behavioral response of C. elegans can be used to detect the TB-specific VOCs methyl p-anisate, methyl nicotinate, methyl phenylacetate and o-phenylanisole, in chemotaxis assays. It is important to note, the breath of a TB patient contains a heterogeneous mixture of TB-VOCs, although the proportionality of this blend remains unknown. To further evaluate the efficacy of C. elegans as a potential TB biosensor, we also investigated C. elegans' behavioral response to combinations of TB-VOCs, both pairwise and collectively. We found that C. elegans detects a mixture of all TB-VOCs, exhibiting a significant avoidance behavior. In addition, dauer larvae, a long-lived stress resistant alternative development state of C. elegans in which the animals can survive for extended periods of time in dry conditions with no food, were also demonstrated to detect the VOCs. Taken together, we propose a potential work-flow for a noninvasive diagnostic test for TB that incorporates collecting VOCs from the breath of the patient, exposing dauer larvae housed in a microfluidic device to the VOCs, and recording their response. These results showcase the potential for C. elegans to be incorporated into a new and affordable diagnostic test device for TB and, perhaps, other disease states, capitalizing upon its innate olfactory responses.

Buechner M et al. (1995) International C. elegans Meeting "EXC MUTATIONS AFFECT APICAL CYTOSKELETON"

Buechner M, Hedgecock EM, Hall DH

[International C. elegans Meeting, 1995]

Epithelia have distinct apical and basal surfaces. The apical surface of the excretory canal cell forms a hollow lumen surrounded by an electron- dense cytoplasmic coat throughout the length of the canal processes. The lumen is connected to myriad thin beaded tubules called canaliculi. Electron microscopy and wheat germ agglutinin staining of mutants defective in excretory canal structure reveals defects at the apical surface of this cell. Four classes of defects affecting the lumenal surface are seen: 1) lumena of exc-1, exc- 2, exc-4, exc-5, exc-9, let-4, and let-653 animals swell into large cysts coincident with separation of the apical membrane from its cytoplasmic coat, which bunches up at the ends of the cyst; 2) exc-7 mutants accumulate large endolysosomes throughout the canal, especially at the tips; 3) in exc-3, exc-6, and exc- 8 animals, the lumen splits into multiple lumena; 4) sma-1 animals have abnormally wide lumen and cytoplasm. In contrast, basolateral surfaces and cytoplasm of the mutants are generally unaffected; the canal's gap junctions to the hypoderm are normal, as are the canal basement membrane and microtubules. We hypothesize that these mutations identify components of the apical membrane and its associated cytoskeleton and extracellular matrix, both of the excretory canal and other epithelial cells. Several of these mutants exhibit pleiotropic epithelial defects, including separation of the terminal web from the membrane of the intestinal brush border and cuticular malformations. The let-653 gene encodes a secreted mucin (S.M. Jones & D. Baillie, p.c.), a large glycosylated protein found at many apical surfaces.

P. Meister et al. (2006) European Worm Meeting "Subnuclear Positioning Changes upon Activation of Transcription in C. elegans"

P. Meister, S.M. Gasser, B. Pike

[European Worm Meeting, 2006]

P. Meister, B. Pike and S.M. Gasser. Active and inactive genes are not randomly positioned in the eukaryotic nucleus, yet the impact of their localization is still not completely understood. Moreover, it is not yet established in the context of a whole organism with differentiated cells whether nuclear positioning of genes precedes or results from functional chromatin characteristics such as timing of replication or transcriptional potential. We use the worm to study nuclear dynamics in the context of a multicellular organism with specialized differentiated cells. We created strains expressing GFP-lacI under transcriptional control of an ubiquitous promoter by injection and integration. Surprisingly, embryos and larvae expressing GFP-lacI show two bright nuclear spots per nucleus. By genetic and FISH analyses, we show that the array itself is recognized by the GFP-lacI protein. This is likely to be due to the presence of a single lacO site in the sequence of the plasmids used to create these arrays. Since most of the arrays created for the last 20 years involve plasmids containing lacO sites, this is a powerful technique to identify arrays localization when genes coded by the arrays are either active or inactive. Indeed, quantitative microscopic analysis shows that the positioning of genes varies in the nuclei of differentiated cells depending on the activity of the integrated promoter.. Using this technique, we will now study the localization of a pharyngeal specific gene array in vivo during differentiation of the pharynx. Future studies will relate the DNA replication timing with expression patterns and subnuclear positioning.

Ellen Nollen et al. (2006) European Worm Meeting "C. elegans Models of Protein Misfolding Diseases"

Ellen Nollen, Tjakko van Ham, Ronald H. A. Plasterk.

[European Worm Meeting, 2006]

Ellen Nollen, Tjakko van Ham & Ronald H. A. Plasterk. Aggregation of misfolded proteins occurs in various age-related neurodegenerative disorders, including Parkinsons, Alzheimers, and Huntingtons disease. To understand how cells protect themselves against misfolded proteins, we search for genes that enhance or prevent protein aggregation. C. elegans strains expressing polyglutamine stretches fused to YFP with visible, age-dependend protein aggregation are used as a genetic model. Using a genome-wide RNAi screen, we have previously identified 186 genes that, when knocked down, cause premature protein aggregation. These genes include genes involved in protein synthesis, folding, degradation and RNA synthesis and processing. 1. Conversely, we performed a forward mutagenesis screen to identify genes that, when mutated, suppress age-dependent polyglutamine aggregation. For one suppressor mutant, in which aggregation is suppressed by more than 75%, we have now identified the responsible mutation. This mutation is a missense mutation in a gene encoding a protein of unknown function that is highly conserved between C. elegans and humans. Knock-down by RNAi of the same gene in wild-type worms yielded a similar reduction in aggregation, suggesting a loss-of-function mutation. We are currently further characterizing this mutant and the remaining suppressor mutants. In addition, to establish whether the genes we have identified are specific for polyglutamine aggregation or whether they comprise of a general protein homeostatic buffer, we have developed a worm model for aggregation of alpha synuclein, which occurs in Parkinson''s disease. Altogether our results will provide insight into cellular protection against misfolded proteins and yield targets for therapy against protein misfolding diseases.. 1Nollen E.A.A., Garcia S.M., van Haaften G., Kim S., Chavez A., Morimoto R.I., Plasterk R.H. (2004) Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation. Proc. Natl. Acad. Sci. U.S.A. 101(17):6403-8.

Teixeira, J. et al. (2019) International Worm Meeting "Identification of the ubiquinone pathway as a suppressor of Myotonic Dystrophy-associated toxicity."

Brandao, A., Garcia, S., Teixeira, J., Harju, A.

[International Worm Meeting, 2019]

Myotonic dystrophy type 1 (DM1) is the most common type of dystrophy in adulthood. It is caused by the accumulation of mutant RNA in the nucleus due to the expression of expanded CUG repeats in the 3' UTR of the myotonic dystrophy protein kinase (DMPK) gene. These RNA-bearing CUG repeats form hairpin structures that interact inappropriately to RNA binding proteins such as splicing factors, namely muscleblind-like (Mbnl) and CUG-binding protein (CUGBP) families, thereby causing aberrant alternative splicing leading to a multisystemic disease. Notably, due to the multisystemic nature of DM1, the full extent of cellular processes affected by these toxic RNAs is still unknown. Our aim is to identify new modulators and mechanisms of RNA toxicity. To address this aim, we performed an RNAi screen by using a previously characterized C. elegans DM1 model[1]. Since this C. elegans model mimics DM1 phenotypes, changes in its motility defect were used as readout for toxicity. We identified the ubiquinone (CoQ) pathway as a suppressor of DM1, as downregulation of this pathway increases DM1 toxicity, whereas CoQ supplementation partially rescues the DM1 motility defect. Furthermore, our data also suggest that complex II of mitochondrial electron transport chain is implicated in DM1 dysfunction. The role of mitochondria in DM1 pathogenesis is further underlined by our preliminary results showing that DM1 animals have an altered mitochondrial morphology. Taken together, we established a genetic connection between DM1, ubiquinone pathway and mitochondrial function and are currently examining the mechanisms of DM1 mitochondrial dysfunction regulation. 1. Garcia, S.M., et al., Identification of genes in toxicity pathways of trinucleotide-repeat RNA in C. elegans. Nat Struct Mol Biol, 2014. 21(8): p. 712-20.

Mandy Smith et al. (2001) International C. elegans Meeting "Expression of a C. elegans Mucin-Like Gene"

Samuel M Politz, Mandy Smith

[International C. elegans Meeting, 2001]

Based on the amino acid sequence of a cDNA encoding the major larval surface protein of the parasitic nematode Toxocara canis 1 , plus studies of T. canis surface proteins, it is likely that nematode surface proteins are structurally similar to the mucins that line and protect epithelial cell layers in vertebrate animals. Mucins have two characteristic features: 1. domains rich in serine and threonine, which are extensively O-glycosylated; 2. domains rich in cysteine, which form inter- or intra-molecular disulfide bonds. These features confer special physical and biochemical properties on mucins, such as high solution viscosity and resistance to proteolysis. The epitope recognized by monoclonal antibody M38 is detected on the surface of live C. elegans L1s, and the timing of its expression is altered in srf-6 mutants, which display it in stages L1 through L4. We demonstrated that the M38 epitope is carried by a 30 kD antigen detected in extracts of C. elegans L1s 2 . The antigenicity of this protein is destroyed by pretreatment with O-glycanase, suggesting that it is an O-linked glycoprotein, like the T. canis TES-120 surface antigen. A nematode-specific gene family in C. elegans has been described, members of which encode adjacent serine-threonine-rich and cysteine-rich (SXC or six-cysteine) domains 3 . Because of the likelihood that these encode surface proteins, we have begun to characterize their expression by RT-PCR. For initial studies, we have chosen the predicted genomic coding sequence F41G3.10, which contains 10 exons, each of which encodes a ser-thr-rich sequence followed by an SXC domain. In initial experiments using gene-specific primers, we have amplified a small cDNA fragment spanning 3' terminal exons 9 and 10 in total RNA from mixed stages of C. elegans. We plan to study the stage-specificity of expression of this RNA, as well as map a full-length cDNA representing this transcript. 1 Gems, D. and Maizels, R.M. (1996) Proc. Natl. Acad. Sci. USA (1996) 93: 1665-1670. 2 Hemmer, R.M., Donkin, S.G., Chin, K.J., Grenache, D.G., Bhatt, H., and Politz, S.M. (1991) J. Cell Biol. 115: 1237-1247. 3 Blaxter, M. (1999) Science 282: 2041-2046.

Lu, T. et al. (2019) International Worm Meeting "Characterizing Hect-family ubiquitin ligase during oocyte formation in C. elegans."

Mains , P., Lu, T., Soueid, H., Smit , R.

[International Worm Meeting, 2019]

Mitotic and meiotic spindles differ from one another. Meiotic spindles are short and localized at the cortex to bud off polar bodies, whereas mitotic spindles extend throughout the cell. After fertilization in C. elegans, the oocyte undergoes a rapid meiosis to mitosis transition, in about 10 minutes. Meiotic spindle formation requires katanin, encoded by mei-1 and mei-2, a microtubule-severing complex. Katanin is down-regulated after meiosis by parallel pathways to ensure that it is not active in the subsequent mitotic divisions, during which its expression is lethal. My research focuses on HECD-1 and its method of regulating katanin. HECD-1/HectD1 is a ubiquitin ligase known to change the localization of proteins, while other ubiquitin ligases (CUL-2, CUL-3, MEL-26) that regulate katanin mark the protein for degradation 1. Using immunofluorescence of FLAG tagged HECD-1, which I showed to retain wild-type function, I will look for changes in localization of HECD-1 in mutant backgrounds for katanin and its regulators. As HECD-1 also interacts with the STRIPAK complex in mammals2, I will make double mutants of STRIPAK orthologs (ccm-3, M4.1, otub-2, cash-1, mob-4, C49H3.6, pptr-1) with katanin regulators to identify new players in the katanin pathway. Preliminary findings using RNAi have shown the two genes, otub-2/OTUD7A, a deubiquitinase, and M4.1/SMAP, a sarcolemma associated protein, respectively enhance and suppress the lethality caused by ectopic katanin activity during mitosis. Results have been replicated for M4.1 suppression using double mutants. STRIPAK acts in excretory canal formation and germline development in C. elegans 3,4, but its subunit composition varies, for example, HECD-1 does not act in these processes. As a result, our research will show the specific components of the STRIPAK complex participating in katanin regulation, as well as the interplay between ubiquitin ligases that affect protein levels and those that change protein localization. 1. Beard, S.M., et al. (2016) Development. G3 (Bethesda). 6:3257-3268 2. Tran, H., et al. (2013) J Biol Chem. 288:3753-3767 3. Lant, B., et al. (2015) Nat Commun. 6:6449 4. Pal, S., et al. (2017). Curr Biol. 27:868-876

Lagido, Cristina et al. (2009) International Worm Meeting "Can environmental chemicals which affect mammalian reproduction have an impact on C. elegans physiology?"

Glover, L. Anne, Frost, Andrew, McLaggan, Debbie, Rhind, Stewart M., Amezaga, Maria R., Fowler, Paul A., Lagido, Cristina

[International Worm Meeting, 2009]

Environmental chemicals (ECs), including endocrine disrupting compounds and heavy metals can persist in the environment and have detrimental effects on reproductive, immune and neurological functions and induce cancers1. Assessment of risk posed by ECs is often based on their known individual toxicity and carcinogenicity rather than their endocrine disrupting effects and yet, effects are often associated with much lower concentrations than those resulting in toxic or carcinogenic effects. For example, prolonged exposure of pregnant sheep to a "real-world", complex mixture of ECs in pastures and soils fertilized with sewage sludge, had an adverse effect on fetal ovary development2. The application of sewage sludge to agricultural soils emphasises the need to better evaluate potential risks to public health. We have developed a new tool to rapidly assess effects of environmental and physiological stress. We generated luminescent ATP sensor C. elegans strains through constitutive expression of the firefly luciferase gene and showed that their light output declines in response to knockdown of respiratory genes or treatment with xenobiotics3. We validated luminescence as a toxicological endpoint by showing that the decline in bioluminescence upon exposure to the model toxicant Cd was a sensitive indicator of toxicity, comparable to other conventional sublethal endpoints, such as reproduction and development4. Using our luminescent ATP sensor C. elegans strain, we have recently determined that a sublethal exposure to sewage sludge extract has a concentration-dependent detrimental effect on the worm''s energy status. We also established that sewage sludge affected worm reproduction. We aim to investigate whether conserved pathways are involved in these responses in order to understand potential exposure effects. 1Rhind, S.M. (2008) Reprod Dom Anim 43 (Suppl. 2), 15-22. 2Fowler, P.A. et al. (2008) Mol Hum Reprod 14 (5), 269-280. 3Lagido et al. (2008) BMC Physiol 8(1) :7; Lagido et al. (2001) FEBS Letters 493, 36-39. 4Lagido et al. (2009) Toxicol Sci.

Julia Grabitzki et al. (2006) European Worm Meeting "Phosphorylcholine-Modified Proteins of Caenorhabditis elegans are Specific to Developmental Stages"

Michael Ahrend, Gunter Lochnit, Julia Grabitzki, Rudolf Geyer

[European Worm Meeting, 2006]

Julia Grabitzki, Michael Ahrend, Rudolf Geyer and Gunter Lochnit. The free-living nematode Caenorhabditis elegans has been found to be an excellent model system for developmental studies [1] investigating parasitic nematodes [2] and drug screening [3]. Structural analyses of glycoconjugates derived from this organism revealed the presence of nematode specific glycosphingolipids of the arthro-series, carrying, in part, phosphorylcholine (PC) substituents [2]. PC, a small haptenic molecule, is found in a wide variety of prokaryotic organisms, i. e. bacteria, and in eukaryotic parasites such as nematodes. There is evidence that PC-substituted proteins glycolipids are assumed to be responsible for a variety of immunological effects including invasion mechanisms and long-term persistence of parasites within the host [4]. In contrast to PC-modified glycosphingolipids [5], only a limited number of PC-carrying (glyco)proteins were identified so far [6-9]. We have analysed the expression of PC-modified proteins of C. elegans during developmental stages using two dimensional SDS-Page separation, 2D-Western-blot and MALDI-TOF mass spectrometry. The pattern of PC-modified proteins was found to be stage specific. The PC-modification on proteins was most abundant in the egg and dauer larvae-stages followed by the adult-stage and L4. Only small amounts of the PC-substitution were found in L3 and L2. In L1 we couldnt detect any PC-Modification. The prediction of the cellular localisation of the identified proteins revealed a predominant cytosolic and mitochondrial occurrence of the PC- modification. Most of the identified proteins are involved in metabolism or in protein synthesis.. 1.. Brenner, S., Genetics, 1974. 77(1): p. 71-94.. 2.. Lochnit, G., R.D. Dennis, and R. Geyer, Biol Chem, 2000. 381(9-10): p. 839-47.. 3.. Lochnit, G., R. Bongaarts, and R. Geyer, Int J Parasitol, 2005. 35(8): p. 911-23.. 4.. Harnett, W. and M.M. Harnett, Mod. Asp. Immunobiol., 2000. 1(2): p. 40-42.. 5.. Friedl, C.H., G. Lochnit, R. Geyer, M. Karas, and U. Bahr, Anal Biochem, 2000. 284(2): p. 279-87.. 6.. Haslam, S.M., H.R. Morris, and A. Dell, Trends Parasitol, 2001. 17(5): p. 231-5.. 7.. Cipollo, J.F., C.E. Costello, and C.B. Hirschberg, J Biol Chem, 2002. 277(51): p. 49143-57.. 8.. Cipollo, J.F., A.M. Awad, C.E. Costello, and C.B. Hirschberg, J Biol Chem, 2005. 280(28): p. 26063-72.

T.J. van Ham et al. (2007) International Worm Meeting "A C. elegans model links novel modifiers of a-synuclein inclusion formation to aging."

K.L. Thijssen, E.A.A. Nollen, T.J. van Ham, R.H.A. Plasterk

[International Worm Meeting, 2007]

Accumulation and aggregation of <font face=symbol>a</font>-synuclein in the brain is characteristic of, and considered to be causatively related to, several age-related neurodegenerative diseases such as Parkinsons disease (PD). To study early stages of <font face=symbol>a</font>S accumulation and to identify modifier genes, we created transgenic C. elegans expressing the human <font face=symbol>a</font>S gene fused to YFP in the body wall muscle. These transgenic animals show localization of <font face=symbol>a</font>S-YFP to discrete foci during aging, which is not seen for YFP alone, indicating specificity to <font face=symbol>a</font>S. These foci are present as early as day two after hatching, and become more abundant during aging. By fluorescence recovery after photobleaching (FRAP) we determined that the foci contain mobile material until day 11. However, at old age also immobilized aggregated protein is present. The amount of <font face=symbol>a</font>S-foci increases to day 11 and decreases thereafter. Interestingly, this decrease in foci formation is associated with a relative increase in the amount of immobilized aggregates. We performed a genome-wide RNAi screen for modifiers of foci formation, and found 108 genes that repeatedly showed increased foci formation when silenced. Remarkably, none of these genes overlaps with genes found in a screen for polyglutamine aggregation we performed in C. elegans1. This might indicate that different mechanisms are involved in these diseases. To further characterize genes found in the screen, deletion strains of a selection of the genes representing different functional classes were crossed into the <font face=symbol>a</font>S-YFP strain. Deletion of a gene - involved in aging and encoding one of the most potent suppressors of foci-formation - resulted in a twofold increase in the amount of foci on day thirteen. We are currently further characterizing the deletion strains. In conclusion, we developed a new animal model that captures features of age-related PD. We used the model to dentify novel modifiers of <font face=symbol>a</font>S-YFP foci formation, of which we are further characterizing the effect of deletion of these genes on <font face=symbol>a</font>S-YFP foci formation. These results can provide insight into the early cellular mechanisms involved in <font face=symbol>a</font>S accumulation, and could thereby yield molecular targets for therapy. 1 Nollen E.A.A., Garcia S.M., van Haaften G., Kim S., Chavez A., Morimoto R.I., Plasterk R.H.A. (2004) Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation. Proc. Natl. Acad. Sci. U.S.A. 101(17):6403-8.