Green, R.A.* et al. (2017) International Worm Meeting "CYK-4 functions independently of kinesin-6 to promote intercellular bridge closure during oocyte formation in the syncytial C. elegans germline."

Lee, K.Y.*, Desai, A.B., Gutierrez, E, Groisman, A, Green, R.A.*, Oegema, K.F., Gomez-Cavasos, J.S.

[International Worm Meeting, 2017]

In metazoa, germ cells undergo incomplete cytokinesis to form syncytia with cells connected by intercellular bridges. Centralspindlin, a complex containing two molecules of kinesin-6 and two of the Rho family GTPase-activating protein (GAP) CYK-4, is a conserved bridge component. Here, we analyze centralspindlin function in the C. elegans oogenic germline, which contains ~1000 cellular compartments connected by intercellular bridges to a cytoplasmic core. In contrast to cytokinesis, where both centralspindlin components are essential for spindle midzone and contractile ring assembly, CYK-4, but not kinesin-6, is required for a germline cytokinesis-like event where intracellular bridges close to bud oocytes off the syncytium. The CYK-4 C1 domain, predicted to interact with the plasma membrane, and GAP domain interface predicted to interact with Rho family GTPases were both required to recruit CYK-4 to intercellular bridges. Knockdown of RhoA, but not Cdc42 or Rac, disrupted germline structure, suggesting that the GAP domain recruits CYK-4 to intercellular bridges by binding RhoA.

Jolanta Polanowska et al. (2006) European Worm Meeting "Regulation of BRC-1 - Dependent Ubiquitylation at DNA Damage Sites"

Jolanta Polanowska, Simon Boulton, Tatiana Garcia-Muse, Julie Martin

[European Worm Meeting, 2006]

Jolanta Polanowska1,2, Julie Martin1, Tatiana Garcia-Muse1 and Simon Boulton1. The BRCA1 tumour suppressor and its heterodimeric partner BARD1 constitute an E3-ubiquitin (Ub) ligase and function in DNA repair by unknown mechanisms. We have previously described C. elegans BRCA1 and BARD1 orthologues (brc-1 and brd-1, respectively) that possess many of the functional domains present in their human counterparts, including RING, ankyrin, and BRCT domains (Boulton et al., 2004). Consistent with conserved roles in DNA repair, BRC-1 and BRD-1 interact to form a heterodimer via their respective RING domains. To explore the mechanistic role of BRC-1 and BRD-1 in DNA repair processes we have characterized a C. elegans BRC-1/BRD-1 complex (CeBCD) purified by tandem immunoaffinity before and at different time points after IR-treatment. This approach is a first for C. elegans and demonstrates that protein complexes purified in this manner are amenable to biochemical analysis and can be used in combination with genetics and cell biology to accelerate functional discoveries. We present evidence that the CeBCD complex possesses an E3-Ub ligase that is activated on chromatin in response to IR-treatment and further demonstrate that the DNA damage checkpoint promotes association of the CeBCD complex with E2-Ub conjugating enzyme, Ubc5(LET-70), to form an active E3-Ub ligase in response to DNA damage. We also show that ubiquitylation events at DNA damage sites require brc-1, brd-1, ubc5(let-70), mre-11 and atl-1, thus providing in vivo evidence to support our biochemical analysis.. Boulton, S.J., Martin, J.S., Polanowska, J., Hill, D.E., Gartner, A. and Vidal, M. (2004) Curr Biol, 14, 33-39.

Lee, J. et al. (2019) International Worm Meeting "Y92H12A.4(ints-3), a subunit of the Integrator complex, acts in AFD thermosensory neurons to support a behavioral preference."

Almoril-Porras, A., Colon-Ramos, D., Lee, J., Hawk, J.

[International Worm Meeting, 2019]

A fundamental question in neuroscience is to elucidate the cellular and molecular changes that occur during learning. To address this, we are using C. elegans thermotaxis behavior as a model to identify molecules that play a role in learning. C. elegans does not have an innate preferred temperature; instead, worms will migrate on a thermal gradient toward their previously experienced temperature conditions. Previously, PKC-1 has been identified as a key molecule that bidirectionally regulates temperature preference independent of experience: pkc-1(lof) animals are constitutively thermophilic, while pkc-1(gof) animals are constitutively cryophilic (Okochi et al. 2005). In order to identify interactors of PKC-1, we conducted a forward mutagenesis screen on pkc-1(gof) animals and isolated one allele, ola295, that robustly suppressed their constitutively cryophilic behavior. Using SNP mapping, whole genome sequencing, and CRISPR rescue, we identified ola295 as a nonsense allele of Y92H12A.4(ints-3), a subunit of the Integrator complex, which was recently shown in C. elegans to play a conserved role in small nuclear RNA (snRNA) processing (Gomez-Orte et al. 2019). Cell-specific rescue experiments suggest Y92H12A.4 acts in AFD thermosensory neurons to support the constitutive cryophilic behavior of pkc-1(gof) animals. Y92H12A.4(ola295) animals show decreased GCaMP amplitudes in AFD neurons and an increased response penetrance in the postsynaptic AIY neurons in response to temperature stimuli, suggesting that the allele results in the disinhibition of AIY neurons to suppress pkc-1(gof) cryophilic behavior. We are now investigating whether this suppression arises from a shift in the gain of AFD sensory responses through snRNA-dependent mechanisms.

REMY J.J. (2003) International Worm Meeting "LONG TERM MEMORY OF THE NATAL CHEMOSENSORY WORLD IN NEMATODES"

REMY J.J.

[International Worm Meeting, 2003]

The nematode C. elegans rely on a limited number of neurons to sense chemicals, each of it expressing several G-coupled chemoreceptors out of more than 800 found in the genome. Since neurons direct behavior and not the receptors they express, worm responses to chemicals will depend on the chemoreceptors expression pattern in the different chemosensory neurons. As in other species, expression of these genes in nematodes can start very early during development, before they play their role in adult sensing and behavior. We found different strains of C. elegans able to stay sensitized to odorants to which they have been shortly exposed from embryogenesis to the L1 larval stage. Sensitization by early exposure must be food or serotonine associated and lasts for the whole reproductive life. It is however not erasable by starvation at the adult stage. This form of experience-dependent sensory adaptation can be highly specific for different odorants concentrations, even if they are sensed by the same olfactory neuron, and is significantly enhanced in daf-22(ml30) worms that does not secrete pheromone, the only known repressor of chemoreceptor expression (1). By contrast, the sensory processing interneurone AIY seems necessary since the two ttx-3(ot22) and ttx-3(ks5) mutants (2) were found defective for odor imprinting, as were found the themosensory mutants sel-12(ar131), sel-12(ar171), and ncs-1(qa406) (3, 4). All together, these data suggest that the nematode natal chemosensory world can be permanently imprinted, if food or serotonine associated, possibly through ligand-dependent chemoreceptors expression mechanisms. The biological significance of this form of sensory plasticity will be discussed. 1) Peckol EL et al., PNAS 2001, 98, 11032-11038 2) Hobert O et al., Neuron 1997, 19, 345-357 3) Levitan D and Greenwald I. Nature 1995, 377, 351-4 4) Gomez M et al., Neuron 2001, 30, 241-248

Daniel C. Koboldt et al. (2007) International Worm Meeting "High-throughput identification of structural variations from C. briggsae sequence traces."

Julia E. Staisch, Raymond D. Miller, Daniel C. Koboldt

[International Worm Meeting, 2007]

Recent reports of widespread copy number differences including large insertion/deletions (indels) have demonstrated that this variation affects such substantial portions of the human genome that it is likely to play a significant role in human disease. C. elegans strains also harbor large indels (Maydan, J.S. et al., 2007, Genome Res 17:337). While comparative genome hybridization and SNP genotyping technologies have been adapted to detect structural variants, doing so at the nucleotide level remains a challenge. We have developed a method that allows for the high-throughput identification of structural variation from sequence trace data. We tested the method using sequence traces from C. briggsae strain HK104, which has a SNP on average every 115 bp when compared to the canonical strain (AF16). Each set of sequence traces was used to create a BLAST database against which the reference genome was aligned using WU-BLAST. The BLAST results were parsed and filtered to identify reads that appear to span an indel. Alignment positions and orientations were analyzed to characterize the size and precise molecular boundaries of the apparent indels. From a database of 13,632 sequencing traces from strain HK104 we identified 399 candidate indel events of 50 - 1,000 bp, 62 indels of 1-10 kbp, and a number of larger indels. We chose an initial test set of two indels for each of the six chromosomes, and designed PCR assays for them. Nine of the 12 PCR assays were successful under uniform conditions and each of the nine reflected the predicted indel. We also designed and successfully used three FP-TDI genotyping assays for the indels. Additional candidates are being tested. Our method is both efficient and sensitive. It has shown that large indels are common in C. briggsae. The large indels are very useful as tools for mapping studies and they raise the question about their effects on gene expression.

Yu, Xinwei et al. (2021) International Worm Meeting "Fast deep learning correspondence for neuron tracking and identification in C. elegans using semi-synthetic training"

Sharma, Anuj K., Linderman, Scott W., Randi, Francesco, Yu, Xinwei, Leifer, Andrew M., Creamer, Matthew S.

[International Worm Meeting, 2021]

The nervous system of the nematode C.elegans is well characterized, such that each of the 302 neurons is named and has stereotyped locations across animals(Witvliet et al., 2020). The capability to find corresponding neurons across animals is essential to investigate neural coding and neural dynamics across animals. Despite the worm's overall stereotypy, the variability in neurons' spatial arrangement is sufficient to make predicting neural correspondence a challenge. We present an automated method to track and identify neurons in C. elegans, called "fast Deep Learning Correspondence" or fDLC, based on the transformer network architecture(Vaswani et al., 2017). The transformer has shown great success in natural language processing tasks by modeling the dependencies between words in a sentence. We reasoned this same architecture would be well-suited to extract spatial relationships between neurons in order to build a representation that facilitates finding correspondence to neurons in a template worm. The model is trained once on empirically derived semi-synthetic data and then predicts neural correspondence across held-out real animals via transfer learning. The same pre-trained model both tracks neurons across time and identifies corresponding neurons across individuals. Performance is evaluated against hand-annotated datasets, including NeuroPAL(Yemini et al., 2020). Using only position information, the method achieves 80.0% accuracy at tracking neurons within an individual and 65.8% accuracy at identifying neurons across individuals. Accuracy is even higher on a published dataset(Chaudhary et al., 2021). Accuracy reaches 76.5% when using color information from NeuroPAL. Unlike previous methods, fDLC does not require straightening or transforming the animal into a canonical coordinate system. The method is fast and predicts correspondence in 10 ms making it suitable for future real-time applications. Witvliet D, Mulcahy B, Mitchell JK, Meirovitch Y, Berger DR, Wu Y, et al.Connectomes across development reveal principles of brain maturation in C.elegans. bioRxiv. 2020;doi:10.1101/2020.04.30.066209. Vaswani A, Shazeer N, Parmar N, Uszkoreit J, Jones L, Gomez AN, et al.Attention is All You Need; 2017.Available from:https://arxiv.org/pdf/1706.03762.pdf. Yemini E, Lin A, Nejatbakhsh A, Varol E, Sun R, Mena GE, et al. NeuroPAL: A multicolor atlas for whole-brain neuronal identification in C. Elegans. Cell.2020;doi:10.1016/j.cell.2020.12.012. Chaudhary S, Lee SA, Li Y, Patel DS, Lu H. Graphical-model framework for automated annotation of cell identities in dense cellular images. eLife.2021;10:e60321. doi:10.7554/eLife.60321.

Paola Jurado et al. (2007) International Worm Meeting "Insights into the molecular mechanisms of decision making."

Paola Jurado, Ikue Mori

[International Worm Meeting, 2007]

Thermotaxis is an elaborated but well characterised behaviour in C. elegans. After cultivation with food at a certain temperature, the worms are able to associate this temperature with the presence or absence of food1,2. If the conditioned animals are then transferred to a new plate with a temperature gradient, they migrate to the previously conditioned temperature, even in the lack of E. coli, where they stay for a limited period of time. Thermotaxis is therefore an ideal scenario to investigate the molecular basis of the integration of sensory inputs that lead to complex neural functions such as memory or learning. Some mutants involved in these processes have been described. The aho (abnormal hunger orientation) animals show defects in learning induced by two paired stimuli, temperature and absence of food2. Over-expression of the neuron-specific calcium sensor-1 (NCS-1) accelerates temperature learning and gives rise to a longer lasting memory3. We are interested in how the integration of sensory inputs leads to memory, and how this memory can be modified by the appearance of a novel input which might cause a new decision to arise. To approach this issue, we are looking for mutants affected in this integration process, especially for mutants with impairments on making a new decision. We are currently searching for animals that once they have been conditioned to find E. coli at a certain temperature, do not, or take longer on leaving this temperature even after an unsuccessful search for food. We have assayed some known mutants for this phenotype. Worms with a mutation in the sensory signal integration gene hen-1, codifying for a secretory protein with an LDL receptor motif4, seem to stay longer than wild type animals at their conditioned temperature. After EMS mutagenesis, we started to perform a screening in which we found several putative mutants presenting the desired phenotype. These mutants are currently under characterisation and we shall report the results in the meeting. 1.Mori, I. and Y. Ohshima (1995). Nature 376(6538): 344-8. 2.Mohri, A., et al. (2005). Genetics 169(3): 1437-50. 3.Gomez, M., et al. (2001). Neuron 30(1): 241-8. 4.Ishihara, T., et al. (2002). Cell 109(5): 639-49.

Ridolfi, Verena Alexia et al. (2021) International Worm Meeting "Downregulation of SEMO-1, a novel hydrogen sulfide-generating C. elegans enzyme, enhances lifespan: role of AAK-1/-2"

Priebs, Josephine, Ridolfi, Verena Alexia, Philipp, Thilo Magnus, Kohnlein, Karl, Klotz, Lars-Oliver, Steinbrenner, Holger

[International Worm Meeting, 2021]

The C. elegans ortholog of human selenium-binding protein 1 (SELENBP1), Y37A1B.5, is a pro-aging factor that confers resistance to high doses of selenite (1). SELENBP1 was recently identified as a methanethiol oxidase (MTO), catalyzing the conversion of methanethiol to hydrogen sulfide (H2S), hydrogen peroxide (H2O2) and formaldehyde. Here, we tested whether Y37A1B.5 has MTO activity and whether the AMPK orthologs AAK-1/-2 are involved in the effects of the protein on C. elegans lifespan. We developed an MTO activity assay that is based on in situ-generation of methanethiol from methionine as catalyzed by a bacterial recombinant L-methionine gamma-lyase, followed by detection of two methanethiol oxidation products, H2S and H2O2. Using this assay, we demonstrate MTO activity of isolated recombinant Y37A1B.5, similar to recombinant human SELENBP1. Moreover, MTO activity was detected in lysates from wild-type nematodes but not in lysates from a newly generated Y37A1B.5-deficient mutant strain, suggesting that the Y37A1B.5 protein is the major C. elegans MTO. Thus, Y37A1B.5 was named SEMO-1 (SELENBP1 ortholog with MTO activity). It is a novel methanethiol oxidase and therefore a novel potential source of H2S and H2O2, two molecules known to affect lifespan in C. elegans. A Y37A1B.5/SEMO-1-deficient mutant strain showed an extended lifespan similar to the previously reported worms exposed to Y37A1B.5-specific RNAi (1). SEMO-1, therefore, is a factor apparently shortening C. elegans lifespan. Interestingly, lifespan extension following SEMO-1 depletion was abrogated in an AAK-deficient strain (NB245; deficient in both isoforms of the catalytic AAK subunit), and vice versa, SEMO-1 depletion through RNAi appeared to enhance AAK phosphorylation in wild-type worms. As AAK activity is known to be related to C. elegans lifespan, we propose that the extended lifespan of SEMO-1-depleted worms is caused by AAK activation. The mode of AAK activation following SEMO-1 depletion remains to be identified. (1) Kohnlein K, Urban N, Guerrero-Gomez D, Steinbrenner H, Urbánek P, Priebs J, Koch P, Kaether C, Miranda-Vizuete A, Klotz LO. Redox Biol. 28:101323 (2020).

Hannes E Buelow et al. (2005) International Worm Meeting "Complex heparan sulfate modifications instructively affect axon guidance choices"

Hannes E Buelow, Dominic Didiano, Oliver Hobert

[International Worm Meeting, 2005]

Heparan sulfate proteoglycans (HSPG) are components of the extracellular matrix through which axons navigate to reach their target cells. The heparan sulfate (HS) side chains of HSPGs show complex and differentially regulated patterns of secondary modifications, including sulfations of distinct hydroxyl groups and epimerization of an asymmetric carbon atom. These modifications endow the HSPG-containing extracellular matrix with the potential to code for an enormous molecular diversity (1). We sought to decode this potential diversity by analyzing mutations in three C.elegans enzymes that catalyze secondary modifications in HS, namely the glucuronyl C5 epimerase, the heparan 6O-sulfotransferase and 2O-sulfotransferase. Each of the mutants are viable yet exhibit distinct as well as overlapping axonal and cellular guidance defects in specific subsets of neuron classes, demonstrating that different modifications are crucial for distinct aspects of neuronal development. We have linked individual HS modifications to two specific guidance systems, the Slit/Robo and kal-1/Anosmin-1 system. We show that the activity of both guidance systems is dependent on different HS modifications in different cellular contexts (2). To distinguish whether combinations of HS modifications have a permissive or instructive role in axon guidance choices we misexpressed specific HS modifying enzymes. These experiments establish that specific misexpression of HS modifying enzymes (presumably leading to aberrant HS modification patterns) elicit defined axonal defects in some neurons but not in others indicating that specifically modified HS has the potential to play an instructive role during neuronal development. Using genetic and molecular approaches we have begun to identify the genes that define this instructive role of HS and that respond to it. Our results demonstrate that the molecular diversity in HS encodes information leading us to propose the existence of a heparan sulfate code that instructively controls different aspects of nervous system development. (1) Lee, J.S., and Chien, C.B. (2004). When sugars guide axons: insights from heparan sulphate proteoglycan mutants. Nat. Rev. Genet. 5, 923-935. (2) Blow, H.E., and Hobert, O. ( 2004). Differential sulfations and epimerization define heparan sulfate specificity in nervous system development. Neuron 41, 723-736.

Karen J Yook et al. (2005) International Worm Meeting "Using Mos1 to catch a bus."

Jonathan Hodgkin, Karen J Yook

[International Worm Meeting, 2005]

All life must defend itself against pathogens. C. elegans does not have an adaptive immune system and homologues of key components of the conserved innate immune Toll pathway are missing from its genome, so how does this animal cope with pathogens? To address this question, we are using forward genetic analysis to identify the molecular components of the worm's response to a bacterial infection caused by the host specific bacterium, Microbacterium nematophilum. Worms that come into contact with M. nem. become constipated, develop a swelling of the post-anal region (the Deformed anal region phenotype) and exhibit a reduced growth rate. Previous screens using EMS mutagenesis and mut-7 activation of endogenous transposons have demonstrated that over 20 loci can be mutated to inhibit the swelling response to M. nem. These loci were named bus for bacterially unswollen. We used Mos1 transposon mutagenesis to molecularly identify 4 additional bus loci. Mos1 insertions in four loci, tax-4, egl-8, bus-19 and bus-20, independently correlate with a reduction in the swelling response to M. nem.. Much work has been done to investigate tax-4 and egl-8 function in the worm, but their role in pathogen-induced anal swelling is not easily interpreted. bus-19 and bus-20 define loci which have not been previously characterised. bus-19 is predicted to encode a highly conserved transmembrane protein. bus-20 is predicted to encode a galactosyltransferase in the Glycosyltransferase 31 family. Other members of this family include bre-5 and bre-2, which have been shown to be required for Cry5B pore-forming toxin susceptibility [1, 2]. An initial analysis of these four bus mutants suggests that the corresponding genes are involved in setting up or maintaining cuticle properties of the worm (e.g. bus-19, bus-20), in signaling the swelling response (e.g. egl-8), or possibly in both processes (e.g. tax-4). More information about these bus genes will be presented at the meeting. 1.Griffitts, J.S., et al.,Science, 2001. 293(5531): p. 860-4. 2.Marroquin, L.D., et al., Genetics, 2000. 155(4): p. 1693-9.