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[
International Worm Meeting,
2017]
Glycosyltransferases (GTs) are a broad class of proteins involved in the transfer of a glycosyl group from a donor molecule to an acceptor. As such, they are involved in a wide range of biological functions through their roles in glycosylation modifications, synthesis of cellular receptors, biosynthesis of polysaccharides, glycolipids and glycoproteins. In C. elegans, GTs have been implicated to play roles in development, cuticle formation, detoxification, signaling and other pathways. However, only around 20% of the more than 260 GTs have been characterized. The large expansion of some of the GT families, presence of unique sugars and pathways in worms and the unknown specific roles of GTs in the implicated pathways pose specific questions and challenges that require a deeper understanding of the functional units of these GTs in C. elegans. We used a Bayesian statistical approach to align and classify more than 250,000 GT sequences across all taxonomic groups into functional categories based on the patterns of conservation and variation in large multiple sequence alignments. We use patterns unique to each GT family as a conceptual starting point for investigating their sequence-structure-function relationships. Implementing these methods, we can further pinpoint contrasting, similar and co-evolved features that differentiate, associate and functionally relate the GT families respectively. We will present an initial analysis that highlights the presence of co-conserved features that distinguish multiple GT families, highlighting GTs in worms. We have generated a phylogenetic classification based on these features and mapped phenotypic associations for these families based on literature. Our analysis reveals several expanded and unique GT families in C. elegans with limited information that we hypothesize might be involved in detoxification, signaling and other pathways unique to worms. Using the co-conserved features as a starting point, we further investigate structural data to pinpoint targets for mutational and metabolomic studies. An initial metabolomic analysis by others in the lab of select GT mutants in C. elegans is revealing specific features that are statistically different, suggesting family specific phenotypic changes. Informatic analysis layered with multiple data sources from literature serve as a tool to identify targets and derive hypotheses to conduct deeper metabolomic studies that can help elucidate the functional changes and biological activity of the associated GT families.
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[
International Worm Meeting,
2021]
The Ras small GTPase is the most mutated oncoprotein, driving both tumorigenesis and metastasis. Oncogenic Ras effectors Raf and PI3 Kinase cascades are well studied and pharmacologically targeted. Ras directly binds a third oncogenic effector, RalGEF, which in turn activates a "Ras-Like" cousin, Ral. Yet the downstream consequences of RalGEF-Ral activation are poorly understood. Consequently, we are studying the functions of RalGEF-Ral in C. elegans development. We found that mutations of RalGEF and Ral enhance migration phenotypes of mutants in genes with established roles in cell migrations. We tested these findings by using the canal associated neurons (CANs) as our model, while validating with HSN cell migration, neurite guidance, and animal locomotion. We found that Ral functions cell autonomously as a permissive developmental signal. Ras, the canonical activator of RalGEF>Ral in cancer, functions as an instructive signal. Unexpectedly, we identified a function for Rap1, a close relative of Ras, in the same process RalGEF>Ral, suggesting that Rap1 and not Ras activates RalGEF>Ral. These findings demonstrate the role of canonical RalGEF>Ral signaling in development of the nervous system, and implicate an unexpected upstream activator, Rap1.
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[
International Worm Meeting,
2021]
Inappropriate activation of the Ras>Raf>MEK>ERK signaling cascade is a common driver in both cancer and RASopathies, and as such represents an attractive target for development of therapeutics. The terminal MAP kinase, extracellular signal-regulated kinase (ERK), has been studied extensively in vitro and ex-vivo with cultured cells but very little in vivo. We utilized C. elegans and CRISPR genome editing to tag the endogenous C. elegans ERK-encoding gene,
mpk-1. We observed endogenous MPK-1::mKate2 protein to be ubiquitously expressed, with elevated expression at L2 and L3 stages and in a distinct set of tissues. We validated our tool as a reporter of upstream activation by observing cytosol-to-nuclear translocation of MPK-1 in the most proximal maturing oocyte, a phenotype that was enhanced by deletion of the negative regulator
rskn-1. We next examined MPK-1 nuclear recruitment during patterning of the vulval precursor cells (VPCs). MPK-1 has been shown to be necessary and sufficient for the central VPC, P6.p, to assume 1° fate during developmental patterning of the six VPCs. Unexpectedly, during VPC induction we observed MPK-1 to translocate into the nuclei of all six VPCs in a temporal and concentration gradient centered on P6.p. This observation contrasts with previous results using the ERK-nKTR reporter of substrate activation. Our reagent raises some interesting questions about mechanisms and indicators of MPK-1 activation and may provide new insights into regulation of MPK-1 activation in vivo.
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Zhang, S., Gouveia, G., Tayyari, F., Bifarin, O.O., Edison, A.S., Taujale, R.
[
International Worm Meeting,
2017]
Glycosyltransferases (GTs) catalyze the transfer of glycosyl groups from sugar substrate donors to a host of acceptor substrates - oligosaccharides, monosaccharides, proteins, lipids, nucleic acids, and other small molecules; effectively GTs catalyze glycosidic bond formation between these multifarious possibilities of molecules. Numerous studies show that GTs are important for developmental and physiological processes in C. elegans (Berninsone, 2006). However, most GTs have not yet been empirically validated. C. elegans utilizes GTs for critical functions, including pheromone signaling with ascarosides and the detoxification pathways, though identities of GTs regulating these functions remain unknown. C. elegans controls much of its behavior and development through the use of ascarosides, which are also present in many free-living and parasitic nematodes (Choe et al 2012). Some of the phenotypes mediated by ascarosides include aggregation, olfactory plasticity, dauer formation, attraction behavior, and hermaphrodite behavior. (Srinivasan et al 2008; Edison, 2009; Ludewig & Schroeder, 2013) Chemically, ascarosides are glycosides of the dideoxy sugar ascarylose, attached to a fatty acid side chain, thus implicating GTs in their biosynthesis. In addition, the innate immune system in C. elegans utilizes a wide range of immune effectors and enzymes (including GTs) for microbial defenses and xenobiotics detoxification (Lindblom & Dodd, 2006; Stupp et al 2012). Stupp et al 2012 showed that C. elegans can detoxify two bacterial toxins, 1-hydroxyphenazine (1-HP), and indole via N- and O-glycosylation. Our research aims to discover the roles of specific GTs in biological processes like these. In this exploratory study, we selected a dozen GT mutant strains, the majority of which belong to the GT-A fold protein (families 2, 7, 21, 27, and 13). 6 replicates of each strains (L1 stage) were cultured to about a population of 100-200 thousand for NMR metabolomics measurements, and animals were randomly selected from each sample for the measurement of population distribution using the large particle flow cytometer COPAS Biosorter. The Biosorter measures the extinction and time of flight of individual nematode which is used as a descriptor of developmental stages. Analysis of metabolic changes of some of the GT mutants and the association with the population distribution will be reported.
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[
International Worm Meeting,
2021]
Lateral specialization of the central nervous system is a well-established feature across species, yet the underlying mechanism through which functional asymmetry arises is largely unknown. EM reconstruction of the C. elegans connectome found that ASE to AWC synapses were stronger on the left than the right. To corroborate and elucidate this asymmetric connection, we generated a reporter strain labelling ASE->AWC connections using in vivo Biotin Labelling of Intercellular Contact (iBLINC). While we observed the same left-sided bias of this connection, we also discovered that the asymmetric fates of the ASEs are a necessary but not sufficient factor in establishing this left-side bias. Furthermore, we found that
ins-6/insulin-like is involved in the establishment of this asymmetric connection. Using a fosmid-based
ins-6::GFP reporter, we observed that
ins-6 expression in ASJ also exhibits left-sided bias. The asymmetry of ASE to AWC iBLINC signal is abolished in cell-specific knock-out animals of
ins-6 in ASJ but not in ASI. Moreover, genetically removing
ins-6 in ASJL by use of
tbx-37p::Cre reversed the asymmetry of the ASE to AWC connection. Meanwhile, removing the putative
ins-6 receptor
daf-2 in ASEL but not ASER symmetrized the ASE to AWC connection. Finally, we observed that mutation on an antagonistic insulin,
ins-22, partially suppressed the phenotype of
ins-6 mutants. These results taken together suggest that the left-side bias of ASE to AWC connection is controlled by insulin signaling, where asymmetrically expressed insulin-like molecules from ASJs act locally to regulate connectivity of the ASE>AWC synaptic connection. We aim to further investigate the effect of insulin signaling on the plasticity of ASE to AWC connection and general synapse dynamics. We also aim to characterize the previously unreported asymmetric gene expression in ASJ
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[
Neuronal Development, Synaptic Function, and Behavior Meeting,
2006]
We obtained in vivo intracellular recordings from the amphid sensory neuron pair AFD. Nematodes whose AFD neurons are laser ablated fail to track their cultivation temperature (Mori & Ohshima, 1995, Nature 376:344) and Ca2+ imaging has revealed increases in intracellular Ca2+ in AFD in response to warming (Kimura et al., 2004, Current Biology 14:1291), identifying AFD as a likely thermosensor. Consistent with this idea, we find that AFD responds to changes in temperature: warming elicits an inward current and cooling an outward current. These thermoreceptor currents (TRCs) activate rapidly and are sufficient to modulate the cell"s membrane potential by more than 20 mV. Both inward and outward TRCs are retained in the absence of intracellular K+, suggesting that K+ channels do not play a role in generating TRCs. TRCs were not detected in AWA, an amphid sensory neuron pair that synapse onto AFD, indicating that TRCs are a specific property of AFD.
Nematodes migrate down temperature gradients when placed above a threshold temperature. This threshold for thermotaxis is set by the worms" cultivation temperature (Tc) and can be reset within ~2 hours by shifting worms to a new Tc. Paralleling the behavior, TRCs in AFD activate above a threshold temperature defined by recent experience. Interestingly, the sensory neuron adapts far more rapidly than the behavior. We estimate that thresholds for TRC activation can be reset in less than 15 minutes. The behavioral and sensory responses also differ in their dependence on feeding state: whereas animals starved for >3 hours cease to perform thermotaxis, TRC"s are preserved in animals starved for >10 hours. Our results support the hypothesis that AFD is a temperature-sensing neuron, and provide direct insight into how temperature is encoded in the C. elegans nervous system
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Herzog, Margareta, Uyar, Bora, Rajewsky, Nikolaus, Akalin, Altuna, Theil, Kathrin, Froehlich, Jonathan, Glazar, Petar
[
International Worm Meeting,
2021]
Understanding how regulatory sequences control gene expression is fundamental to explain how phenotypes arise in health and disease. The functions of regulatory elements must ultimately be understood within their genomic environment and developmental- or tissue-specific contexts. Here, we used induced Cas9 expression and multiplexed guide RNAs in C. elegans to create hundreds of mutations in enhancers, promoters and 3′ UTRs of 16 genes in parallel. We then analyzed the resulting complex populations by either selecting for phenotypic traits or reporter expression, or by DNA- or RNA amplicon sequencing of bulk samples. We developed a software pipeline, crispr-DART, to analyze targeted sequencing and describe the characteristics of >12,000 dsDNA break-induced indel mutations. We also analyzed the
lin-41 3′ UTR and found that the two
let-7 miRNA binding sites can function independently and that one of the sites is more important for mRNA repression and phenotype. In summary, our approach enables highly parallelized functional analysis of regulatory sequences in vivo.
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[
International Worm Meeting,
2021]
What if we could rapidly and reliably homozygose every locus in the C. elegans genome during genetic manipulations? What if we could combine the full nuclear information from one C. elegans strain with cytoplasmic contents from a second strain? We can do both, and it is remarkably easy! Besseling and Bringmann (2016) identified a molecular intervention for C. elegans in which premature segregation of maternal and paternal chromosomes in the fertilized oocyte can produce viable animals exhibiting a non-Mendelian inheritance pattern. Overexpression in embryos of a single protein regulating chromosome segregation (GPR-1) provides a germline derived clonally from a single parental gamete. We present a collection of strains and cytological assays to consistently generate and track non-Mendelian inheritance. These tools allow reproducible and high-frequency (>80%) production of non-Mendelian inheritance, the facile and simultaneous homozygosis for all nuclear chromosomes in a single generation, the precise exchange of nuclear and mitochondrial genomes between strains, and the assessments of non-canonical mitosis events.
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[
Neuronal Development, Synaptic Function, and Behavior Meeting,
2006]
Mechanotransduction underlies an array of physiological processes, including hearing, pain, and touch. In C. elegans, mechanical stimulation of the body wall opens MEC-4 DEG/ENaC transduction channels in touch receptor neurons (TRN). Touch sensation also requires components of a characteristic 15-protofilament (pf) microtubule bundle and extracellular matrix (ECM). Genetic interactions between the genes encoding the MEC-4 channel, 15-pf microtubules, and ECM led to the hypothesis that MEC-4 channel gating occurs in a tethered mode. We tested this hypothesis by using post-embedding immunoelectron microscopy (IEM) to establish the subcellular distribution of the MEC-4 channel relative to the 15-pf microtubules and the ECM.
First, we developed antibodies against the MEC-4 channel subunits, MEC-2 and MEC-4. At the light level, these antibodies label the TRNs of adult animals with a punctate pattern similar in organization and spacing to previously published findings1. The MEC-2 and MEC-4 antibodies fail to label TRNs in their respective null mutants, confirming the specificity of both antibodies. Second, we used these antibodies to label TRNs in 50 nm serial sections. Consistent with published genetic, molecular, and functional observations, post-embedding IEM reveals that the MEC-2 and MEC-4 antibodies label the 15-pf microtubules and plasma membrane of TRNs. Microtubule-associated MEC-2 and MEC-4 label is observed on 15-pf microtubules located near the center and the periphery of the microtubule bundle. Label near the periphery is >50 nm from the plasma membrane. These observations suggest that some MEC-2 and MEC-4 puncta observed at the light level represent channels in transit. Membrane-associated MEC-2 and MEC-4 label is distributed around the circumference of the TRN. All membrane-associated label is >100nm from the nearest intracellular contact point between the membrane and the 15-pf microtubule ends. Only a fraction of the membrane-associated label is in register with hemidesmosome-like structures. Thus, MEC-4 channels are not exclusively aligned with these structures. These findings indicate that MEC-4 channels are not consistently associated with either 15-pf microtubule termination points or hemidesmosome-like structures. We speculate, therefore, that MEC-4 channels can be opened independently of intracellular and extracellular tethers.
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[
2008]
"H11 was best characterized as "Hidden antigen", inducing very high levels of antibody-mediated protective immunity, on average a greater than 90% reduction in faecal egg counts (FECs) and >75% reduction in worm burdens. However, recombinant forms of H11, expressed in E. coli and insect cells using recombinant baculovirus, have so far failed to induce protection level similar to that achieved using native antigen. Studies indicate that conformational epitopes and/or glycosylation may be involved in protection.In our study, cDNA of H11 of Haemonchus contortus was cloned and identified via RT-PCR. 2960 bp of cDNA sequence of H11 were amplified from Haemonchus contortus ZJ strain, containing 2919 bp of the integrated open reading frame sharing 99.5% identity with that in database (Smith, 1997). The predicted protein possessed 972 amino acids containing five significant amino acid substitutions of Phe-184 to Leu, Lys-526 to Arg, Glu-561 to Lys, Ser-815 to Phe and Gly-881 to Glu.Due to the probable role of conformational epitopes and glycosylation in protection, a typical region with 347 amino acids from Y224 to E570 was chosen to further study, containing two N-glycosylation sites and one zinc-binding region, corresponding to partial cDNA of H11 from T670 to G1710 expressed in BL21 (DE3).By the alignment analysis between H11 and its homolog in C. elegans and C. briggsae, a partial genomic DNA sequence spanning 4806 bp was amplified using specific primers (HC-GSF and HC-GSR), referring to the patterns of introns and exons of its homologous genes in C. elegans and C. briggsae.Sequence analysis demonstrated typically GT-AG consensus sequences at each intron-exon splice junction and 11 exons were separated by 10 introns in this gemomic DNA sequence, corresponding to 1162 bp cDNA of H11. The 2810 bp 5' flanking region was amplified using the "genome walker" protocol. Except normal transcriptional factors such as TATA box, CCAAT box, more than nine GATA boxes were present in this region, which probably played a certain role in transcription of H11 gene. The antecedent characterizations of H11 may interpret why recombinant antigen failed to induce active protection. "