Duong T et al. (2020) Development "The Rheb-TORC1 signaling axis functions as a developmental checkpoint."

Reiner DJ, Mote FS, Duong T, Rasmussen NR, Ballato E

[Development, 2020]

In many eukaryotes, the small GTPase Rheb functions as a switch to toggle activity of TOR complex 1 (TORC1) between anabolism and catabolism, thus controlling lifespan, development, and autophagy. Our CRISPR-generated, fluorescently tagged endogenous <i>C. elegans</i> RHEB-1 and DAF-15/Raptor are expressed ubiquitously and localize to lysosomes. Disruption of LET-363/TOR and DAF-15/Raptor are required for development past the third larval stage (L3). We observed that deletion of RHEB-1 similarly conferred L3 arrest. Unexpectedly, robust RNAi-mediated depletion of TORC1 components caused arrest at stages prior to L3. Accordingly, conditional depletion of endogenous DAF-15/Raptor in the soma revealed that TORC1 is required at each stage of the life cycle to progress to the next stage. Reversal of DAF-15 depletion permits arrested animals to recover to continue development. Our results are consistent with TORC1 functioning as a developmental checkpoint that governs at each stage the decision of the animal to progress through development.

Duong T et al. (2020) Int J Mol Sci "Insulated Switches: Dual-Function Protein RalGEFRGL-1 Promotes Developmental Fidelity."

Rasmussen NR, Reiner DJ, Duong T

[Int J Mol Sci, 2020]

The <i>C. elegans</i> vulva is an excellent model for the study of developmental biology and cell-cell signaling. The developmental induction of vulval precursor cells (VPCs) to assume the 3-3-2-1-2-3 patterning of cell fates occurs with 99.8% accuracy. During <i>C. elegans</i> vulval development, an EGF signal from the anchor cell initiates the activation of Ras^LET-60 &gt; Raf^LIN-45 &gt; MEK^MEK-2 &gt; ERK^MPK-1 signaling cascade to induce the 1 cell. The presumptive 1 cell signals its two neighboring cells via Notch^LIN-12 to develop 2 cells. In addition, Ras^LET-60 switches effectors to RalGEF^RGL-1 &gt; Ral^RAL-1 to promote 2 fate. Shin et al. (2019) showed that RalGEF^RGL-1 is a dual-function protein in VPCs fate patterning. RalGEF^RGL-1 functions as a scaffold for PDK^PDK-1 &gt; Akt^AKT-1/2 modulatory signaling to promote 1 fate in addition to propagating the Ras^LET-60 modulatory signal through Ral^RAL-1 to promote 2 fate. The deletion of RalGEF^RGL-1 increases the frequency of VPC patterning errors 15-fold compared to the wild-type control. We speculate that RalGEF^RGL-1 represents an &quot;insulated switch&quot;, whereby the promotion of one signaling activity curtails the promotion of the opposing activity. This property might increase the impact of the switch on fidelity more than two separately encoded proteins could. Understanding how developmental fidelity is controlled will help us to better understand the origins of cancer and birth defects, which occur in part due to the misspecification of cell fates.

Duong, Dung T. et al. (2011) Worm Breeder's Gazette "The sneeze response: worms avoid piperine, the active component of black pepper"

Sadaiappen, Rebekah R., Barrett, Peter L., Duong, Dung T.

[Worm Breeder's Gazette, 2011]

Samuel ADT et al. (2003) J Neurosci "Synaptic activity of the AFD neuron in Caenorhabditis elegans correlates with thermotactic memory."

Murthy VN, Samuel ADT, Silva RA

[J Neurosci, 2003]

Thermotactic behavior in Caenorhabditis elegans is sensitive to both a worm's ambient temperature (T-amb) and its memory of the temperature of its cultivation (T-cult). The AFD neuron is part of a neural circuit that underlies thermotactic behavior. By monitoring the fluorescence of pH-sensitive green fluorescent protein localized to synaptic vesicles, we measured the rate of the synaptic release of AFD in worms cultivated at temperatures between 15 and 25degreesC, and subjected to fixed, ambient temperatures in the same range. We found that the rate of AFD synaptic release is high if either T-amb > T-cult or T-amb > T-cult, but AFD synaptic release is low if T-amb congruent to T-cult. This suggests that AFD encodes a direct comparison between T-amb and T-cult.

Bommireddy R et al. (2007) Trends Mol Med "TGFbeta1 and Treg cells: alliance for tolerance."

Bommireddy R, Doetschman T

[Trends Mol Med, 2007]

Transforming growth factor beta1 (TGFbeta1), an important pleiotropic, immunoregulatory cytokine, uses distinct signaling mechanisms in lymphocytes to affect T-cell homeostasis, regulatory T (T(reg))-cell and effector-cell function and tumorigenesis. Defects in TGFbeta1 expression or its signaling in T cells correlate with the onset of several autoimmune diseases. TGFbeta1 prevents abnormal T-cell activation through the modulation of Ca(2+)-calcineurin signaling in a Caenorhabditis elegans Sma and Drosophila Mad proteins (SMAD)3 and SMAD4-independent manner; however, in T(reg) cells, its effects are mediated, at least in part, through SMAD signaling. TGFbeta1 also acts as a pro-inflammatory cytokine and induces interleukin (IL)-17-producing pathogenic T-helper cells (T(h) IL-17 cells) synergistically during an inflammatory response in which IL-6 is produced. Here, we will review TGFbeta1 and its signaling in T cells with an emphasis on the regulatory arm of immune tolerance.

Agulnik SI et al. (1995) Genomics "Conservation of the T-box gene family from Mus musculus to Caenorhabditis elegans."

Bollag RJ, Silver LM, Agulnik SI

[Genomics, 1995]

Recently, a novel family of genes with a region of homology to the mouse T locus, which is known to play a crucial, and conserved, role in vertebrate development, has been discovered. The region of homology has been named the T-box. The T-box domain of the prototypical T locus product is associated with sequence-specific DNA binding activity. In this report, we have characterized four members of the T-box gene family from the nematode Caenorhabditis elegans. All lie in close proximity to each other in the middle of chromosome III. Homology analysis among all completely sequenced T-box products indicates a larger size for the conserved T-box domain (166 to 203 residues) than previously reported. Phylogenetic analysis suggests that one C. elegans T-box gene may be a direct ortholog of the mouse Tbx2 and Drosophila omb genes. The accumulated data demonstrate the ancient nature of the T-box gene family and suggest the existence of at least three separate T-box-containing genes in a common early metazoan ancestor to nematodes and vertebrates.

Shin, H. et al. (2017) International Worm Meeting "Identification of a novel Ral signal transduction cascade in C. elegans 2 deg vulval fate patterning."

Duong, T., Reiner, D., Kaplan, R., Shin, H.

[International Worm Meeting, 2017]

Vulval precursor cells (VPCs) are patterned through graded action of EGF from the anchor cell (AC) in conjunction with LIN-12/Notch-mediated lateral signaling. Together these signals control the highly reproducible 3 deg -3 deg -2 deg -1 deg -2 deg -3 deg pattern that develops to form the vulva. A key protein in this process is LET-60/Ras, which in mammals is the most mutated oncoprotein. LET-23/EGFR promotes LET-60/Ras activation to induce 1 deg fate via the canonical Raf-MEK-ERK MAP kinase cascade. We previously demonstrated that LET-60/Ras also switches effectors to activate its effector RalGEF-Ral to induce 2 deg fate in support of Notch. In general, 1 deg - and 2 deg -promoting signals are strongly antagonistic. Thus, in equipotent cells Ras switches effectors between Raf and RalGEF-Ral to promote mutually antagonistic 1 deg and 2 deg cell fates, respectively. However, the Ral signaling cascade is poorly understood in all systems. We therefore screened candidate Ral binding partners from the literature. Two subunits of the Exocyst complex have been previously shown to bind Ral in other systems. We found that loss of Exo84 but not Sec5 caused the same phenotype as loss of Ral, suggesting that Ral signals through Exo84 to promote 2 deg fate. To move further downstream we analyzed a little known family of MAP4 kinases. In Drosophila Sec5 and Msn/MAP4K, the ortholog of C. elegans MIG-15, physically interact and promote JNK signaling, but function in opposition to Ral signaling. Consistent with the Drosophila genetics, our preliminary data in vulval development do not support the model that Sec5 and Msn function as a Ral effector. We hypothesize that the paralogous MAP4K subfamily, C. elegans GCK-2 and Drosophila Hppy, constitutes the real Ral effector. We found loss of GCK-2 phenocopies loss of Exo84, and GCK-2 functions cell autonomously downstream of Ral in the vulva. Using CRISPR/Cas9, we tagged the N-terminus of endogenous GCK-2 with mNeonGreen (mNG). mNG::GCK-2 is localized in the cytoplasm of 1 deg and 2 deg cells. The mechanism by which Ral and Exo84 activate GCK-2 remains unclear. We tagged endogenous Ral with mKate2 and observed red signal at plasma membrane and junctions. We also knocked a constitutively activating mutation into endogenous tagged Ral. We further found that Ral-Exo84-GCK-2 signals through a conserved MAP3K/MLK-1-MAP2K/MEK-1-p38/PMK-1 cascade. This cascade may further activate downstream MAPKAP kinase, MAK-2. We generated PMK-1::mNeonGreen to measure the activity of the Ral signaling cascade through activation-dependent cytoplasmic-to-nuclear translocation of p38. Our results support a model where Ral promotes 2 deg fate in support of Notch via MAP4K activation of a p38 MAP kinase cascade.

Ju T et al. (2006) Glycobiology "Identification of core 1 O-glycan T-synthase from Caenorhabditis elegans."

Ju T, Zheng Q, Cummings RD

[Glycobiology, 2006]

The common O-glycan core structure in animal glycoproteins is the core 1 disaccharide Galbeta1-3GalNAcalpha1-Ser/Thr, which is generated by addition of Gal to GalNAcalpha1-Ser/Thr by core 1 UDP-Gal:GalNAcalpha1-Ser/Thr beta1,3-galactosyltransferase (core 1 beta3-Gal-T or T-synthase, EC2.4.1.122)(2). Although O-glycans play important roles in vertebrates, much remains to be learned from model organisms such as the free-living nematode Caenorhabditis elegans, which offer many advantages in exploring O-glycan structure/function. Here we report the cloning and enzymatic characterization of T-synthase from C. elegans (Ce-T-synthase). A putative C. elegans gene for T-synthase, C38H2.2, was identified in GenBank by a BlastP search using the human T-synthase protein sequence. The full-length cDNA for Ce-T-synthase, which was generated by PCR using a C. elegans cDNA library as the template, contains 1,170 bp including the stop TAA. The cDNA encodes a protein of 389 amino acids with typical type-II membrane topology and a remarkable 42.7% identity to the human T-synthase. Ce-T-synthase has 7 Cys residues in the lumenal domain including 6 conserved Cys residues in all of the orthologs. The Ce-T-synthase has 4 potential N-glycosylation sequons, whereas the mammalian orthologs lack N-glycosylation sequons. Only one gene for Ce-T-synthase was identified in the genome-wide search and it contains 8 exons. Promoter analysis of the Ce-T-synthase using green fluorescent protein constructs show that the gene is expressed at all developmental stages and appears to be in all cells. Unexpectedly, only minimal activity was recovered in the recombinant, soluble Ce-T-synthase secreted from a wide variety of mammalian cell lines, whereas robust enzyme activity was recovered in the soluble Ce-T-synthase expressed in Hi-5 insect cells. Vertebrate T-synthase requires the molecular chaperone Cosmc, but our results show that Ce-T-synthase does not require Cosmc, and might require invertebrate-specific factors for formation of the optimally active enzyme. These results show that the Ce-T-synthase is a functional ortholog to the human T-synthase in generating core 1 O-glycans and opens new avenues to explore O-glycan function in this model organism.

Muir RE et al. (2007) Int J Syst Evol Microbiol "Leucobacter chromiireducens subsp. solipictus subsp. nov., a pigmented bacterium isolated from the nematode Caenorhabditis elegans, and emended description of L. chromiireducens."

Muir RE, Tan MW

[Int J Syst Evol Microbiol, 2007]

A yellow-pigmented, Gram-positive, aerobic, non-motile, non-spore-forming, irregular rod-shaped bacterium (strain TAN 31504(T)) was isolated from the bacteriophagous nematode Caenorhabditis elegans. Based on 16S rRNA gene sequence similarity, DNA G+C content of 69.5 mol%, 2,4-diaminobutyric acid in the cell-wall peptidoglycan, major menaquinone MK-11, abundance of anteiso- and iso-fatty acids, polar lipids diphosphatidylglycerol and phosphatidylglycerol and a number of shared biochemical characteristics, strain TAN 31504(T) was placed in the genus Leucobacter. DNA-DNA hybridization comparisons demonstrated a 91 % DNA-DNA relatedness between strain TAN 31504(T) and Leucobacter chromiireducens LMG 22506(T) indicating that these two strains belong to the same species, when the recommended threshold value of 70 % DNA-DNA relatedness for the definition of a bacterial species by the ad hoc committee on reconciliation of approaches to bacterial systematics is considered. Based on distinct differences in morphology, physiology, chemotaxonomic markers and various biochemical characteristics, it is proposed to split the species L. chromiireducens into two novel subspecies, Leucobacter chromiireducens subsp. chromiireducens subsp. nov. (type strain L-1(T)=CIP 108389(T)=LMG 22506(T)) and Leucobacter chromiireducens subsp. solipictus subsp. nov. (type strain TAN 31504(T)=DSM 18340(T)=ATCC BAA-1336(T)).

Agulnik SI et al. (1997) Genome "Three novel T-box genes in Caenorhabditis elegans."

Ruvinsky I, Agulnik SI, Silver LM

[Genome, 1997]

The T-box gene family consists of members that share a unique DNA binding domain. The best characterized T-box gene, Brachyury or T, encodes a transcription factor that plays an important role in early vertebrate development. Seven other recently described mouse T-box genes are also expressed during development. In the nematode Caenorhabditis elegans, four T-box genes have been characterized to date. In this study, we describe three new C. elegans T-box genes, named Ce-tbx-11, Ce-tbx-12, and Ce-tbx-17. Ce-tbx-11 and Ce-tbx-17 were uncovered through the sequencing efforts of the C. elegans Genome Project. Ce-tbx-12 was uncovered through degenerate PCR analysis of C. elegans genomic DNA. Ce-tbx-11 and Ce-tbx-17 are located in close proximity to the four other previously described T-box genes in the central region of chromosome III. In contrast, Ce-tbx-12 maps alone to chromosome II. Phylogenetic analysis of all known T-box domain sequences provides evidence of an ancient origin for this gene family.