Rog, O. et al. (2011) International Worm Meeting "Quantitative analysis of chromosome dynamics during C. elegans meiosis."

Rog, O., Dernburg, A. F., Wynne, D. J.

[International Worm Meeting, 2011]

Meiosis, the specialized cell division that produces gametes and enables sexual reproduction, involves the separation of homologous chromosomes, the two copies of each chromosome inherited from each parent. This segregation process requires that each chromosome first physically pair with its homolog and precisely exchange genetic information. These complex events are coordinated both spatially and temporally by synapsis - the regulated assembly of a protein polymer called the synaptonemal complex, between paired homologs. C. elegans presents an exceptional opportunity to visualize the process of pairing and synapsis in real-time in living animals. Our analysis of chromosome pairing reveals the existence of coordinate, dynein-driven motion, that dramatically translocates one end of each chromosome in order to assist in correct pairing. Surprisingly, we also identify a constraint on movement that is removed upon meiotic entry, enabling chromosomes to pair. We will also present our progress in developing tools to visualize synapsis and the concomitant changes in chromosome morphology. Quantitative analysis using these tools will provide rigorous, in depth characterization of chromosome dynamics.

Daniel C. Bennett et al. (2007) International Worm Meeting "Identifying Interactors of ROG-1, a C. elegans FRS2 Homolog."

Michael J. Stern, Isaac E. Sasson, Daniel C. Bennett

[International Worm Meeting, 2007]

Signal transduction by mammalian receptor tyrosine kinases (RTKs) often requires intracellular signaling adaptors. One such adaptor is FRS2/snt-1, which mediates signaling through the neurotrophin (Trk) and FGF receptors to the Ras/MAPK cascade (Kouhara, et al.</I> (1997)). FRS2 interacts with these RTKs through a PTB protein-protein interaction domain, originally named for its affinity for phosphorylated tyrosine residues (Forman-Kay and Pawson (1999)). The C. elegans</I> genome contains one FRS2-like gene, rog-1</I>. ROG-1 is required in the germ line for meiotic progression and interacts genetically with let-60</I> Ras, suggesting that it transduces signals to the Ras-MAP Kinase cascade in a manner similar to its mammalian counterpart FRS2 (Matsubara, et al.</I> (2007)). Thus, ROG-1 may represent the link between an unidentified transmembrane RTK and the established Ras-MAP Kinase signaling core required for germ line meiotic progression (Church, et al.</I>(1995)). However work in our lab suggests that, unlike mammalian FRS2, ROG-1 does not interact with the C. elegans</I> FGFR pathway. Homozygotes for a putative null mutant, rog-1(tm1031)</I>, are sterile and display embryonic lethality (Matsubara, et al.</I> (2007)) but do not show any sex myoblast migration defects, and tm1031</I> fails to suppress hyperactivation of the FGFR pathway in the hypodermis. Consistent with this, we have not detected a yeast two-hybrid interaction between ROG-1 and the C. elegans</I> FGFR, EGL-15. Thus, to identify the pathway in which ROG-1 functions, we are pursuing two complementary yeast two-hybrid approaches. The first of these approaches is a traditional yeast two-hybrid screen to identify proteins which bind to the PTB domain of ROG-1. To this end, we have screened 1.65 x 107 cDNA clones from the RB2 cDNA library and have identified 29 candidate interactors. We are identifying and confirming these candidate interactors. The second of these approaches is a directed two-hybrid test with the intracellular domains of the entire set of 38 C. elegans</I> RTKs. To facilitate this, we are forcing the auto-phosphorylation of these 38 C. elegans</I> RTKs in yeast by creating fusions to both the DNA binding domain of LexA and the N-terminal coiled-coil domain of human TPR, which has been shown to constitutively activate receptor tyrosine kinases. We hope that these studies will provide a more complete model for ROG-1 function and help elucidate signal transduction through MAP Kinase in the C. elegans</I> germ line, as well as provide insights into the mechanism of signal transduction through receptor tyrosine kinases in C. elegans</I>.

Wang, Peng et al. (2011) International Worm Meeting "Caenorhabditis elegans O-GlcNAc cycling mutants alter the proteotoxicity of models of human neurodegenerative disorders."

Forsythe, Michele, Wang, Peng, Lazarus, Brooke, Hanover, John, Love, Dona, Krause, Michael

[International Worm Meeting, 2011]

O-linked N-acetylglucosamine (O-GlcNAc) addition is an important post-translational modification that occurs on hundreds of proteins, including nuclear pore proteins, transcription factors, proteasome components and neuronal proteins. O-GlcNAc can be added onto and removed from serine or threonine residue by two evolutionally conserved enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. O-GlcNAcylation is abundant in the brain and it has been linked to human neurodegenerative disease. We have exploited viable null alleles of the enzymes of O-GlcNAc cycling to examine the role of O-GlcNAcylation in well-characterized C. elegans models of neurodegenerative proteotoxicity. O-GlcNAc cycling dramatically modulated the severity of the proteotoxic phenotype in transgenic models of tauopathy, b-amyloid peptide and polyglutamine expansion. Intriguingly, loss-of-function of OGT alleviated, while loss of OGA enhanced these proteotoxicity phenotype. Consistent with these observations, the O-GlcNAc cycling mutants exhibit altered stress responses and changes in the protein degradation machinery. These findings suggest that modulators of O-GlcNAc cycling may prove useful for anti-neurodegenerative disease therapies.

Lee, J. et al. (2009) International Worm Meeting "Proteomic analysis of O-GlcNAcylation during dauer formation in C. elegans."

Paik, Y., Kim, K., Lee, J.

[International Worm Meeting, 2009]

Modification of proteins at serine or threonine residues with N acetylglucosamine (O-GlcNAcylation) plays a regulatory role in many model organisms. Here, we investigated the mechanism by which O-GlcNAcylation regulates entry into the stress-induced dormant state dauer in Caenorhabditis elegans. We confirmed that ogt-1 (O-GlcNAc transferase) mutants exhibited a dauer-defective phenotype whereas oga-1 (O-GlcNAcase, catalyzes O-GlcNAc removal) mutants exhibited a dauer-prone phenotype when treated with daumone. Consistent with these findings, treatment with low levels of daumone and the O-GlcNAcase inhibitor PUGNAc enhanced the frequency of dauer entry. Treatment of daf-2 with PUGNAc increased the frequency of dauer entry, providing additional evidence that O-GlcNAcylation promotes dauer formation. Alterations to the proteome as a result of induction of O-GlcNAcylation were analyzed by two-dimensional electrophoresis (2DE) using lysates of N2 and oga-1 worms. Seven differentially expressed protein spots were further analyzed using LC-MS/MS. The identities of these proteins suggest that O-GlcNAcylation influences stress resistance, protein folding, and mitochondrial function. To identify potential target proteins that are O-GlcNAcylated during dauer formation, we specifically labeled O-GlcNAc with fluorescent dye using O-GlcNAc labeling system containing TAMRA fluorescence dye targeting O-GlcNAcylated proteins. The fluorescently labeled samples were resolved by 2DE and 20 protein candidates were selected. Our data suggest that O-GlcNAcylation may regulate a shift in some proteins including cytoskeletal and protein turnover during dauer formation. Thus, these data may be valuable in identifying the mechanism by which high levels of O-GlcNAcylation enhance dauer formation. (This study was supported by a grant from the Korea Health 21 R&D project, Ministry of Health and Welfare of Republic of Korea [A030003 to YKP].).

Mahaffey, M. et al. (2017) International Worm Meeting "O-GlcNAc cycling and mitochondrial function."

Mahaffey, M., Austin, B., Berninsone, P., Sacoman, J.

[International Worm Meeting, 2017]

O-linked- beta -N-acetylglucosamine (O-GlcNAc) modified proteins are critical in myriad cellular processes and functions. Abnormal O-GlcNAcylation is associated with a spectrum of diseases, including Alzheimer's disease, cancer, cardiovascular disease, and diabetes. Mitochondrial protein O-GlcNAcylation is emerging as a key regulator of cellular energetic metabolism, redox signaling and cell survival pathways, but the mechanisms involved are largely unknown. O-GlcNAc transferase (OGT) is the enzyme responsible for the addition of O-GlcNAc to target proteins while O-GlcNAcase (OGA) catalyzes the removal of the modification from target proteins. OGT and OGA are encoded by single genes in C. elegans (ogt-1 and oga-1, respectively). OGT and OGA modulate lifespan, stress susceptibility and oxidative stress resistance. We hypothesize that O-GlcNAc cycling mediated by OGT and OGA plays a role in regulating mitochondrial metabolism and morphology. To that end, we measured oxygen consumption rate (OCR), a proxy for mitochondrial oxidative phosphorylation function, in N2, ogt-1 and oga-1 null mutants. OCR was measured using the Seahorse Bioscience XFe 24 Extracellular Flux Analyzer and normalized by number of worms for each individual well. Our results suggest that altered O-GlcNAc cycling reduces oxygen consumption by negatively impacting mitochondrial oxidative metabolism. In addition, O-GlcNAc cycling mutants have altered sensitivity to rotenone, an inhibitor of NADH ubiquitone reductase (Complex I). These results suggest that modulation of mitochondrial function by O-GlcNAc cycling may underlie some of the phenotypes of O-GlcNAc cycling mutants.

Scott Everet Baird et al. (2004) East Coast Worm Meeting "Association of Oscheius species with millipedes in the Wright State University Woods."

Scott Everet Baird, Christine M Spice

[East Coast Worm Meeting, 2004]

Rhabditid nematodes commonly form phoretic or necromenic associations with other soil invertebrates. These associations typically are as dauers although although associations of other larval stages also have been reported. Two hermaphroditic species of Oscheius , O. myriophila and an isolate tenatively identified as O. necromena , have been reported as necromenic associates of millipedes. Both of these species have been found in association with the millipede Pseudopolydesmus serratus in the Biology Preserve on the campus of Wright State University. A third as yet unnamed hermaphroditic species of Oscheius was found in association with a second millipede, Oxidus gracilis . O. myriophila , O. necromena , and O. sp. were found at the same collection site, i.e. they were sympatric. O. myriophila never was found in association with Ox. gracilis despite this millipede being its type host. When first observed, all collected nematodes were L4s, consistant with a previous report of the association of O. myriophila L4s with millipedes. Relationships among these species were investigated through sequence comparisons of 18S rDNA. O. myriophila , O. necromena , and O. sp. clustered together as a monophyletic clade within the insectivora -group of Oscheius . Thus, association with millipedes and their hermaphroditic mode of reproduction may be ancestral characters of this group of species.

Rahman, Mohammad M. et al. (2009) International Worm Meeting "Elevated O-GlcNAc modification can extend Caenorhabditis elegans lifespan."

Rahman, Mohammad M., Wells, Lance, Karim, Enas, Stuchlik, Olga, Kipreos, Edward

[International Worm Meeting, 2009]

O-linked beta-N-acetylglucosamine (O-GlcNAc) modification is an abundant nucleo-cytoplasmic post-translational glycosylation of proteins associated with age-related diseases like Alzheimer''s, Parkinson''s, and type II diabetes. However a link between O-GlcNAc modification of proteins and organismal aging has not been demonstrated. This work uses the nematode C. elegans to establish a link between nutrient availability, O-GlcNAc cycling, and longevity. We found that O-GlcNAc modification of protein(s) is critical for normal lifespan in adult animals, while unchecked O-GlcNAc modification of protein(s) increases adult lifespan in C. elegans. We demonstrate that the adult lifespan extension arising from an elevated level of O-GlcNAc modification is dependent on the DAF-16/FoxO transcription factor. DAF-16 is a key factor in insulin-like signal transduction, which regulates reproductive development, lifespan, and dauer formation in response to nutrient availability. Our current data indicates that O-GlcNAc cycling influences a subset of insulin-like signaling-mediated functions that regulate adult lifespan, without affecting other downstream aspects of the insulin-like signaling pathway.

Taub, D.G. et al. (2019) International Worm Meeting "O-GlcNAc signaling enhances neuronal regeneration through modulation of cellular metabolism."

Awal, M.R., Taub, D.G., Gabel, C.V.

[International Worm Meeting, 2019]

The lack of neuronal regeneration after damage to the central nervous system remains a critical deficiency in modern medicine. While regrowth of a severed axon is inherently energetically demanding, the mechanisms of metabolic regulation in a damaged neuron remain largely unknown. This represents a critical gap in our understanding of neuronal repair that is fundamental to therapeutic strategies. We have recently discovered that alterations in O-GlcNAc signaling can shift cellular metabolism to dramatically potentiate the regenerative capacity of a damaged neuron in vivo. O-linked ?- N-acetylglucosamine (O-GlcNAc) is a post-translational modification of serines/threonines that functions as a sensor of cellular nutrients. Performing in vivo laser axotomies in C.elegans, we find that neuronal regeneration is substantially increased by disruptions of either the O-GlcNAc Transferase or the O-GlcNAcase that decrease and increase O-GlcNAc levels, respectively. A lack of O-GlcNAc acts through the AKT-1 branch in the insulin-signaling pathway to utilize glycolysis. In contrast, increased O-GlcNAc levels activate an opposing branch of the insulin-signaling pathway whereby SGK-1 modulates the FOXO transcription factor DAF-16 to influence mitochondrial function. Exploiting the effects of O-GlcNAc signaling on metabolism and regeneration, we are determining the metabolic response within a damaged neuron, how cellular metabolism acts as limiting factor in neuronal regeneration and how it might be altered for neuro-therapeutic benefits.

Carta LR et al. (1995) International C. elegans Meeting "NEW HERMAPHRODITIC SPECIES OF OSCHEIUS (ANDRASSY, 1976) (RHABDITIDAE, NEMATODA)"

Thomas WK, Carta LR, Sternberg PW

[International C. elegans Meeting, 1995]

Morphological, life cycle and molecular characteristics are used to describe seven hermaphroditic species of Oscheius (Andrassy, 1976) including O. brevesophaga, n. sp. O. mesoesophaga n. sp., O. fijiensis n. sp., O. spiculunca n. sp. O. longirecta n. sp., O. subvulva n. sp., and O. longimascula n. sp. These were compared with other living isolates of the group provided through David Fitch. Starvation induced males were mated among the isolates to define distinct biological species. These species were sequenced over a 310 nucleotide bp region of the nuclear 28S rRNA. Vulval anatomy was observed in the early L4 stage. Life cycle was determined from egg to egg at 20 C. to within half a day. The only non-fertile hybrids produced were those of O. mesoesophaga hermaphrodites mated with O. tipulae males. With just 1 base pair difference between each sp. in the series, O. brevesophaga, mesoesophaga and tipulae are best distinguished by esophageal length. By contrast, three interbreeding isolate groups of O. brevesophaga are defined by 1 or 2 bp changes. Thus, at least 2 bp changes may be necessary to distinguish a biological species in this group, while no change may be sufficient (e.g. as in Rhabditis anomala and a new hermaphroditic sibling species, Rhabditis heranamaloides). This can help define species where males are nearly impossible to find or mate. Vulval anatomy revealed that 2o lineages generated 4 cells and 3o generated 4 cells (4:4) in the three non- Dolichura groups, and 4:2 cells in three of the four species of the Dolichura group examined. (Caenorhabditis has a 7:2 pattern.) A significant number of O. subvulva individuals showed an interesting asymmetry with 4 cells resulting from the 3o P4.p lineage, while the 3o P8.p lineage gave only two cells. Four major groups are defined by larger base pair difference gaps between than within groups. These correspond to somewhat distinct groups morphologically and developmentally as follows: Tipulae group: Spicules <30 micron with tips not visibly swollen, Life cycle < 5 days at 20 C, 2o:3o = 4:4. (O. brevesophaga, mesoesophaga, tipulae) Insectivora group: Spicule tips hooked or acute, Spicule > 30 micron, Bursa usually pseudopeloderan, >= 4 lateral lines, life cycle < 5 days at 20 C, 2o3o = 4:4. (O. longirecta, spiculunca) Fijiensis group: Spicule tips swollen, life cycle < 5 days at 20o C, Medial vulva, 2o:3o = 4:4. (O. fijiensis) Dolichura group: Spicules with swollen tips, Life cycle > 5 days at 20o C, Vulva usually > 50%, 2o:3o usually = 4:2. (O. dolichura, dolichuroides, subvulva, longimascula)

Eustice, Moriah et al. (2015) International Worm Meeting "O-GlcNAc cycling plays a crucial role in germline plasticity and acts in coordination with fatty acid beta-oxidation to regulate ARD entry in C. elegans."

Hanover, John, Eustice, Moriah

[International Worm Meeting, 2015]

Adult reproductive diapause (ARD), or the oogenic germline starvation response, is triggered when C. elegans are starved at the mid-L4 stage of development. ARD is characterized by extended lifespan and reproductive potential as well as germline plasticity, wherein the germline shrinks back to approximately 35 germ cell nuclei per gonad arm during starvation but regenerates following refeeding. Previous studies found that the fatty acid beta-oxidation protein NHR-49 plays a key role in the establishment of ARD. However, questions remain about the way in which changes in metabolism are sensed and trigger the plasticity observed with adult diapause.To address these questions, we aimed to examine the complex relationships between ARD and metabolism, specifically focusing on the modification O-N-acetyl-glucosamine (O-GlcNAc). O-GlcNAc is a dynamic monosaccharide post-translational modification that integrates signals from several metabolic pathways, including glucose and fat metabolism. Key enzymes in the cycling of O-GlcNAc are O-GlcNAc transferase (OGT), which is responsible for the addition of O-GlcNAc, and O-GlcNAcase (OGA), which removes the modification. Due to the fact that the substrate of OGT, UDP-GlcNAc, is produced as the final step of the hexosamine biosynthetic pathway, the regulation of O-GlcNAc cycling is uniquely situated to link metabolic signaling with growth, development, and longevity. Using mutants of OGT and OGA we discovered that O-GlcNAc cycling plays a prominent role in both ARD entry and exit. Furhter, we found that the role of O-GlcNAc in the establishment of ARD is genetically linked to NHR-49 function, such that impairment of O-GlcNAc cycling suppresses the NHR-49 entry defect.These findings suggest that perturbation of nutrient-sensitive O-GlcNAc is crucial to the developmental dynamics observed in ARD and acts in concert with NHR-49 to regulate entry into reproductive diapause. Understanding ARD dynamics is important as it provides a unique model in which to explore the relationship between nutrient status and the regulation of reproduction and aging. .