Ogawa H et al. (1997) International C. elegans Meeting "UNC-18 MEDIATES BINDING OF SYNTAXIN TO THE SYNAPTIC VESICLES"

Harada S-i, Sassa T, Yamamoto H, Rand JB, Hosono R, Ogawa H

[International C. elegans Meeting, 1997]

Genetic studies have revealed that the essential function of the unc-18 gene is the synaptic transmission. n-Sec-1, the mammalian UNC-18 homolog is believed to be associated with a synaptic plasma membrane protein prior to synaptic vesicle docking. If this were the case for the C. elegans UNC-18, the gene mutations would result in constitutive fusions of synaptic vesicles. However, the gene mutations result in the accumulation of synaptic vesicles and neurotransmitters. To examine the conflicting observations, we analyzed the binding of wild-type and mutant UNC-18 to syntaxin. UNC-18 is a hydrophilic, globular, and neutral protein (pI 6.95). In the Sf21 cells the protein is predominantly localized in the cytoplasm, indicating its intrinsic solublility. To investigate further the intracellular distribution of UNC-18, we isolated synaptic vesicles by the Percoll gradient fractionation. UNC-18 was detected on synaptic vesicles by Western blotting. We cloned C. elegans cDNAs encoding the mammalian syntaxin1A homolog (Ce syntaxin). We tested the binding of UNC-18 to GST-Ce syntaxin fusion protein. UNC-18 binds to the recombinant syntaxin in vitro with high affinity. These findings indicate that the protein is periplasmic associating with both synaptic vesicles and plasma membrane, although intrinsically soluble. In vesicle traffic UNC-18 may be a regulator with the plasma membrane through syntaxin. UNC-18 may cause a conformational change of syntaxin and allowing it to bind to synaptic vesicles. To test the hypothesis, we sequenced cDNAs of 15 unc-18 mutants. We found eight missense mutations. We are examing the ability of the mutant UNC-18 proteins to bind to Ce syntaxin and to associate with synaptic vesicles.

Dana L. Miller et al. (2007) International Worm Meeting "Adaptation to hydrogen sulfide increases thermotolerance and lifespan."

Mark B. Roth, Dana L. Miller

[International Worm Meeting, 2007]

Hydrogen sulfide (H₂S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H₂S to begin to elucidate the molecular mechanisms of H₂S action. We show that nematodes exposed to H₂S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H₂S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H₂S. Increased thermotolerance and lifespan is not observed in the sir-2.1(ok434) deletion mutant exposed to H₂S. However, mutants in the insulin signaling pathway (both daf-2 and daf-16), animals with mitochondrial dysfunction (isp-1 and clk-1) and a genetic model of caloric restriction (eat-2) all exhibit H₂S-induced increased thermotolerance. These data suggest that H₂S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H₂S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H₂S.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

Harada S-i et al. (1997) International C. elegans Meeting "A CIS-ACTING ELEMENT REQUIRED FOR NEURON-SPECIFIC EXPRESSION OF THE UNC-18 GENE"

Hosono R, Ogawa H, Harada S-i, Kobayashi T, Yamamoto H

[International C. elegans Meeting, 1997]

In synaptic transmission, synaptic vesicles form complexes with molecules constituting the plasma membrane. To clarify the molecular mechanisms of this step, we examined the regulation of the unc-18 gene, a synaptic gene. The unc-18 gene encodes a protein that modulates synaptic vesicles at the presynaptic terminal and is expressed in a highly specific pattern in the C. elegans nervous system. We are searching for cis-acting elements and trans-factors binding these elements which control neuron-specific expression of the unc-18 gene. To determine the elements essential for neuron-specific expression of the gene, we have coupled the unc-18 gene to the reporter gene lacZ or GFP, and examined the pattern of expression of the transgenic organism. The unc-18 gene includes a 10kb genome sequence. We cloned a 2.8kb upstream region in expression vector pPD21.28 and pPD95.75(provided by A.Fire). The two constructs including 2.8kb upstream region of the initiator ATG were also expressed in the ventral nerve code(VNC) and head region. We analyzed the sequence necessary and sufficient for unc-18 gene expression using deletion constructs. We have identified a 312 bp 5' flanking sequence, which is sufficient to produce GFP expression in VNC and head ganglions. We also found the sequence in C.briggsae corresponding to the sequence in C.elegans, indicating that the unc-18 cis-acting element is functionally conserved. We are now making site-specific mutations and deletions of this enhancer region.

Haradal S et al. (1995) International C. elegans Meeting "IDENTIFICATION OF A CIS-ACTING ELEMENT DICTATING NEURON-SPECIFIC EXPRESSION OF UNC-18"

Haradal S, Yamamoto H, Hosono R, Kobayashi T, Ogawa H

[International C. elegans Meeting, 1995]

Mutations in the gene unc-18 cause defectiveness in the release of neurotransmitters. The UNC-18 protein is present at the presynaptic terminal. The predicted amino acid sequence for UNC-18 is significantly similar to that for the yeast SEC1, one of the essential elements of the yeast secretory pathway from the Golgi body to the plasma membrane. The mammalian counterpart, n-Secl or Munc-18 also interacts with the plasma membrane. Therefore, UNC-18 may contribute to the release of nellrotranmitters. To understand the molecular mechanisms by which the release of neurotransmitters is regulated, we are searching for a possible regulatory region of the unc-18 gene. We fused the upstreamregulatory sequence of unc-18 to the pPD21 28 vector to study the expression of unc-18 and showed that transcriptional regulation of the unc-18 gene could account for the pattern of lacZ fusion protein expression. The construct including 3.2-kb upstream region of the initiation codon ATG was expressed in a highly specific pattern in the C. elegans nervous system. The unc-18-lacZ fusion first expressed in a few cells at the embryollic comma stage and in motor neurons at the 3-fold larval stage. The expression pattern of the Beta-galactosidase reporter gene in embryos, larvae, and adults was generally consistent with the results from staining with anti-UNC-18 antibody, that is, the unc-18 was mainly expressed in motor neurons and a limited number of neurons constituting nerve ganglia. We have identified a functional sequence in the upper stream within 700 bp of the unc-18 gene that drives primarily lacZ expression in the nervous system, especially in motor neurons. We have cloned a homologue of unc-18 from a C. briggsae genomic DNA. Sequence copmparison of C. elegans unc-l8 to its C. briggsae homologue reveals that the predicted amino acid sequence is almost identical. However, the upperstream sequence is greatly divergent although the SLI trans-splice site is conserved.

Rasika Kalamegham et al. (2004) East Coast Worm Meeting "EGL-26 belongs to the NlpC/P60 superfamily of putative enzymes and is closely related to a mammalian acyl transferase"

Wendy Hanna-Rose, Rasika Kalamegham

[East Coast Worm Meeting, 2004]

egl-26 was identified in a genetic screen to uncover mutants with vulval morphology defects. In egl-26 mutants the morphology of a single vulval toroid (vulF) is abnormal and a proper connection to the uterus is not made leading to the egg-laying defect. EGL-26 is a member of the NlpC/P60 superfamily of enzymes, which is characterized by a Histidine containing domain and a Cysteine containing domain (H-box and NC domain, respectively). EGL-26 along with other eukaryotic proteins belongs to a distinct subclass of NlpC/P60-related putative enzymes. The mammalian proteins lecithin: retinol acyltransferase or LRAT and H-ras revertant 107 or H-Rev107 are the most closely related to EGL-26. Both LRAT and H-Rev107 contain putative transmembrane domains in addition to the H-box and NC domains. Although EGL-26 contains no putative transmembrane domains, it is localized at the apical membrane of cells where it is expressed. Proper localization of LRAT within the retinal pigment epithelium is essential for its function. Significantly, an S-F substitution at amino acid 275 of EGL-26 found in the egl-26 (n481) allele causes mislocalization of an EGL-26::GFP fusion leading to general cytoplasmic expression as opposed to normal apical membrane localization. The corresponding Serine residue is conserved in both LRAT and H-Rev107. We are attempting to analyze the relationship between the mammalian proteins and EGL-26 by attempting a rescue of egl-26 mutants by expression of either LRAT or H-Rev107 or both. We plan to test the importance of membrane localization by restoring membrane localization to EGL-26n481 via addition of alternative membrane localization signals.

Schwartz, Hillel et al. (2013) International Worm Meeting "Developing Heterorhabditis nematodes as an experimental system for the study of mutualistic symbiosis."

Schwartz, Hillel, Sternberg, Paul

[International Worm Meeting, 2013]

Entomopathogenic nematodes of the genus Heterorhabditis are insect killers that live in mutually beneficial symbiosis with pathogenic Photorhabdus bacteria. Photorhabdus is rapidly lethal to insects and to other nematodes, including C. elegans, but is required for Heterorhabditis growth in culture and for the insect-killing that defines the entomopathogenic lifestyle. The symbiosis between Heterorhabditis and Photorhabdus offers the potential to study the molecular genetic basis of their cooperative relationship. We developing tools to make such studies more feasible: we have been studying multiple nematodes of the genus Heterorhabditis and developing tools for the molecular genetic analysis of Heterorhabditis bacteriophora. Many species of Heterorhabditis and variants of Photorhabdus have been isolated; some pairings show specificity in their ability to establish a symbiotic relationship. To better understand these interactions and other variations in the lifestyles of Heterorhabditis, we have sequenced H. indica, H. megidis, H. sonorensis, and H. zealandica; a H. bacteriophora genome sequence is available. A comparison of these closely related species may help us to identify mechanisms that regulate the response to bacterial interactions and to find variations that correlate with differences in lifestyle or bacterial compatibility. In addition to genomics, we are developing H. bacteriophora as a laboratory organism. H. bacteriophora grows well on plates, has been reported to be susceptible to RNAi and transgenesis, and can develop as a selfing hermaphrodite, and so should be a powerful system for the molecular genetic study of the aspects of biology to which it is uniquely well suited, most prominently symbiosis. This potential is severely diminished by inconvenient sex determination: the self-progeny of hermaphrodites are mostly females with some males; at low density, their progeny are almost exclusively females. We have screened for and isolated a constitutively hermaphroditic mutant for use in molecular genetic studies of symbiosis. This mutant also offers the opportunity to explore the basis of hermaphrodite sex determination in H. bacteriophora.

Fujita, T. et al. (2019) International Worm Meeting "Identification of novel autophagy negative regulator modulating C. elegans reproductive lifespan."

Yoshimori, T., Fujita, T., Nakamura, S., Hamasaki, M., Kubo, S., Ogawa, H.

[International Worm Meeting, 2019]

In many species reproductive capacity is negatively correlated with animal lifespan. How the balance between these two is regulated is still unclear. The knowledge regarding the factors or processes specifically regulating reproductive capacity are still limited. So far it has been shown that TGF-b Sma/Mab signaling pathway non-cell autonomously regulates reproductive lifespan (Luo et al., Cell, 2010) but the gonad intrinsic factor is not poorly characterized. By using siRNA based screening in mammalian cell culture we identified novel nuclear protein, which negatively regulates an evolutionally conserved cytoplasmic degradation process, autophagy. Knockdown of the protein function induced autophagy via transcriptional upregulation of some autophagy related genes. The expression study of C. elegans homologs revealed that this nuclear protein is highly expressed in germline and/ or somatic gonadal cells including distal tip cells and sheath cells. Moreover, knockdown of these protein function significantly extended reproductive lifespan in an autophagy dependent manner, but not extended animal lifespan. These results indicate that this novel autophagy negative regulators uncouple the balance between reproduction and animal lifespan and thus are key factors to elucidate the underlying mechanism. We would like to discuss the possible mechanisms with our updated data.

Schwartz H et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Towards a SNP map for Heterorhabditis bacteriophora"

Sternberg P, Schwartz H

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Heterorhabditis bacteriophora is a species of insect-parasitic nematode that lives in mutually beneficial symbiosis with pathogenic Photorhabdus luminescens bacteria. P. luminescens bacteria are lethal to insects and to other nematodes, including the soil nematode Caenorhabditis elegans, but are required for H. bacteriophora growth. The symbiosis between H. bacteriophora and P. luminescens therefore offers the potential to study the molecular genetic basis of their cooperative relationship. We are interested in developing tools to make such studies more feasible; in particular, we propose to generate tools for genetic mapping in H. bacteriophora. We will test independent isolates of H. bacteriophora to ensure that the isolates are cross-fertile. We will then examine the abilities of these isolates to grow on and to become infected with different wild-type and mutant Photorhabdus bacteria. From these tests we will select an isolate on which we will use next-generation high-throughput sequencing technology for the purpose of refining the existing draft genome sequence and for the creation of a SNP map. We anticipate that this SNP map will enable us and the wider insect-parasitic nematode community to identify induced mutations and natural variations affecting the interactions between H. bacteriophora nematodes and pathogenic Photorhabdus bacteria.

Yamakawa A et al. (1997) International C. elegans Meeting "EXPRESSION OF THE C.ELEGANS RABGDI"

Hosono R, Yamakawa A, Harada S-i, Sassa T, Ogawa H

[International C. elegans Meeting, 1997]

The proper targeting of secretory vesicles to the plasma membrane by transport vesicles leads to docking, fusion and release of secretory products into the external milieu through both constitutive and regulated secretion. The Rab proteins on the transport vesicles enable the membrane vesicle to identify and/or fuse with their targets. Rab proteins interconvert between two conformations. RabGDP dissociate inhibitor (GDI) delivers Rab protein to its specific compartment and retrieves Rab from its fusion target after it has completed a catalytic cycle. GDI distinguishes GTP- and GDP-bound conformations of Rabs. GDI retrieves spent Rab proteins from target membranes into the cytosol, allowing their return to the correct organelle for subsequent rounds of intracellular transport. To further understand the role of the C. elegans RabGDI (CeRabGDI) in the synaptic vesicle transport, we analyzed the gene, the cDNA structure and localization of the protein. The CeRabGDI cDNA encodes a polypeptide of 444 amino acid residues which contain a domain with high sequence similarity to mammalian RabGDI. We have located the CeGDI encoding gene (gdi-1) on LGIV near sup-24. Analyses of genomic and cDNA sequence reveals that gdi-1 spans 5.9 kb and contains 3 exons encoding a 1.7 kb transcript. Northern blot analysis showed that the gdi-1 transcript is present at the embryonic and adult stages at high levels. In situ hybridization revealed the CeRabGDI transcript around the pharynx, vulva and tail regions. We made antisera against the CeGDI protein expressed in Sf21 cells. With the antisera, we detected a single 50 kDa band on SDS-PAGE.