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[
International Worm Meeting,
2011]
To better understand the neural basis that regulates a worm's sensory behavior and its modulation by learning, we are studying avoidance behavioral responses to 2-nonanone. We previously reported that the avoidance behavior to 2-nonanone is enhanced, rather than reduced, after preexposure to the odor, and this enhancement is acquired as a non-associative dopamine-dependent learning (Kimura et al., J. Neurosci., 2010; Fujita and Kimura, this abstract). In addition, we observed that worms respond to a spatial gradient of 2-nonanone (Yamazoe and Kimura, CE Neuro, 2010), which cannot be simply explained by the pirouette or weathervane strategies.
2-nonanone is mainly sensed by the AWB neurons, which have been shown to exhibit odor-OFF response in aqueous step stimulation with 2-nonanone (Troemel et al., Cell 1997; Ha et al., Neuron 2010). To understand how the neuronal circuits of worms regulate the characteristic 2-nonanone behavioral response, we are monitoring calcium changes in the AWB and downstream neurons using G-CaMP 4 (Shindo et al., PLoS ONE, 2010). We thank Drs. S. Oda, K. Yoshida, and Y. Iino (U. Tokyo) for suggestions on microfluidics; M. Hendricks and Y. Zhang (Harvard) for aqueous 2-nonanone stimulation; and E. Busch and M. de Bono (MRC) for gaseous microfluidic stimulation.
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[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
The intracellular positioning of organelles and other components is important for cells to function. The one-cell stage embryo of C. elegans is a good and well-studied model to address the mechanics underlying organelle positioning. We have been focusing on the positioning of the centrosomes. By calculating the expected forces exerted by microtubules and comparing the forces with the position and velocity of the movements of centrosomes in vivo, we were able to propose feasible models to explain the positioning of the centrosomes during pronuclear centration [1], asymmetric spindle displacement [2], and spindle elongation [3]. For better understanding of the organelle positioning inside the cell, the physical properties of the cytoplasm is critical. We are starting to construct a physical model to describe dynamic behaviors of the cytoplasm. [References] 1. A. Kimura & S. Onami (2005) Computer simulations and image processing reveal length-dependent pulling force as the primary mechanism for C. elegans male pronuclear migration. Dev. Cell., 8: 765-775. 2. A. Kimura & S. Onami (2007) Local cortical pulling-force repression switches centrosomal centration and posterior displacement in C. elegans. J. Cell Biol., 179: 1347-1354. 3. Y. Hara & A. Kimura (2009) Cell-size-dependent spindle elongation in the Caenorhabditis elegans early embryo. Curr. Biol., 19: 1549-1554.
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[
International Worm Meeting,
2007]
Hydrogen sulfide (H<sub>2</sub>S), 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 H<sub>2</sub>S to begin to elucidate the molecular mechanisms of H<sub>2</sub>S action. We show that nematodes exposed to H<sub>2</sub>S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H<sub>2</sub>S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H<sub>2</sub>S. Increased thermotolerance and lifespan is not observed in the
sir-2.1(
ok434) deletion mutant exposed to H<sub>2</sub>S. 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 H<sub>2</sub>S-induced increased thermotolerance. These data suggest that H<sub>2</sub>S 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 H<sub>2</sub>S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H<sub>2</sub>S.
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[
International C. elegans Meeting,
1999]
Understanding how the aging process can be regulated is a fascinating and fundamental problem in biology. We have found that signals from the reproductive system influence the lifespan of the nematode C. elegans . If the cells that give rise to the germline are killed with a laser microbeam, the lifespan of the animal is extended (Hsin and Kenyon, Nature , in press). Our findings suggest that germline signals act by modulating the activity of an insulin/IGF-1-like pathway known to regulate the aging of this organism. Mutants with reduced activity of the insulin/IGF-1 receptor homolog DAF-2 have been shown to live twice as long as normal (1,2,3), and their longevity requires the activity of DAF-16, a member of the forkhead/winged-helix family of transcriptional regulators (1,2,4,5). We find that in order for germline ablation to extend lifespan, DAF-16 is required, as well as a putative nuclear hormone receptor, DAF-12 (6,7). In addition, our findings suggest that signals from the somatic gonad have an opposite effect on lifespan, and that this effect appears to require DAF-2 activity. Together our findings imply that the C. elegans insulin/IGF-1 system integrates multiple signals to define the rate of aging of the animal. This study demonstrates an inherent relationship between the reproductive state of this animal and its lifespan, and may have implications for the co-evolution of reproductive capability and longevity. 1. C. Kenyon, et al., Nature 366 , 461-4 (1993). 2. P. Larsen, P. Albert, D. Riddle, Genetics 139 , 1567-83 (1995). 3. K. Kimura, H. Tissenbaum, Y. Liu, G. Ruvkun, Science 277 , 942-6 (1997). 4. K. Lin, J. Dorman, A. Rodan, C. Kenyon, Science 278 , 1319-22 (1997). 5. S. Ogg, et al., Nature 389 , 994-9 (1997). 6. W-H. Yeh. Ph.D. Thesis, University of Missouri, Columbia (1991). 7. D. Riddle, P. Albert, in C. elegans II D. Riddle, T. Blumenthal, B. Meyer, J. Priess, Eds. (Cold Spring Harbor Laboratory Press, 1997) pp. 739-68.
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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.
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[
International Worm Meeting,
2019]
Animals experience species specific life-stages during lifespan, for example infants, adolescence, adulthood and elderly in humans. Generally, the order of life stage that animals experience is deterministic, whereas the timing of the transition varies among individuals, or is stochastic. In each life stage, animals move in a characteristic behavioral mode. The behavioral modes can be considered to be a proxy of the internal variables such as skeletal structure of the body, nerve-muscular connections, and amount of hormone in the body. In this study, we developed a microfluidic device (WormFlo), which has 108 microchambers for culturing individual adult C. elegans by continuously exchanging buffer in the chambers. WormFlo allowed us to monitor the motion of individual C.elegans to identify the life stage of C. elegans at the adult stage. We succeeded in recording C. elegans swimming motion at video rate for 6 days using WormFlo. We found that C. elegans repeated active and inactive states, which corresponds to the previously reported episodic swimming. Interestingly, the residence time of the actively moving state of C. elegans distributed in an exponential manner, even when the time scale for observation was changed from sub-second to several tens of minute. This result indicates that the residence time of active state in C. elegans swimming motion is self-similar in a broad range of time scales, implying the presence of fractal nature in C. elegans swimming motions. We are studying the behavioral mode change at the adult stage of C. elegans during ageing by using our system.
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[
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.
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[
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.
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[
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.
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[
International Worm Meeting,
2013]
MicroRNAs (miRNAs) are small non-coding regulatory RNAs that regulate gene expression at the post-transcriptional level and are involved in a broad spectrum of biological processes. Rearrangements of inter- or intra-molecular RNA structures and conformational changes of ribonucleoprotein complexes in the multiple processes of the miRNA pathway have led to the involvement of RNA helicase activities but little is known so far. In eukaryotes, RNA helicases generally belong to the superfamily 2 (SF2) in helicase classification, especially the DExD/H-box helicase family. The DExD/H-box proteins have been shown in association with many cellular processes involving RNA. To better understand the possible roles of DExD/H-box RNA helicases in miRNA function, we employed RNAi screen to identify genetic interaction between C. elegans DExD/H-box RNA helicases and the
let-7 miRNA, which controls the timing of cell cycle exit and terminal differentiation. In addition to the RNA helicase
p72, a component of Drosha Microprocessor complex, and CGH-1 that has been reported to facilitate the function of miRNA-induced silencing complex (miRISC), we found several DExD/H-box RNA helicases, which are involved in ribosomal RNA processing, pre-mRNA splicing and mRNA surveillance, may also take part in miRNA biogenesis and/or function. (Support: National Science Council, Taiwan. NSC 100-2311-B-002-006-MY3).