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[
Neuronal Development, Synaptic Function and Behavior, Madison, WI,
2010]
There is increasing evidence that sleep may play a synaptic function, yet little is known about the mechanisms of synaptic alterations during sleep. We used pharmacologic and optogenetic studies of synaptic transmission at the neuromuscular junction during lethargus, a sleep-like state in C. elegans. Our observations suggest a surprising decrease in GABAergic signaling during lethargus. Worms in lethargus demonstrate hyper-sensitivity to aldicarb. During adult satiety, another quiescent behavioral state, worms were also aldicarb-hypersensitive, demonstrating another similarity between lethargus quiescence and adult quiescence. The aldicarb hypersensitive
unc-25 mutants, which lack GABA, demonstrate a wild-type time-course to paralysis during L4 lethargus. This suggests that the mechanism of hypersensitivity involves reduced GABAergic neurotransmission. In support of this mechanism, we found a reduced behavioral response (body elongation) to optogenetic stimulation of GABAergic motor neurons during lethargus. We observed that worms in lethargus are resistant to muscimol a GABA agonist, suggesting that a post-synaptic mechanism is responsible for the decreased GABAergic signaling. In a pilot genetic screen, we obtained one mutant with resistance to aldicarb during lethargus yet normal sensitivity as an adult. This shows that hypersensitivity to aldicarb during lethargus is genetically regulated.
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[
International Worm Meeting,
2019]
When animals are sick or injured from an exposure that results in cell stress, they respond by sleeping; such behavior is called sickness or stress induced sleep (SIS). The pathway regulating SIS may be conserved from nematodes to vertebrates, and is relevant to fatigue behavior in humans during sickness. Cellular stress leads to activation of the epidermal growth factor (EGF) receptor on the ALA neuron, which releases neuropeptides. ALA neuropeptides induce SIS behavior, which consists of movment and feeding quiescence, elevated arousal threshold, and rapid reversibility. While much is known about ALA activation and its down stream mechanisms, the SIS pathway upstream of EGF remains poorly understood. This is the focus of our work. Mutants disrupting the cuticular collagens DPY-5, DPY-10, or DPY-13 showed impaired feeding and movement quiescence following exposures to ultraviolet (UV) irradiation, heat shock, or viral infection. Each of the three dpy mutants showed normal or enhanced SIS following EGF overexpression, suggesting that these genes act upstream of or in parallel to EGF. To determine if mutants with a Dpy phenotype due to mutations other than collagen genes are important for SIS, we tested mutants in
dpy-19, which encodes a C-mannosyltransferase.
dpy-19 mutants had normal SIS following both UV and heat shock, suggesting that specifically collagen gene disruption and not the Dpy phenotype explains the impairment in SIS in the
dpy-5, -10, and -13 mutants. To determine whether disruption of the cuticle without disrupting collagens affects SIS, we tested
bus-8 mutants, which encodes a glycosyltransferase that is important for cuticle integrity.
bus-8 mutants are not Dpy.
bus-8 mutants were not defective in SIS, suggesting that disrupting the cuticle is not sufficient to impair SIS, but that collagen disruption specifically is important. We propose that effects of skin collagen disruption on sleepiness are relevant to commmon complaint of fatigue in patients with connective tissue disorders.
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[
Neuronal Development, Synaptic Function and Behavior, Madison, WI,
2010]
Molting lethargus is a developmentally regulated period of quiescent behavior that has physiological and genetic similarities to sleep, including a homeostatic response to deprivation. We hypothesized that metabolic signaling by the insulin-like signaling pathway would be affected by deprivation of lethargus quiescence. A GFP::DAF-16 fusion protein was used to monitor the subcellular location of DAF-16 in response to forced swimming for 30 minutes. We found an increase in nuclear GFP::DAF-16 after deprivation compared to time-matched controls during L4 lethargus but not during the adult stage. To test in a different fashion whether deprivation causes reduced insulin signaling, we deprived
daf-8 mutants of L1 lethargus quiescence. Daf-C mutants have a temperature sensitive period at the end of the L1 stage (Swanson and Riddle, 1981), suggesting that the dauer decision is made during L1 lethargus. Following quiescence deprivation during L1 lethargus but not earlier or later, there was an increase in dauer formation in comparison to control animals. Thus, insulin signaling is reduced by deprivation of lethargus quiescence. To test for a role for
daf-16 in the behavioral response to deprivation of lethargus quiescence, we examined the behavior of
daf-16 mutants following forced swimming during lethargus. Wild-type animals show elevated response latencies to octanol or to blue light stimulation following deprivation of lethargus quiescence. In contrast, four
daf-16 alleles behaved the same after deprivation as they had in the absence of deprivation, indicating a defective homeostatic response to deprivation of lethargus quiescence. Introduction of
daf-16 in several tissues rescues this defective homeostatic response, suggesting a non-cell autonomous role for
daf-16 in this response. In contrast to
daf-16 mutants,
daf-2 mutants showed an exaggerated homeostatic response, which was partially
daf-16 dependent, to deprivation of lethargus quiescence.
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[
International Worm Meeting,
2009]
Interactions between proteins are a key component of most or all biological processes. A key challenge in biology is to generate comprehensive and accurate maps (interactomes) of all possible protein interactions in an organism. This will require iterative rounds of interaction mapping using complementary technologies, as well as technological improvements to the approaches used. For example, we recently developed a novel yeast two-hybrid approach that adds a new level of detail to interaction maps by defining interaction domains(1). Currently, I am working to generate an interaction map of proteins involved in controlling cell polarity in C. elegans to improve our understanding of the molecular mechanisms that establish and maintain cell polarity in multicellular organisms. I will combine two fundamentally different interaction mapping techniques: the yeast two-hybrid system (Y2H) and affinity purification/mass spectrometry (AP/MS). This will provide more detail by identifying both direct interactions between pairs of proteins by Y2H, and the composition of protein complexes by AP/MS. Moreover, interactions missed by one technology may be detected by the other, leading to a more complete interaction map. I will integrate the physical interactions with phenotypic characterizations. To this end I will systematically characterize the interaction network in vivo using two distinct models of polarity: asymmetric division of the one-cell embryo, and stem-cell-like divisions of a multicellular epithelium (in collaboration with M. Wildwater and S. van den Heuvel). M. Boxem, Z. Maliga, N. Klitgord, N. Li, I. Lemmens, M. Mana, L. de Lichtervelde, J. D. Mul, D. van de Peut, M. Devos, N. Simonis, M. A. Yildirim, M. Cokol, H. L. Kao, A. S. de Smet, H. Wang, A. L. Schlaitz, T. Hao, S. Milstein, C. Fan, M. Tipsword, K. Drew, M. Galli, K. Rhrissorrakrai, D. Drechsel, D. Koller, F. P. Roth, L. M. Iakoucheva, A. K. Dunker, R. Bonneau, K. C. Gunsalus, D. E. Hill, F. Piano, J. Tavernier, S. van den Heuvel, A. A. Hyman, and M. Vidal, A protein domain-based interactome network for C. elegans early embryogenesis. Cell, 2008. 134(3): p. 534-545. .
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Yousuke Seida, Kazuhiro Maeda, Tomonori Kawata, Masumi Katane, Hiroyuki Kobuna, Takao Inoue, Yasuaki Saitoh, Hiroyuki Arai, Yasuhito Nakagawa, Masae Sekine, Taro Sakamoto, Hiroshi Homma, Takemitsu Furuchi
[
East Asia Worm Meeting,
2010]
Among free D-amino acids existing in living organisms, D-serine (D-Ser) and D-aspartate (D-Asp) are the most actively studied. D-Ser has been proposed as a neuromodulator that regulates L-glutamate-mediated activation of the N-methyl-D-Asp (NMDA) receptor by acting as a co-agonist. On the other hand, several lines of evidence suggest that D-Asp plays important roles in regulating developmental processes, hormone secretion and steroidogenesis. D-Amino acid oxidase (DAO) and D-Asp oxidase (DDO) are known as stereospecific degradative enzymes that catalyze the oxidative deamination of D-amino acids. DAO displays broad substrate specificity and acts on several neutral and basic D-amino acids, while DDO is highly specific for acidic D-amino acids. DAO and DDO are presumed to regulate endogenous D-Ser and D-Asp levels, respectively, as well as mediate the elimination of accumulated exogenous D-amino acids in various organs. Previously, we demonstrated that nematode Caenorhabditis elegans, a multicellular model animal has at least one active DAO gene and three active DDO genes, while it had been thought that most organisms bear only one copy of each DAO and DDO gene. In addition, our previous study revealed that the spatiotemporal distributions of these enzymes in the body of C. elegans are different from one another. In this study, to elucidate the physiological roles of the C. elegans DAO and DDOs, we characterized several phenotypes of four C. elegans mutants in which each gene is partially deleted and inactivated. We also determined free D-amino acid contents in several worm samples using high-performance liquid chromatography (HPLC) techniques. We will report the phenotypes of the C. elegans mutants in comparison with those of wild-type C. elegans, as well as alterations in D-amino acid levels within the body.
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Inoue, Takao, Sekine, Masae, Saitoh, Yasuaki, Arai, Hiroyuki, Furuchi, Takemitsu, Sakamoto, Taro, Okutsu, Mari, Homma, Hiroshi, Katane, Masumi
[
International Worm Meeting,
2013]
Among free D-amino acids existing in living organisms, D-serine (D-Ser) and D-aspartate (D-Asp) are the most intensively studied. In mammals, D-Ser has been proposed as a neuromodulator that regulates L-glutamate (L-Glu)-mediated activation of the N-methyl-D-Asp (NMDA) receptor by acting as a co-agonist. On the other hand, several lines of evidence suggest that D-Asp plays important roles in regulating hormone secretion and steroidogenesis. D-Amino acid oxidase (DAO) and D-Asp oxidase (DDO) are known as stereospecific degradative enzymes that catalyze the oxidative deamination of D-amino acids. Mammalian DAO and DDO are presumed to regulate endogenous D-Ser and D-Asp levels, respectively. Previously, we demonstrated that D-Ser, D-Asp, D-Glu and D-alanine (D-Ala) are present in nematode Caenorhabditis elegans, a multicellular model animal. We also found that C. elegans has at least one active DAO gene and three active DDO genes (DDO-1, DDO-2 and DDO-3), and that the spatiotemporal distributions of these enzymes in the body of C. elegans differ from one another. Furthermore, our previous study showed that alterations of brood size and hatching rate are observed in four C. elegans mutants lacking each gene for the DAO and DDOs. Interestingly, lifespan extension was observed in the DDO-3 mutant. To characterize the mechanism of lifespan extension in the DDO-3 mutant, we performed genetic epistasis experiments to test interactions between the DDO-3 gene and other known longevity pathways. The results suggest that DDO-3 is involved in caloric restriction-induced lifespan extension but not in insulin/IGF signaling pathway, NAD/sir2 pathway nor mitochondrial electron transport system. We also found that D-Glu and L-tryptophan (L-Trp) accumulate throughout life in the DDO-3 mutant. Now we are investigating the relationship between aging and the accumulations of D-Glu and L-Trp.
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[
International Worm Meeting,
2017]
Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.
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[
International Worm Meeting,
2021]
Microbiome-derived metabolites are able to impact the host's nervous system through the gut-brain axis. D-amino acids (D-AAs), the D-enantiomers of prevalent L-amino acids, have unique functions such as neuromodulators in animals including mollusks, rodents, and primates, while microbiota is a known contributor to the source of D-AAs in animals. Among all D-AAs, D-Ala is a potent agonist of the glycine binding site of N-methyl-D-aspartate (NMDA) glutamate receptors in vitro. Additionally, D-Ala immunoreactivity in pancreatic beta-cells and adrenocorticotropic hormone (ACTH)-secreting cells suggests its involvement in glucose homeostasis. While several researches showed microbiota as a major source of D-Ala in animals, the effect of bacterial D-Ala synthesis and metabolism on host physiology has not been studied. In this study, we fed wild type Caenorhabditis elegans N2 with Escherichia coli mutants with knockout of genes relevant to D-Ala biosynthesis (dadX, alr) and D-Ala metabolism (ddlA), and then examined changes in C. elegans phenotypes with or without the presence of high glucose. All three bacterial mutants displayed similar growth curves in nutrient-rich liquid media, but decreased D-Ala/total alanine ratio compared to the parental E. coli strain. To prevent the introduction of exogenous D-Ala, we cultured C. elegans on peptone-free NGM media. We found no statistically significant differences in life span when fed on the selected E. coli mutants. However, we observed a slight avoidance to all three bacterial mutants compared to the parental strain. When amended with 40 mM glucose, deltaddlA significantly decreased the life span of N2. No food preference was observed on glucose-amended plates. These results indicate that the combination of deficiency in bacterial DdlA activity and high glucose led to a decreased life span in C. elegans.
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[
International Worm Meeting,
2003]
The ion channel formed by MEC-4 and MEC-10 is proposed to mediate sensory mechanotransduction in C. elegans touch cells. Initial efforts to reconstitute this channel in heterologous cells have shown that MEC-4 and MEC-10 form an amiloride-sensitive, Na+-selective ion channel complex together with MEC-2 stomatin and MEC-6 paraoxonase (1, 2). We are using double mutant cycle analysis to map the interaction surface between the pore-forming subunits, MEC-4 and MEC-10. Our general approach is to co-express wild-type and mutant forms of MEC-4 and MEC-10 and identify interacting amino acid residues using the antagonist, amiloride, as a probe. Preliminary findings have already identified one such interaction site: the d position. The importance of the d position was first highlighted by gain-of-function mutations in genes encoding ASC proteins in C. elegans (3, 4) that cause neuronal degeneration in vivo. In both MEC-4 and MEC-10, the wild-type residue is an alanine. Thus far, we have analyzed mutations that cause neuronal degeneration in vivo (T, V, and D) and mutations that we predict will be similar to wild-type (S, C). Our initial results are consistent with the idea that d position forms part the interaction surface between MEC-4 and MEC-10. We speculate that residues at this position contribute to a gate that regulates access to the amiloride binding site in the ion channel pore. 1. M. B. Goodman et al., Nature 415, 1039-42. (2002); 2. D. S. Chelur et al., Nature 420, 669-673 (2002); 3. M. Driscoll, M. Chalfie, Nature 349, 588-593 (1991); 4. M. Chalfie, E. Wolinsky, Nature 345, 410-416 (1990).
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[
International C. elegans Meeting,
1999]
Bodywall muscles are derived from four of the embryonic founder cells as well as the postembryonic M blast cell. We are studying patterning of both the embryonic and postembryonic muscles in order to understand how myogenic cell fates are specified during development. As an entry point to study muscle patterning during embryonic development, we characterized enhancer elements from the
hlh-1 regulatory region that are able to drive reporter expression in muscle precursors of the MS, D and C lineages. Analyses of these elements suggested regulatory roles of the Hox genes and the
hlh-1 gene itself. These analyses also suggested regulatory mechanisms involving unknown bZIP, bHLH and novel transcription factors. We are currently in the process of genetically identifying these factors. The postembryonic M lineage provides an alternative model to study myogenic fate specification. The M lineage gives rise to 14 bodywall muscles, 2 coelomocytes and 16 sex muscles. A number of genes had previously been identified to function in patterning the M lineage, including the Hox gene
mab-5 and the bHLH transcription factor twist (1, 2, 3). The role of the Hox factors in patterning the M lineage has been something of a mystery:
mab-5 is expressed during the entire M lineage, but
mab-5 mutants cause only limited lineage transformation. We will describe a series of experiments indicating a more central role for the Hox genes in activating twist and specifying the M lineage. We found that
lin-39 mab-5 double mutants fail to activate twist and completely lack products of the M lineage. Expression (either ectopic or in a normal pattern) of either Hox gene is sufficient to activate twist expression. However, twist activation is not sufficient for specification of the M lineage. Current efforts are directed towards identification of other factors involved in specifying the M lineage. 1. Kenyon, C. (1986), Cell 46: 477-487 2. Harfe B. D., Vaz Gomes A., Kenyon C., Liu J., Krause M., Fire A. (1998) Genes & Dev. 12: 2623-2635 3. A. Corsi, S. Kostas, E. Jorgensen, A. Fire, and M. Krause (poster)