Nukazuka, Akira et al. (2009) International Worm Meeting "Up- and down-regulation of TOR-Raptor and TOR-Rictor complexes by semaphorin in C. elegans."

Nukazuka, Akira, Takagi, Shin, Oda, Yoichi, Tamaki, Shusaku, Fujisawa, Hajime

[International Worm Meeting, 2009]

Semaphorins regulate a wide range of developmental events. In C. elegans, the signal controls morphogenesis of epidermal cells, as shown by the finding that semaphorin mutants display aberrant arrangement of epidermis-derived tissues, rays. To gain insight into semaphorin signaling, we conducted a screen and isolated a loss-of-function mutation in Rictor, which suppressed the ray phenotype in semaphorin mutants. Rictor is known to make a complex TORC2 with TOR. Independently of TORC2, TOR also makes a complex TORC1 with Raptor. Signaling via TORC1 promotes mRNA translation, whereas TORC2 stabilizes F-actin formation. Consistently with previous reports on an antagonistic relationship between two TORC pathways, we found that Raptor overexpression, similarly to Rictor deficiency, suppressed the ray phenotype in semaphorin mutants. Conversely, TORC1 inhibition and TORC2 activation phenocopied semaphorin mutants. Thus, our data indicate that TORC1 cooperates while TORC2 antagonizes with semaphorin signaling. In addition, knockdown of 4EBP, which is a TORC1 substrate and a translation repressor, suppressed the semaphorin mutant phenotype, suggesting that TORC1 mediates the signal by repressing 4EBP and thus activating translation. Likewise, deficiency of PKCa, which is a TORC2 substrate, also suppressed the mutant phenotype, implicating that TORC2 inhibition, which probably results in actin destabilization, also mediates the signal. Therefore, our data imply that TOR serves to integrate the semaphorin input and subsequently transduce it into divergent downstream cascades.

Suzuki, Motoshi et al. (2009) International Worm Meeting "Infrared laser-mediated gene induction in targeted single cells in C. elegans."

Kamei, Yasuhiro, Yuba, Syunsuke, Takagi, Shin, Oda, Yoichi, Suzuki, Motoshi

[International Worm Meeting, 2009]

Methods for manipulating gene expression at the experimentalist''s discretion would provide a useful means to analyze diverse biological processes. Heat shock promoters drive the expression of downstream genes in response to heat stimuli. Previous studies showed that irradiation with the visible laser light (440 nm) can induced heat shock response-mediated expression of transgene in targeted cells. However, irradiation with a 440 nm laser, which was originally used for cell ablation, may have detrimental effects on cells. We have developed a novel microscope system called infrared laser-evoked gene operator (IR-LEGO)(Kamei et al., Nat Methods 2009 Jan;6(1):79-81.). Because the absorption coefficient of water is higher in the infrared (IR) region, the IR light would heat cells more efficiently than the visible light. Application of IR-LEGO to C. elegans revealed that irradiation for 1s was sufficient for gene induction in a targeted single seam cell. Under appropriate conditions, 40% of irradiated seam cells expressed GFP under the control of a heat shock promoter, and all irradiated cells developed normally, indicating that the IR irradiation did not cause cell damage. We also found that the expression of MAB-5 and MIG-24 protein induced with IR-LEGO each rescued the phenotypes of the corresponding mutants. We also report on current attempts to induce gene expression in targeted cells of early embryos and in single neurons of the nerve ring.

Ahringer, Julie et al. (2011) International Worm Meeting "A role for H4K20me1 in X chromosome dosage compensation."

Lieb, Jason, Dong, Yan, Vielle, Anne, Kotwaliwale, Chitra, Strome, Susan, Lang, Jackie, Dernburg, Abby, Rechtsteiner, Andreas, Ahringer, Julie, Ercan, Sevinc, Liu, Shirley, Liu, Tao, Dose, Andrea, Egelhofer, Thea

[International Worm Meeting, 2011]

In C. elegans, the dosage compensation complex (DCC) equalizes X chromosome gene dosage between XO males and XX hermaphrodites by repressing X transcription by two-fold specifically in hermaphrodites. The DCC is comprised of proteins homologous to the condensin complex, and it localizes to the X chromosomes in hermaphrodites but not in males. The mechanism by which the DCC downregulates expression in XX animals remains unclear. A genome-wide map of 19 histone modifications in two developmental stages of C. elegans (Liu et al, 2011) showed that the monomethylation of histone H4 at lysine 20 (H4K20me1) is enriched on the X chromosome of XX embryos and L3 larvae. H4K20me1 is also associated with actively transcribed genes on all chromosomes. The X-enrichment of H4K20me1 suggests a possible role for this modification in dosage compensation. Here, we report that H4K20me1 is enriched on the X chromosomes of XX hermaphrodites, but not in XO males. Enrichment of H4K20me1 on the X occurs after the localization of the DCC to the X and depends on the DCC function. In mammals, PR-Set7 catalyzes monomethylation of H4K20. RNAi knockdown of set-1, the likely C. elegans ortholog of PR-Set7, results in synthetic lethality with a number of dosage compensation mutations. These results support a function for H4K20me1 in dosage compensation. The X chromosome is also silenced in the germline in a DCC-independent manner. We found that in the mitotically proliferating zone of the germline, H4K20me1 is abundant on all chromosomes, but as nuclei enter meiosis, H4K20me1 levels are globally reduced. In contrast to somatic nuclei, H4K20me1 is specifically absent from the X chromosome during the meiotic prophase. H4K20me1 was recently shown to be important for chromosome condensation in mammals (Oda et al, 2009), possibly by recruiting condensin II (Liu et al, 2010). Our data indicate that the condensin-like C. elegans DCC preferentially increases H4K20me1 levels on the active X-linked genes. One plausible model for the role of H4K20me1 in dosage compensation is that preferential increase of H4K20me1 to the X may result in further recruitment of condensins to lower access of the transcription machinery to active X-linked genes. Liu et al (2011) Genome Res. 21, 227-36. Oda et al (2009) Mol Cell Biol. 29, 2278-95. Liu et al (2010) Nature. 466(7305):508-12.

Miyanishi, Yosuke et al. (2011) International Worm Meeting "Functional imaging of neuronal activity for 2-nonanone stimulation."

Nakai, Junichi, Kimura, Kotaro, Miyanishi, Yosuke

[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.

Pooranachithra M et al. (2019) Life Sci "Unravelling the wound healing ability and mode of action of pyridine carboxamide oxime using Caenorhabditis elegans as potential prescreen wound model."

Pooranachithra M, Venkateswaran K, Sekhar DS, Gnanasekaran J, Balamurugan K, Deepa M, Prabhanand BJ

[Life Sci, 2019]

AIM: In the current scenario of ethical issues related to animal usage in research, the present study was intended to explore the proficient utility of nematode, Caenorhabditis elegans as wound model in preliminary screening of wound healing therapeutics. MAIN METHODS: In this study, a new wounding protocol and quantitative assessment strategies for various healing parameters [survival, Reactive Oxygen Species (ROS), calcium signals, F-actin dynamics, new collagen synthesis and wound induced anti-microbial peptides] were developed and used for preliminary screening of wound healing actives from natural sources. Wound healing ability of positive lead Tridax procumbens (TP) and its major phytocompounds [Octa decenoic acid (ODA), Pyridine carboxamide oxime, known as Nicotinamide (NA) and Dimethyl Benz[c]acridine (DMB)] were assessed using C. elegans wound model and cell lines scratch wound healing assay. Mode of action of active lead was elucidated using metabolome analysis coupled with MALDI-MS followed by molecular docking. KEY FINDINGS: From the four tested methanolic extracts, TP was chosen as positive lead compared to control, Benzalkonium chloride (BKC) based on survival and new collagen synthesis analyses. Results indicated that the wound healing ability of TP was majorly contributed by NA. Further, it was found that NA acts in chloromethyl nicotinamide derivative form by interacting with the known wound healing biomarker, glycogen synthase kinase 3 (GSK-3) to exert wound healing ability. SIGNIFICANCE: The study evidenced that C. elegans, could be a reliable wound model for high-throughput screening of wound healing actives and to identify their possible mode of action.

Hirabayashi J et al. (2000) J Chromatogr A "Reinforcement of frontal affinity chromatography for effective analysis of lectin-oligosaccharide interactions."

Hirabayashi J, Kasai K-I, Arata Y

[J Chromatogr A, 2000]

Frontal affinity chromatography is a method for quantitative analysis of biomolecular interactions. We reinforced it by incorporating various merits of a contemporary liquid chromatography system. As a model study, the interaction between an immobilized Caenorhabditis elegans galectin (LEC-6) and fluorescently labeled oligosaccharides (pyridylaminated sugars) was analyzed. LEC-6 was coupled to N-hydroxysuccinimide-activated Sepharose 4 Fast Flow (100 microm diameter), and packed into a miniature column (e.g., 10 x 4.0 mm, 0.126 ml). Twelve pyridylaminated oligosaccharides were applied to the column through a 2-ml sample loop, and their elution patterns were monitored by fluorescence. The volume of the elution front (V) determined graphically for each sample was compared with that obtained in the presence of an excess amount of hapten saccharide, lactose (V0); and the dissociation constant, Kd, was calculated according to the literature [K. Kasai, Y. Oda, M. Nishikawa, S. Ishii, J. Chromatogr. 376 (1986) 33]. This system also proved to be useful for an inverse confirmation; that is, application of galectins to an immobilized glycan column (in the present case, asialofetuin was immobilized on Sepharose 4 Fast Flow), and the elution profiles were monitored by fluorescence based on tryptophan. The relative affinity of various galectins for asialofetuin could be easily compared in terms of the extent of retardation. The newly constructed system proved to be extremely versatile. It enabled rapid (analysis time 12 min/cycle) and sensitive (20 nM for pyridylaminated derivatives, and 1 microg/ml for protein) analyses of lectin-carbohydrate interactions. It should become a powerful tool for elucidation of biomolecular interactions, in particular for functional analysis of a large number of proteins that should be the essential issues of post-genome projects.