Kimura Y et al. (2008) Z Naturforsch C "Nematicidal activities of acetylene compounds from Coreopsis lanceolata L."

Kimura Y, Fujioka S, Shimada A, Hiraoka K, Kawano T

[Z Naturforsch C, 2008]

1-Phenylhepta-1,3,5-triyne (1), 5-phenyl-2-(1'-propynyl)-thiophene (2), and 2-(3'-acetoxy-1'-propynyl)-5-phenylthiophene (3) were isolated from Coreopsis lanceolata L., and their structures identified by spectroscopic methods. Compounds 1 and 2 showed effective nematicidal activities against Bursaphelenchus xylophilus and Caenorhabditis elegans, but had hardly any effect against Pratylenchus penetrans. Compound 3 did not show any effective nematicidal activity.

Kimura Y et al. (2007) Z Naturforsch C "Nematicidal activity of 5-hydroxymethyl-2-furoic acid against plant-parasitic nematodes."

Hayashi A, Shimada A, Tani S, Kawano T, Fujioka S, Kimura Y, Ohtani K

[Z Naturforsch C, 2007]

A nematicide, 5-hydroxymethyl-2-furoic acid (1), was isolated from cultures of the fungus Aspergillus sp. and its structure was identified by spectroscopic analysis. Compound 1 showed effective nematicidal activities against the pine wood nematode Bursaphelenchus xylophilus and the free-living nematode Caenorhabditis elegans without inhibitory activity against plant growth, but 1 did not show any effective nematicidal activity against Pratylenchus penetrans.

Kimura, Y. et al. (2011) International Worm Meeting "Tubulin polyglutamylation in the sensory cilia is flexibly regulated in response to the environmental stresses."

Kunitomo, H., Hameed, S., Setou, M., Iino, Y., Kimura, Y., Blacque, O. E., Kaplan, O. I.

[International Worm Meeting, 2011]

The primary cilium is a microtubule (MT)-based organelle that is significant for sensory signal transduction. Sensory channels and receptors are transported and condensed on the sensory cilia through intraflagellar transport (IFT) along MT. Polyglutamylation is a reversible post-translational modification (PTM) seen in a specific glutamate residue near the carboxyl terminus of a- and b-tubulins. We employed a genetic approach using C. elegans to analyze the regulatory mechanisms and physiological roles of this unique PTM. We recently identified the enzyme genes for tubulin polyglutamylation genetically and biochemically. Adding glutamates is accomplished by a member of tubulin tyrosine ligase-like (TTLL) family ttll-4, whereas shortening polyglutamate chains is performed by ccpp-6, a gene encoding a cytosolic carboxypeptidase. Since in C. elegans, tubulin polyglutamylation is exclusively detected in the sensory cilia, we studied the phenotypes observed in ciliated sensory neurons using ttll-4 and ccpp-6 mutants. The modification is completely lost in ttll-4 mutant worms. In contrast, ccpp-6 mutant showed a mild increase of the modification. Both mutants did not have severe morphological defects in sensory cilia, however, whose anterogade movement of IFT particles was affected. In ttll-4 mutants, the speed of particle movement was decreased. Conversely, ccpp-6 mutants worms showed an increase of the movement velocity. Thus, the efficiency of IFT could be controlled by the extent of polyglutamylation on MT. Additionally, ttll-4 mutants showed the sensitivity reduction measured by behavioral analyses such as chemotaxis for NaCl and osmotic avoidance, indicating tubulin polyglutamylation is required for chemosensory behavior. Based on the results above, we hypothesized that this bidirectional PTM could be used to modify the sensitivity of sensory neurons. Intriguingly, the extent of tubulin polyglutamylation is increased by the treatment of various environmental stresses such as physical vibration, temperature, and high osmotic pressure. We also identified the signaling pathways to control this stress-responsive enhancement. Taken together, tubulin polyglutamylation in sensory cilia is not a rigid system but a flexible modification in response to environmental stimulations and could be used for the modulation of sensory plasticity.

Kimura Y et al. (2000) Japanese Worm Meeting "CaM-K-mediated signal cascade is conserved in some neurons of C. elegans in vivo"

Kimura Y, Tokumitsu H, Eto K

[Japanese Worm Meeting, 2000]

Ca2+/CaM protein kinases (CaM-K) is kown to have important roles for Ca2+-dependent signal transduction. CaM-K-mediated signal cascade consists of the upstream CaM-kinase kinase (CaM-KK) and downstream CaM kinase I (CaM-KI) and CaM-kinase IV (CaM-KIV). In addition, a transcription factor cAMP-responsive element binding protein (CREB) is activated by phosphorylation with downstream CaM-KIV. We characterized C. elegans orthologues of CaM-KK (ckk-1) and CaM-KI (cmk-1) and found that CaM-KK-CaM-KI-CREB cascade is conserved and operated both in vitro and in transfected cells. CMK-1is highly homologous to mammalian CaM-KI and is activated by CKK-1 through phosphorylation of Thr179 in a Ca2+ /CaM-dependent manner. An activated CMK-1 can phosphorylate CREB protein and activate its transcription activity. We constructed ckk::gfp and cmk::gfp fusion genes and analyzed the expression patterns of both genes. A ckk-1::gfp reporter was expressed small number of head neruons after L3 stage. On the other hand, cmk-1::gfpwas seen in the most of the neurons from embryo to adult. To see the localization of activated CREB in vivo, we made a reporter worm that conains 4XCRE::gfp fusion gene. GFP expression cannot be seen in normal development but the expression in small number of neurons in head and tail can be induced by cAMP soaking. This induction can also occur by the expression of constitutively active form of CMK-1. Interestingly, this expression can be seen only after L3 stage and the expression was decreased or disappeared in adult. We also found that the constitutively active form of CMK-1 induced the malformaion of vulva and Egl phenotype. These results suggest that CaM-KI-CREB cascade of C. elegans is also conserved in vivo but it works only in small number of neurons.

Kimura Y et al. (2010) J Biol Chem "Identification of tubulin deglutamylase among Caenorhabditis elegans and mammalian cytosolic carboxypeptidases (CCPs)."

Konishi Y, Kurabe N, Kimura Y, Iino Y, Setou M, Kunitomo H, Blacque OE, Kaplan OI, Tsutsumi K, Ikegami K

[J Biol Chem, 2010]

Tubulin polyglutamylation is a reversible post-translational modification, serving important roles in microtubule (MT)-related processes. Polyglutamylases of the tubulin tyrosine ligase-like (TTLL) family add glutamate moieties to specific tubulin glutamate residues, whereas as yet unknown deglutamylases shorten polyglutamate chains. First we investigated regulatory machinery of tubulin glutamylation in MT-based sensory cilia of the roundworm Caenorhabditis elegans. We found that ciliary MTs were polyglutamylated by a process requiring ttll-4. Conversely, loss of ccpp-6 gene function, which encodes one of two cytosolic carboxypeptidases (CCPs), resulted in elevated levels of ciliary MT polyglutamylation. Consistent with a deglutamylase function for ccpp-6, overexpression of this gene in ciliated cells decreased polyglutamylation signals. Similarly, we confirmed that overexpression of murine CCP5, one of two sequence orthologs of nematode ccpp-6, caused a dramatic loss of MT polyglutamylation in cultured mammalian cells. Finally, using an in vitro assay for tubulin glutamylation, we found that recombinantly expressed Myc-tagged CCP5 exhibited deglutamylase biochemical activities. Together, these data from two evolutionarily divergent systems identify C. elegans CCPP-6 and its mammalian ortholog CCP5 as a tubulin deglutamylase.

Kimura Y et al. (2018) Sci Rep "Environmental responsiveness of tubulin glutamylation in sensory cilia is regulated by the p38 MAPK pathway."

Ikegami K, Tsutsumi K, Kaplan OI, Setou M, Fujiwara M, Kaneko T, Blacque OE, Kimura Y, Teramoto T, Hameed S, Konno A, Ishihara T

[Sci Rep, 2018]

Glutamylation is a post-translational modification found on tubulin that can alter the interaction between microtubules (MTs) and associated proteins. The molecular mechanisms regulating tubulin glutamylation in response to the environment are not well understood. Here, we show that in the sensory cilia of Caenorhabditis elegans, tubulin glutamylation is upregulated in response to various signals such as temperature, osmolality, and dietary conditions. Similarly, tubulin glutamylation is modified in mammalian photoreceptor cells following light adaptation. A tubulin glutamate ligase gene ttll-4, which is essential for tubulin glutamylation of axonemal MTs in sensory cilia, is activated by p38 MAPK. Amino acid substitution of TTLL-4 has revealed that a Thr residue (a putative MAPK-phosphorylation site) is required for enhancement of tubulin glutamylation. Intraflagellar transport (IFT), a bidirectional trafficking system specifically observed along axonemal MTs, is required for the formation, maintenance, and function of sensory cilia. Measurement of the velocity of IFT particles revealed that starvation accelerates IFT, which was also dependent on the Thr residue of TTLL-4. Similarly, starvation-induced attenuation of avoidance behaviour from high osmolality conditions was also dependent on ttll-4. Our data suggest that a novel evolutionarily conserved regulatory system exists for tubulin glutamylation in sensory cilia in response to the environment.

Kimura Y et al. (2002) EMBO Rep "A CaMK cascade activates CRE-mediated transcription in neurons of Caenorhabditis elegans."

Mitani S, Freedman JH, Kobayashi R, Corcoran EE, Muramatsu M, Muramatsu MA, Kimura Y, Tokumitsu H, Means AR, Hagiwara M, Gengyo-Ando K, Eto K

[EMBO Rep, 2002]

Calcium (Ca2+) signals regulate a diverse set of cellular responses, from proliferation to muscular contraction and neuro-endocrine secretion. The ubiquitous Ca2+ sensor, calmodulin (CaM), translates changes in local intracellular Ca2+ concentrations into changes in enzyme activities. Among its targets, the Ca2+/CaM-dependent protein kinases I and IV (CaMKs) are capable of transducing intraneuronal signals, and these kinases are implicated in neuronal gene regulation that mediates synaptic plasticity in mammals. Recently, the cyclic AMP response element binding protein (CREB) has been proposed as a target for a CaMK cascade involving not only CaMKI or CaMKIV, but also an upstream kinase kinase that is also CaM regulated (CaMKK). Here, we report that all components of this pathway are coexpressed in head neurons of Caenorhabditis elegans. Utilizing a transgenic approach to visualize CREB-dependent transcription in vivo, we show that this CaMK cascade regulates CRE-mediated transcription in a subset of head

Y Kimura et al. (1999) International C. elegans Meeting "Ca2+/CaM-dependent protein kinases (CaM-K)-mediated signal cascade is conserved in C. elegans"

K Eto, H Tokumitsu, Y Kimura, M Muramatsu

[International C. elegans Meeting, 1999]

In mammals, Ca 2+ /CaM protein kinases (CaM-K) is thought to be important for Ca 2+ -dependent signal transduction. This cascade consists of the upstream CaM-kinase kinase (CaM-KK) and downstream CaM kinase I (CaM-KI) and CaM-kinase IV (CaM-KIV). In the present study, we have identified CaM-KK and CaM-KI orthologue of C. elegans (named Ce CaM-KK and Ce CaM-KI, respectively) and analyzed their biochemical activities. Ce CaM-KK has two unusual features in the catalytic domain which is conserved between species, that is, a lack of acidic residues important for substrate recognition and an Arg-Pro rich insert (RP-domain) which is essential for the recognition and activation of CaM-KIV and CaM-KI. Indeed, Ce CaM-KK activated mammalian CaM-KIV in vitro . Ce CaM-KI is highly homologous to mammalian CaM-KI and is activated by Ce CaM-KK through phosphorylation of Thr179 in a Ca 2+ /CaM-dependent manner. Truncation at residue 295 in CeCaM-KI generates a constitutively active enzyme, suggesting that COOH-terminal at residue 295 contains autoinhibitory and CaM-binding domains. Unlike mammalian CaM-KI, Ce CaM-KI mainly localized in the nucleus of transfected mammalian cells by the virtue of the NH 2 -terminal six residues containing a functional nuclear localization signal. Taken together, these results suggest that CaM-KK/CaM-KI cascade in C. elegans is conserved and operated both in vitro and intact cells. Current research aims to the physiological functions of Ce CaM-KK and Ce CaM-KI cascade in vivo . Analyses for the expression patterns and isolation of mutant worms for both genes are underway.

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.

Perreault J et al. (2007) Mol Biol Evol "Ro-associated Y RNAs in metazoans: evolution and diversification."

Perreault J, Perreault JP, Boire G

[Mol Biol Evol, 2007]

The Y genes encode small non-coding RNAs whose functions remain elusive, whose numbers vary between species, and whose major property is to be bound by the Ro60 protein (or its ortholog in other species). To better understand the evolution of the Y gene family, we performed a homology search in 27 different genomes along with a structural search using Y RNA specific motifs. These searches confirmed that Y RNAs are well conserved in the animal kingdom and resulted in the detection of several new Y RNA genes, including the first Y RNAs in insects and a second Y RNA detected in Caenorhabditis elegans. Unexpectedly, Y5 genes were retrieved almost as frequently as Y1 and Y3 genes, and, consequently are not the result of a relatively recent apparition as is generally believed. Investigation of the organization of the Y genes demonstrated that the synteny was conserved among species. Interestingly, it revealed the presence of six putative "fossil" Y genes, all of which were Y4 and Y5 related. Sequence analysis led to inference of the ancestral sequences for all Y RNAs. In addition, the evolution of existing Y RNAs was deduced for many families, orders and classes. Moreover, a consensus sequence and secondary structure for each Y species was determined. Further evolutionary insight was obtained from the analysis of several thousand Y retropseudogenes among various species. Taken together, these results confirm the rich and diversified evolution history of Y RNAs.