Silimon, Ruth L. et al. (2013) International Worm Meeting "Overexpression, purification and characterization of C. elegans glyceraldehyde-3-phosphate dehydrogenase isozymes."

Walstrom, Katherine M., Spengler, Justin W., Valbuena, Valeria S. M., Gautier, Megan K., Silimon, Ruth L., Schwabe-Warf, Derek

[International Worm Meeting, 2013]

Glyceraldehyde-3-phosphate (G3P) dehydrogenase (GPD) is the glycolytic enzyme that adds inorganic phosphate to its substrate so that net ATP production is possible. C. elegans has 4 different GPD isozymes. Embryonic GPD-1 and GPD-4 are nearly identical, while the homologous GPD-2 and GPD-3 are expressed in postembryonic worms (Yarbrough and Hetch, JBC 259, 14711, 1984; Huang et al., 1989, JMB 206, 411). GPD-3 is involved in protection from anoxia (Mendenhall et al., 2006, Genetics 174, 1173) and upregulated in dauers and daf-2 mutants (McElwee et al. 2006, Mech. Age. Dev. 127, 922). We subcloned, overexpressed, and purified both GPD-1/4 and GPD-3 and performed enzyme kinetics studies with all three substrates under the optimal reaction conditions at pH 8.0-8.5. Both isozymes exhibited substrate inhibition at high concentrations of phosphate and NAD+, but the embryonic form was inhibited at lower concentrations of phosphate or NAD+ than GPD-3. GPD-1/4 appeared to have a higher specific activity than GPD-3. We determined that the KM values for NAD+, phosphate, and G3P for GPD-3 were 0.3 mM, 0.4 mM, and 2.6 mM, respectively. The KM values for NAD+ and phosphate for GPD-1/4 were 1 mM and ~0.6 mM. The KM values from a partially purified GPD preparation from a mixed population of C. elegans had NAD+ and G3P KM values of 1 mM and 0.3 mM, respectively. Based on the results of Yarbrough and Hetch, we expected the enzyme activity of the adult GPD-2 and GPD-3 enzymes to predominate in the endogenous GPD mixture purified from the worms. Based on our current kinetics results, the endogenous protein mixture had a KM for NAD+ more similar to the GPD-1/4 enzyme. We are continuing to compare the activities of the C. elegans GPD enzymes to determine which isozyme(s) are active in the endogenous GPD mixture.

Katherine M Walstrom et al. (2001) International C. elegans Meeting "Biochemical Characterization of C. elegans Malate Dehydrogenase"

Danny Gonzalez, Katherine M Walstrom

[International C. elegans Meeting, 2001]

In my laboratory, we are overexpressing and biochemically characterizing C. elegans malate dehydrogenase (MDH), the last enzyme in the citric acid cycle. This enzyme, encoded by putative gene F20H11.3, appears to be the mitochondrial form of MDH, since it contains a mitochondrial import presequence. We have overexpressed and purified a version of this enzyme with the mitochondrial import presequence removed at the predicted cleavage site of the mitochondrial processing protease. The purified MDH enzyme has malate dehydrogenase activity that follows Michaelis-Menten kinetics when plotted versus oxaloacetate concentration, and the resulting Km is similar to the Km values for other MDH enzymes. Interestingly, the temperature dependence of the enzyme is a bell-shaped curve with a peak at approximately 30degC, suggesting that the enzyme is adapted to the growing temperature of C. elegans . We are grateful to Yuji Kohara for providing the cDNA clone of MDH.

Jacobs D et al. (1998) Midwest Worm Meeting "LIN-1 ETS IS PHOSPHORYLATED BY ERK MAP KINASE"

Jacobs D, Kornfeld K

[Midwest Worm Meeting, 1998]

LIN-1 encodes a 441 amino acid protein that is likely to be a DNA-binding transcription factor, since it contains an N-terminal ETS domain. lin-1(lf) mutations cause a multivulva phenotype, and lin-1(gf) mutations cause a vulvaless phenotype, suggesting lin-1 is a switch molecule and lin-1 activity prevents Pn.p cells from adopting the primary vulval fate. Genes in the Ras signaling pathway promote the primary fate, and epistasis analyses indicate lin-1 functions downstream of these genes, including mpk-1 Erk MAP kinase, suggesting lin-1 is negatively regulated by the action of this signaling pathway. To investigate whether LIN-1 is directly regulated by MAP kinase, we produced LIN-1 protein in E. coli and assayed partially purified protein for phosphorylation by murine Erk2 MAP kinase in vitro. Full-length LIN-1 protein was an excellent substrate with a Km of 0.2 uM, significantly lower that the 3.3 uM Km of myelin basic protein, a protein frequently used to assay Erk activity. The C-terminal region of LIN-1 (residues 281-441) was necessary for phosphorylation and sufficient to function as a good Erk2 substrate. The lin-1(gf) mutations all affect the C-terminal region of LIN-1, suggesting this region is required for negative regulation of LIN-1. These observation indicate that phosphorylation of the C-terminal region of LIN-1 by Erk negatively regulates LIN-1 activity. We produced and assayed two mutant LIN-1 proteins that contained the alterations caused by two different lin-1(gf) mutations. These proteins had Km values that were significantly higher the Km of wild-type LIN-1, suggesting the lin-1(gf) mutations function by diminishing LIN-1 phosphorylation by Erk. The lin-1(gf) mutations may affect a recognition motif for Erk MAP kinase.

Isaac RE et al. (1991) International C. elegans Meeting "BIOGENIC AMINE METABOLISM IN C. ELEGANS AND PARASITIC NEMATODES: N- ACETYLATION OF 5-HT, DOPAMINE AND OCTOPAMINE."

Isaac RE, Muimo R, Eaves L

[International C. elegans Meeting, 1991]

Oxidative deamination and O-methylation are important reactions in the catabolism of biogenic amines in vertebrates. Although, oxidative deamination is generally considered to be a major pathway for the inactivation of 5-HT and catecholamines in animal parasitic nematodes, this reaction has not been detected in a number of nematode species, including C. eleaans. We have shown, by incubating cut nematodes (C. eleaans, Ascaridia aalli, Brugia pahanqi and Nippostronaylus brasiliensis) with radiolabelled amine, that 5-HT, octopamine and dopamine are taken up by nematode tissues and are metabolised to their N-acetylated derivatives. The N-acetylation reaction requires acetyl-coenzyme A as a co-factor and the activity is localised in a soluble fraction. As yet, we have found no evidence for the involvement of oxidative deamination in the metabolism of these amines in nematodes. No monoamine oxidase activity could be detected in mitochondrial fractions prepared from whole nematodes. Some of the properties of the amine Nacetyltransferase from the parasitic nematode, A. aalli, are described. A single enzyme appears to be responsible for the N-acetylation of 5-HT and octopamine and this activity requires acetyl CoA as a co-factor. The N- acetyltransferase is a soluble enzyme and has an Mr of around 23,000. It has a higher affinity for octopamine (Km, 33uM) compared to 5-HT ( Km, 550uM). The activity towards octopamine is competitively inhibited by tyramine, dopamine and noradrenaline suggesting that these catecholamines may also serve as substrates for this arylalkylamine N- acetyltransferase.

Gu, Wei et al. (2011) International Worm Meeting "Purification and characterization of C. elegans cytoplasmic malate dehydrogenase."

Spengler, Justin W., Walstrom, Katherine M., Gu, Wei, Lindsay, Penelope L.

[International Worm Meeting, 2011]

Malate dehydrogenase (MDH) is the last enzyme in the citric acid cycle. MDH catalyzes the conversion of NAD+ and malate to NADH and oxaloacetate. Since the forward reaction is energetically unfavorable, the reverse reaction is usually studied. Eukaryotes have two versions of this enzyme, one that is imported into the mitochondria and one that remains in the cytoplasm. The cytoplasmic MDH in C. elegans (F46E10.10) was originally mis-classified as lactate dehydrogenase. Hold and Riddle (Mech. Aging Dev. 124, 779, 2003) realized that the amino acids in the active site were compatible with malate binding rather than lactate. We named this enzyme MDH-1 and renamed the mitochondrial enzyme (F20H11.3) MDH-2 so that the naming convention corresponded to that used for other eukaryotes. We overexpressed MDH-1 in E. coli and removed the chitin binding domain tag used for the purification. In kinetic assays with the purified enzyme, MDH-1 had malate dehydrogenase activity that followed Michaelis-Menten kinetics. For the reverse reaction, we determined that the KM for oxaloacetate was 37 mM, and the KM for NADH was 70 mM. Our gel filtration results indicated that MDH-1 was active as a dimer, which is similar to the quaternary structure of most MDH enzymes (Minarik, P. et al., Gen. Physiol. Biophys. 21, 257, 2002). We are in the process of purifying endogenous MDH-1 from worms to compare its activity to our recombinant enzyme.

Muimo R et al. (1995) International C. elegans Meeting "NEMATODE ARYLALKYLAMINE N-ACETYL- TRANSFERASES: PURIFICATION AND CHARACTERISATION FROM ASCARIDIA GALLI"

Muimo R, Isaac RE

[International C. elegans Meeting, 1995]

We have previously shown that nematodes have high levels of arylalkylamine N-acetyltransferase activity and that N-acetylation could be a primary route for regulating biogenic amine levels in nematodes. Three arylalkylamine N-acetyltransferase enzymes have recently been identified and purified to electrophoretic homogeneity from a whole worm homogenate preparation of Ascaridia galli using ammonium sulphate precipitation followed by hydrophobic interaction, metal chelate and ion exchange chromatography. A major peak of activity was associated with a 40 kDa protein band on SDS-PAGE and we now report the characterisation of this enzyme. The enzyme activity was very labile and showed a strong dependency on the presence of dithiothreitol for activity during purification. The enzyme had ability to N-acetylate arylalkyl-, phenyl-, and catechol-amines and ,furthermore, the N-acetylation of 5-HT was inhibited in a dose-dependent manner by amines from these groups. Enzyme activity was maximal at pH 6.5 when measured at 37 oC with tyramine and acetyl CoA as substrates. The enzyme had a high affinity for tyramine (Km 6.6 *M) and was inhibited by Cu2+, Zn2+ and Hg2+ions (1mM). The Km for acetyl CoA was 18.8 *M. Arylamines and hydrazines, substrates for the mammalian liver acetyltransferase enzymes, did not inhibit or markedly affect the enzyme activity towards tyramine indicating that the A. galli arylalkylamine N-acetyltransferase enzyme (40 kDa) is not similar to the mammalian liver arylamine N-acetyltransferase enzymes. The other two arylalkylamine N-acetyltransferase enzyme activities are being characterised and their properties will also be reported.

Mark S Lucanic et al. (2005) International Worm Meeting "max-2 is Required for Proper Targeting of Motor Neurons in C. elegans"

Mark S Lucanic, Hwai-Jong Cheng

[International Worm Meeting, 2005]

We have isolated max-2, a mutant with major defects in motor neuron connectivity using a forward genetic screen designed to identify genes that guide the dorsal migrations of the ventral cord commissural motor neurons of C. elegans. During development the ventral cord commissural motor neurons of C. elegans send processes circumferentially towards the dorsal cord in response to the chemorepellent unc-6/netrin which is present on the ventral side of the animal. These axon guidance events are mediated by the cell surface receptors unc-5 and unc-40/DCC. We are particularly interested in identifying signaling molecules that function downstream of these receptor/ligand interactions. The signaling downstream of these receptor/ligand interactions which guides these neurons occurs through parallel and partially redundant pathways (Huang et al., 2002). Therefore, we expect that loss of function mutations in many of the genes involved in these pathways will not result in major locomotion defects but may still result in significant defects in the guidance of these neurons. To identify genes that function in these signaling pathways we have implemented a sensitive genetic screen. In the preliminary stages of this screen we have identified max-2 (motor axon defective). Max-2 animals have severe defects in circumferential motor neuron axon guidance. The axon guidance defects have varying penetrance among the different subsets of circumferential motor neurons. We have positionally cloned max-2 and found that it is an intracellular signaling molecule. The positional cloning and molecular characterization of max-2 will be presented. Huang, X., Cheng, H.-J., Tessier-Lavigne, M., and Jin, Y. (2002). MAX-1, a Novel PH/MyTH4/FERM Domain Cytoplasmic Protein Implicated in Netrin-Mediated Axon Repulsion. Neuron 34, 563-576.

P Appleford et al. (2004) European Worm Meeting "MUTAGENESIS OF CONSERVED ELEMENTS IN THE PROMOTER OF THE C. ELEGANS T-BOX GENE MAB-9 WITH A VIEW TO IDENTIFYING UPSTREAM REGULATORS."

P Appleford, A Woollard

[European Worm Meeting, 2004]

The Caenorhabditis elegans T-box gene mab-9 has a well-defined role in the cell fate specification of posterior structures. MAB-9 is required in order for two cells in the hindgut to assume their correct fates; in mab-9 mutants these cells adopt the fates of their anterior neighbours. Adult male mutants develop tail abnormalities and hermaphrodite worms have hindgut defects. Both sexes also display a weak uncoordinated phenotype during backwards movement and mab-9 has recently been shown to have a role in axon migration of a subset of motor neurons (Huang et al, 2002). We are interested in identifying cis-regulatory elements upstream of mab-9 . A comparison of the 5' regulatory sequence and introns of mab-9 with that of its closest homologue in the related nematode C. briggsae has been used to identify five regions of close homology, each containing at least one putative T-box binding site. The conserved nature of these regions suggests that they might be of importance in directing cell-specific expression of mab-9. The fact that all five homology regions contain possible T-box binding sites may indicate that mab-9 is autoregulatory or regulated by other T-box genes. To establish the importance or otherwise of each homology region in directing MAB-9 expression, we have used a GFP-tagged rescuing construct with each of the homology regions removed by site-directed mutagenesis. The ability of mutated constructs to rescue mab-9 mutants and to be properly expressed is being investigated. Regulatory sequences which appear important in the control of MAB-9 expression will be chosen for a yeast 1-hybrid screen to identify upstream regulators. Huang X, Cheng H-J, Tessier-Lavigne M, Jin Y (2002); Neuron, 34, 563-576.

Peter J Appleford et al. (2003) International Worm Meeting "The C. elegans T-box gene mab-9: Searching for regulators and interactors."

Julian Seago, Peter J Appleford, Alison Woollard

[International Worm Meeting, 2003]

The Caenorhabditis elegans T-box gene mab-9 has a well-defined role in the cell fate specification of posterior structures. MAB-9 is required in order for two cells in the hindgut to assume their correct fates; in mab-9 mutants these cells adopt the fates of their anterior neighbours. Adult male mutants develop tail abnormalities that prevent mating and hermaphrodite worms are constipated due to rectal defects. Both sexes also display a weak uncoordinated phenotype during backwards movement and mab-9 has recently been shown to have a role in axon migration of a subset of motor neurons (Huang et al, 2002).We are interested in identifying regulators of, and interactors with mab-9, as well as genes which are downstream targets. A C. elegans mixed-stage cDNA library was used in a yeast two-hybrid screen to find candidate proteins which appear to interact with MAB-9 in vitro. The significance of these interactions is currently being examined in vivo.A comparison of the 5-prime regulatory sequence of mab-9 with that of its closest homologue in the related nematode C. briggsae has identified five regions of close homology, each containing at least one putative T-box binding site. The conserved nature of these regions suggests that they might be of importance in directing cell-specific expression of mab-9. The fact that all five homology regions contain possible T-box binding sites may indicate that mab-9 is autoregulatory or regulated by other T-box genes. To ascertain the importance of each of the homology regions in directing correct MAB-9 expression, we have used a GFP-tagged rescuing construct with each of the homology regions removed by site-directed mutagenesis. The ability of mutated constructs to rescue mab-9 mutants and to be properly expressed is being investigated. Regulatory sequences which appear important in the control of MAB-9 expression will be chosen for a yeast 1-hybrid screen to identify upstream regulators. Huang X, Cheng H-J, Tessier-Lavigne M, Jin Y (2002); Neuron, 34, 563-576.

Nassar, Layla et al. (2015) International Worm Meeting "Understanding how sex modulates the female nervous system to drive distinct reproductive behavior states."

Collins, Kevin, Nassar, Layla, Bode, Addys

[International Worm Meeting, 2015]

We are interested in understanding how mating and reproductive behaviors are coordinated in the female nervous system. Specifically, we are identifying the neural signaling systems that drive two mutually exclusive vulval motor behaviors: mating with males or the release of progeny during egg laying [1]. We hypothesize that: 1. female vulval muscle twitching contractions facilitate male spicule insertion during mating; 2. specific mechanical and chemical signals report successful copulation and insemination; and 3. mating initiates reproductive behaviors including oocyte production and egg release. To investigate these hypotheses, we are using calcium imaging techniques to record vulval signaling events during mating with males, after successful insemination, and during the resumption of normal egg laying behavior. We have found that hermaphrodites with sperm have reduced vulval muscle twitching behaviors resulting in inefficient mating. In contrast, hermaphrodites depleted of sperm have increased vulval muscle twitching that facilitates male spicule insertion and mating. After mating is complete, hermaphrodites have sustained vulval muscle twitching that can result in release of sperm from the uterus. This behavior may act as a mechanism for competition with self-sperm. We are now examining how the other cells in the egg-laying circuit, including the HSN and VC neurons and uv1 neuroendocrine cells, respond during steps of male mating. During egg laying behavior, we found that the uv1 cells are mechanically activated by passage of eggs through the vulva, driving release of tyramine that inhibits egg laying [2]. We predict that mechanical stimulation by male spicules may similarly activate the uv1 cells to inhibit egg release during mating. However, once mating is complete, we expect egg laying to resume or even increase. Together, these results will explain how internal and external signals modulate activity in the same neural circuit to drive distinct behavior states.1. Collins, KM, and Koelle, MR (2013). Postsynaptic ERG Potassium Channels Limit Muscle Excitability to Allow Distinct Egg-Laying Behavior States in Caenorhabditis elegans. J. Neurosci. 33, 761-775.2. Collins KM, Bode A, Fernandez R, Tanis JE, Creamer M, Koelle MR (2015). Unpublished results.