Bercher MR et al. (2000) Midwest Worm Meeting "MUA-3 localizes to fibrous organelles and interacts with IFA2"

Wahl J, Plenefisch JD, Bercher MR

[Midwest Worm Meeting, 2000]

Normal locomotion of the nematode Caenorhabditis elegans requires transmission of contractile force from the myofibrillar lattice of the body wall muscles through a series of mechanical linkages between the muscle cells, the surrounding extracellular matrix, the hypodermis (epidermis), to the collagenous cuticle (the exoskeleton). Mutations in mua-3 result in a progressive flaccid paralysis of all or part of the body due to detachment of the body wall muscles from the cuticle, thus is apparently required for the normal maintenance of mechanical linkage between muscle and cuticle as the animal grows in size postembryonically. MUA-3 is a novel 3623 amino acid matrix receptor with a large extracellular domain, a single transmembrane helix, and a smaller cytoplasmic domain. The extracellular domain contains four distinct protein-folding motifs, i.e., 5 LDL, 52 EGF, 1 vWFA and 2 SEA modules. These motifs suggest it may help tether the hypodermal cells to the collagenous structures of the basal lamina and/or cuticle. No vertebrate homologs have been identified. A search of GenBank revealed a second C. elegans gene, initially designated mrp-1, homologous to mua-3., and a nearly identical C. briggsae ortholog, cbmua-3. It has been subsequently demonstrated by E. Bucher that the mrp-1 sequence encodes the muscle attachment gene mup-4 (E. Bucher, personal communication). In order to determine the cellular localization and expression patterns of MUA-3, monoclonal antibodies to the cytoplasmic domain have been raised. Immunofluorecence using anti-MUA-3 antibodies shows localization to the fibrous organelles and other sites where cells form mechanical trans-hypodermal attachments to cuticle. MUA-3 expression is seen in the hypodermis bordering body wall muscle, the ALM and PLM, the rectum, and the vulva. MUA-3 colocalizes with IFA2, encoded by the mua-6 gene product (see abstract by Hresko et al). The pattern is similar to that reported for MH5 and myotactin, components of the fibrous organelles. Expression is seen as early as the two-fold stage of development. The identification of MUA-3 and MUA-6 as fibrous organelle components suggests that other mua genes may also be involved in fibrous organelle structure, function, or regulation. We are currently examining the localization of MUA-3 in mua mutant strains, looking for informative perturbations, and continuing our efforts to clone additional mua genes to confirm this proposal. Abnormal MUA-3 expression patterns in the mua-3 mutants rh195 and rh169 as well as basement membrane component unc-52 have been already observed. In addition, genetic and molecular screens to identify other proteins that interact with MUA-3 are in progress.

MR Bercher et al. (1999) International C. elegans Meeting "Molecular characterization of the C2H2 zinc finger protein MUA-1"

MR Bercher, J Plenefisch, X Zhou

[International C. elegans Meeting, 1999]

MUA-1 is required for postembryonic muscle development in C. elegans . Although normal in appearance, the mechanical links between muscles and exoskeleton(cuticle) are exceptionally weak in MUA-1 mutant animals resulting in a progressive detachment of the body wall muscles from each other and from the hypodermis. The predicted MUA-1 protein contains three C-terminal C2H2 zinc finger domains that have high sequence homology to zinc finger transcription factors of the EKLF/BTEB2 family. While the majority of proteins containing C2H2 zinc fingers are transcription factors that bind GC rich enhancer sequences. A putative N-terminal activation domain in MUA-1 remains uncharacterized. It has some compositional similarity but no sequence homology to other activation domains. Based on the zinc finger homologies and the presence of a putative activator domain, MUA-1 is predicted to act as a transcription factor driving muscle specific protein expression. A fusion protein containing the MUA-1 activation domain fused to the GAL-4 DNA binding domain was constructed. When expressed, the GAL-4-DNA binding domain of this fusion protein binds to a 17mer enhancer sequence upstream of a CAT gene in the reporter construct. If the MUA-1 activation domain is an authentic activation domain, it will drive transcription of CAT in a Cos cell line. CAT expression was assayed in the Cos cell extracts, showing that MUA-1 has modest transcriptional activity. Further experimentation is now needed to further characterize the transcriptional effects of MUA-1. We are also examining MUA-1 expression during muscle development and will be ßattempting to identify the target genes activated in vivo.

Erika Darrah et al. (2003) International Worm Meeting "Expression analysis of unc-13 and identification of proteins interacting with its products"

Erika Darrah, Bethany Stitt, Cristina Polinsky, Rebecca Kohn

[International Worm Meeting, 2003]

We are analyzing the expression of different forms of UNC-13 protein and are identifying other proteins that interact with UNC-13. Some forms of UNC-13 protein are involved in regulating neurotransmitter release. The most abundant protein product expressed from this gene, UNC-13 LR, localizes to synapses (Kohn, 2000). UNC-13 interacts with other presynaptic proteins and affects vesicle priming (Richmond, 2001). The C. elegans unc-13 gene is predicted to express several different protein products including UNC-13 LR, UNC-13 LMR, and UNC-13 MR. L represents the left region of the protein, M represents the middle, and R represents the right. Mutations in the unc-13 L region of the gene alter expression of some protein products and result in uncoordinated movement. A large deletion in the unc-13 R region eliminates all protein products and results in lethality (Kohn, 2000). We are analyzing regulation of expression of the different protein products. Specifically, we are exploring the possibility that an internal promoter is present in the gene that regulates expression of the UNC-13 MR product. A large 7.5 kb intron separates the unc-13 L region of the gene from the unc-13 M region. A cosmid including most of the 7.5 kb intron as well as the unc-13 M and unc-13 R regions can partially rescue deletion mutants. We are fusing part of this intron to GFP in order to determine if a promoter within the intron can drive expression of the gene and to identify cells in which the promoter is active. We are also using a two hybrid screen to identify proteins that interact with the different UNC-13 protein products.

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.

Koelle MR (1997) International C. elegans Meeting "THREE OF THE 11 RGS PROTEINS IN C. ELEGANS REGULATE G PROTEIN SIGNALING IN THE EGG-LAYING SYSTEM"

Koelle MR

[International C. elegans Meeting, 1997]

The recently identified family of RGS proteins inhibit signaling by heterotrimeric G proteins (1). RGS proteins bind to G protein alpha subunits and accelerate their GTPase activity, thereby driving G proteins toward their inactive GDP-bound form (2). In C. elegans, the prototypical RGS protein EGL-10 regulates neurotransmission through the G protein GOA-1: loss-of-function egl-10 mutants are egg-laying defective due to increased GOA-1 signaling (3). We would like to know why there are so many RGS proteins: we found that mammals have at least 17 RGS homologs, and the C. elegans genome sequence predicts 10 RGS proteins besides EGL-10. To begin to address this issue we separately overexpressed each C. elegans RGS gene in transgenic worms by microinjecting genomic DNA for each gene into wild-type worms to form multicopy transgenes. Based on our results with egl-10 and also on studies of RGS genes in yeast and Aspergillus nidulans, we expect that overexpressing an RGS gene in this manner will generally block signaling by the G protein(s) that the RGS protein normally partially inhibits. We assayed egg-laying behavior in our transgenic strains to see if the G protein signaling pathways that control the frequency of this behavior were affected. Overexpression of eight of the RGS genes had no effect on egg laying. Overexpressing either of two RGS genes, egl-10 or C05B5.7, strongly increased the frequency of egg laying and of several other behaviors in the same manner as GOA-1 loss-of-function mutations. These two RGS genes thus both appear to inhibit the G protein GOA-1. Overexpressing an RGS homolog on cosmid C16C2 mildly inhibited egg laying, suggesting that this RGS gene might inhibit the G protein EGL-30, whose activity increases the frequency of egg laying. We are investigating the logic by which three RGS proteins function together to control one behavior. 1. Koelle MR. (1997). Current Opinion in Cell Biol., in press. 2. Berman DM, Wilkie TM, and Gilman AG. (1996). Cell 86:445-452. 3. Koelle MR and Horvitz HR. (1996). Cell 84: 115-15.

Chiang, Y. et al. (2017) International Worm Meeting "Wild-isolates of C. elegans exhibit variable attraction to the sex-pheromone mimicry compound produced by the nematode-trapping fungus A. oligospora."

Chiang, Y., Hsueh, Y.

[International Worm Meeting, 2017]

The nematode-trapping fungus A. oligospora produces odors that mimic sex and food cues to attract many nematode species (Hsueh et al. 2017). One of the compounds (MMB) that likely mimics male pheromone in Canerhbiditis nematodes is highly attractive to two lab strains of C. elegans (N2 and Hawaiian). To investigate whether this attraction is highly conserved among the wild-isolates, we analyzed the MMB chemotaxis behavior among the C. elegans wild isolates from the CeNDR collection. We found that MMB-attraction is highly polymorphic trait. Six out of forty wild-isolates tested exhibited weak attraction to MMB. Genetic analysis showed that this trait is controlled by QTL in some strains but a single locus in others. The high incidence of MMB-insensitive strains among the wild-isolates suggests that lost of MMB attraction might offer benefit to C. elegans in the natural environments. Hsueh YP, Gronquist MR, Schwarz EM, Nath RD, Lee CH, Gharib S, Schroeder FC, Sternberg PW. 2017. Nematophagous fungus Arthrobotrys oligospora mimics olfactory cues of sex and food to lure its nematode prey. eLife 6.

Jonathan Boetto et al. (2006) Development & Evolution Meeting "The Role of Proteolytic Processing of TRA-1A in Sex Determination in C. elegans"

Jonathan Boetto, Mara Schvarzstein, Andrew Spence, Kathleen Dawson

[Development & Evolution Meeting, 2006]

The transcription factor TRA-1 is the terminal global regulator of the sex determination pathway in C. elegans. Hermaphrodites require TRA-1 activity to repress the transcription of genes that specify male cell fates. In the absence of TRA-1 activity, males develop. We have observed that while both sexes accumulate low levels of full-length TRA-1A (Mr ≈ 125kD), hermaphrodites but not males accumulate higher levels of a series of TRA-1 phosphoisoforms of Mr ≈ 90-100kD. We have evidence that these isoforms (TRA-1-100) result from proteolytic cleavage of TRA-1A and are sufficient to repress several known TRA-1 target genes. The transcription factor, Cubitus interruptus (Ci) is the D. melanogaster homologue of TRA-1. Ci undergoes a similar truncation that is necessary to produce the repressor form of the protein. We hypothesize that the proteolytic processing of TRA-1A is similarly required to create the functional form of the protein. I will test this hypothesis by creating transgenes encoding an altered version of TRA-1A inherently resistant to processing. Testing the feminizing activity of these transgenes in vivo will enable me to discern if the full-length precursor is functional. I have attempted to create this variant by designing deletion constructs. Deleted sequences include motifs associated with protein processing, the putative site of proteolytic cleavage and a conserved region of 62% identity with C. briggsae. I have created this third deletion as a cDNA construct, linked to a heat-shock promoter. I have injected this construct into tra-1(e1834) null mutants to test its feminizing activity. Because overexpression might compensate for reduced activity of the unprocessed protein, I have fabricated the additional deletion constructs from a rescuing construct, created in our lab, containing the full genomic tra-1 sequence and regulatory regions. This method enables me to create deletion constructs that more accurately mimic endogenous conditions. As an assay for feminization, I have co-injected the transgenic animals carrying the cDNA construct with vit-2::dsRed, which serves as a reporter of yolk production in the gut. I have observed that heat-shock activation of the deletion contruct induces feminization. I will subsequently be subjecting these animals to Western blotting to evaluate the effect that deletion of the conserved sequence has had on TRA-1-100 accumulation.

Kathleen G Dawson et al. (2005) International Worm Meeting "The Role of Phosphorylation and Proteolytic Cleavage in TRA-1 Regulation of Sex Determination in Caenorhabditis elegans"

Kathleen G Dawson, Andrew M Spence, Jonathan F Boetto, Mara Schvarzstein

[International Worm Meeting, 2005]

The transcription factor TRA-1 is the terminal global regulator of the sex determination pathway in C. elegans. Hermaphrodites require TRA-1 activity to repress the transcription of genes that specify male cell fates. In the absence of TRA-1 activity, males develop. We have observed that while both sexes accumulate low levels of TRA-1A (Mr ≈ 125kD), hermaphrodites but not males accumulate higher levels of a series of TRA-1 phosphoisoforms of Mr ≈ 90-100kD. We have evidence that these isoforms, which we believe to result from proteolytic cleavage of TRA-1A, are sufficient to repress several known TRA-1 target genes. The transcription factor Cubitus interruptus (Ci) is the Drosophila homologue of TRA-1 and both proteins bind similar DNA sequences. Protein kinase A (PKA) phosphorylates Ci to stimulate its proteolytic processing to produce a repressor isoform. We hypothesize that PKA may similarly regulate the production of hermaphrodite-specific cleaved isoforms of TRA-1. This leads us to predict that increased PKA activity would feminize male animals. To test whether PKA may play a role in sex determination, a heat-inducible transgene was used to overexpress the catalytic subunit (PKAc) in C. elegans. A vitellogenin gene reporter (vit-2::gfp) provides a marker of feminization of the intestine. Expression of PKAc induced vit-2::gfp expression in the intestinal cells of male worms. Worms lacking tra-1, however, did not express vit-2::gfp in response to PKAc overexpression. These results suggest that PKAc has TRA-1-dependent feminizing activity in the intestine. Western blot analysis will be carried out to determine whether PKAc overexpression results in accumulation of truncated phosphoisoforms of TRA-1. As a further test of the involvement of PKA in TRA-1 regulation, we have deleted sequences encoding a cluster of potential PKA target sites from a rescuing tra-1 transgene that includes the entire tra-1 locus (see abstract by Cooperstock, Sassi, et al.). Preliminary analysis has revealed that the deletion construct has reduced feminizing activity in tra-1(o) animals. We are now testing whether this reduced ability to rescue hermaphrodite development correlates with a change in the accumulation of the hermaphrodite-specific phosphoisoforms. To test whether truncation of TRA-1A is necessary for the greater accumulation of TRA-1 isoforms in hermaphrodites and thus for the feminizing activity of tra-1, we are producing a derivative of our rescuing tra-1 transgene that is resistant to cleavage. We will present a comparison of its activity to that of the wild-type transgene.

Hyun-Ah Sun et al. (2007) International Worm Meeting "Molecular Cloning of the HMG-CoA Synthase Gene and Its Regulation during the Dauer Stage of Caenorhabditis elegans."

Young-Ki Paik, Nan-Young Yoon, Jeeyong Lee, Hyun-Ah Sun

[International Worm Meeting, 2007]

HMG-CoA synthase (HMGS) has been known to be one of the key enzymes involved in cholesterol biosynthesis (in the cytosol) and ketone body formation (in mitochondria) in animals, depending on the physiological conditions. In C. elegans, neither the presence of this enzyme nor its regulation under various physiological states has been well established. In the course of our study of daumone-related biology, we were interested in the cloning and characterization of HMGS gene. Since one of the characteristic physiological features of dauer larvae of C. elegans is believed to be efficient fat mobilization and ketone body formation to meet the energy demands for survival, we wanted to investigate how this enzyme might be involved in energy metabolism in the daumone-induced dauer larvae. To understand the functional role of this enzyme in the dauer state, we first isolated the F25B4.6 clone, an ortholog of the human HMGS gene, by searching the C. elegans genome database. Our data from DNA sequencing, RNAi and RT-PCR experiments suggest that this clone indeed encodes an authentic C. elegans HMGS and its mRNA level appears to be regulated developmentally, which peaks in the early L2 stage. With the availability of the over-expressed recombinant protein (Mr 51.4 kDa), functional characterization and molecular regulation of HMGS is now underway. (Supported by a grant from the BioGreen 21 Project to YKP.).

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.