Hamilton, Scott et al. (2009) International Worm Meeting "Phosphodiesterases in the C. elegans AWC Neuron."

Hamilton, Scott, L'Etoile, Noelle

[International Worm Meeting, 2009]

Similar to calcium signaling, cyclic nucleotides have been shown to transduce complex intracellular signals that are temporally and spatially discrete. Phosphodiesterases (PDEs) are important regulators of intracellular levels of these second messenger cyclic nucleotides (cGMP and cAMP) that act by hydrolyzing the cyclic nucleotide to its corresponding monophosphate. We propose a central role for one or more PDEs in the AWC for chemosensation and adaptation signal transduction pathways. This is based on evidence that key cyclic nucleotide regulated proteins are required for these processes. For example EGL-4, a cGMP-dependent kinase, has been shown to be necessary and sufficient for adaptation (Lee et al., submitted 2009). DAF-11 and ODR-1, receptor-like guanylate cyclases, produce cGMP in the AWC and TAX-2/TAX-4 form cGMP-dependent channels that provide for calcium signaling. All of these factors are required for odor sensation by the AWC neurons. Thus, a role for cyclic nucleotide regulation by PDEs in AWC neurons is postulated. We are currently examining six of the seven known PDE and PDE-like genes (pdl-1, pde-1, pde-2, pde-4, pde-5 and pde-6) by conducting chemotaxis and olfactory adaptation behavioral screens on single and double mutants. We have isolated two mutants with interesting phenotypes: pde-2 (tm3098) is adaptation defective and pde-4 (ce268) is chemotaxis defective. Additionally, we will present morphological analysis for developmental defects in AWC neurites. Further behavioral, genetic, physiological and molecular analyses will clarify the central role of cyclic nucleotides in the biochemical processes of olfaction and adaptation in the AWC. A better understanding of PDEs and their effect on cyclic nucleotide levels will help shed light on how key neuronal and cardiac processes are regulated temporally and spatially.

Cain, Scott et al. (2015) International Worm Meeting "JBrowse: A new genome browsing tool for WormBase."

Cain, Scott, Harris, Todd, Gao, Sibyl, Stein, Lincoln

[International Worm Meeting, 2015]

JBrowse is a next generation genome browsing tool that is part of the Generic Model Organism Database project (GMOD) and is being developed as a replacement for the Generic Genome Browser (GBrowse) which currently powers the genome browser at WormBase. JBrowse has several advantages as a genome browser, including a very fast user interface, with "Google Maps" style, on-demand rendering; local, in the browser rendering of many file formats (GFF, BigWig, BigBed, BAM, VCF) so that the files don't have to be uploaded to a server; combination tracks that allow users to combine data in tracks using arithmetic and set operations; and a highly customizable user interface. While JBrowse is not expected to replace GBrowse at WormBase any time soon, we are rolling JBrowse out to users now to try, use and give feedback on development directions. Please try out JBrowse athttp://jbrowse.wormbase.org/.

Hamilton, O. Scott et al. (2011) International Worm Meeting "WincG: a cyclic GMP biosensor for worms."

L'Etoile, Noelle D., Brueggemann, Chantal, Juang, Bi-Tzen, O'Halloran, Damien M., Hamilton, O. Scott

[International Worm Meeting, 2011]

Cyclic 3',5'-guanosine monophosphate (cGMP) is a second messenger that has many cellular functions including regulating ion channels and protein kinases. cGMP is catalyzed from guanosine triphosphate (GTP) by guanylyl cyclases and hydrolyzed by phosphodiesterases to guanosine monophosphate (GMP). Given the diversity of cellular functions mediated by cGMP, a means of monitoring and measuring in vivo cGMP dynamics in C. elegans would provide the worm community with better resolution to probe many cellular pathways. Within the past few years a number of cGMP biosensors have been developed for mammalian cell cultures including FRET based reporters and, more recently, a non-FRET based single-fluorophore reporter that combines a circularly permutated EGFP with the cGMP binding sites of the regulatory domain of the Bos taurus PKG I that the authors (Nausch et al., PNAS, 2008) named FlincG (Fluorescent indicator of cyclic GMP). Since there are currently no available cGMP biosensors for nematodes, we have expressed a modified version of the FlincG in C. elegans that we call WincG (Worm indicator of cyclic GMP). By cloning the FlincG into the worm expression vector pPD95.75, removing a non-coding region of the 3'UTR and enhancing the circularly permutated EGFP portion of the construct, we have been able to successfully express the cGMP biosensor under C. elegans promoters. Here we show that, as a proof of concept, when we express the WincG in phasmid neurons and introduce SDS as a repellent stimulus to the tail of a worm (that is immobilized in a worm trap) we are able to consistently measure cGMP changes (deltaF/F0%). We are also currently examining cGMP dynamics in amphid neurons.

Neal, Scott et al. (2015) International Worm Meeting "Food-dependent regulation of developmental plasticity via CaMKI and neuroendocrine signaling."

Sengupta, Piali, Butcher, Rebecca, Neal, Scott, Takeishi, Asuka, Kim, Kyuhyung

[International Worm Meeting, 2015]

Many animals integrate information about the environment, as well as internal state, to choose between alternate developmental trajectories. Environmental conditions, including food availability, inform the choice of C. elegans larvae to enter into the reproductive cycle or the growth arrested dauer stage. This polyphenic developmental decision is mediated via regulation of insulin and TGF-beta signaling. However, the molecular and neuronal mechanisms by which food signals are assessed and integrated to regulate neuroendocrine signaling and this binary developmental decision are not well understood. Here we show that food information is integrated into the dauer pathway by the activity of the CMK-1 CaMKI enzyme in the AWC and ASI sensory neurons. AWC is hyperactive and exhibits enhanced odor responsiveness in both starved wild-type and fed cmk-1 mutants, suggesting that the state of AWC in fed cmk-1 larvae resembles that in starved wild-type animals. Food deprivation also regulates nuclear localization of CMK-1 in AWC in a dynamic manner, and we show that CMK-1 regulates the expression of insulin-like peptide (ILP) genes in AWC as a function of feeding state. CMK-1-regulated ILP signaling from AWC in turn regulates expression of the growth promoting daf-28 ILP gene in the ASJ neurons. CMK-1 also acts in parallel in ASI to regulate expression of the daf-7 TGF-beta gene. Downregulation of daf-28 and TGF-beta expression in cmk-1 mutants drives enhanced dauer formation under conditions of ample food. Together, these results identify mechanisms by which information regarding nutrient availability converges with other sensory cues in a small neuronal network to modulate neuroendocrine signaling and developmental plasticity.

Daniel D. Scott et al. (2007) First Australian C. elegans Meeting "Identification and Characterisation of Mechanistic Partners of ctbp-1 in Caenorhabditis elegans"

Hannah R. Nicholas, Daniel D. Scott, Poh S. Khoo

[First Australian C. elegans Meeting, 2007]

The CtBP family of transcriptional corepressors has been widely investigated in murine and Drosophila model systems, in which it plays essential roles in development, cellular proliferation and apoptosis; however the Caenorhabditis elegans family member ctbp-1 remains relatively uncharacterised. Yeast-2-hybrid screens in our laboratory have identified a number of putative binding partners of ctbp-1 including transcription factors pag-3, zag-1 and egl-13, and the SUMOylation enzyme ubc-9. Other research has also suggested the histone deacetylase sir-2 and the C. elegans Rb homologue lin-35 as cofactors of ctbp-1. We aim to confirm and investigate these proposed partners by screening for genetic interactions between ctbp-1 and each candidate. These shall be identified in pseudo-double mutants constructed by knockdown of ctbp-1 expression by RNA interference within a genetic background containing a mutant candidate. Further, we shall conduct an unbiased mutagenesis screen for genetic interactions with a view to identifying hitherto unknown mechanistic partners of ctbp-1. These data shall significantly expand our knowledge of how ctbp-1 functions in nematodes and may provide insights into the activities of the CtBP family as a whole..

Davis, Scott et al. (2013) International Worm Meeting "Uncovering the molecular basis for ethanol action on the BK channel using genetic screens."

Hu, Kevin, Pierce-Shimomura, Jon, Davis, Scott

[International Worm Meeting, 2013]

Development of alcohol abuse has a strong genetic component. People who are innately resistant to intoxication are at high risk to abuse alcohol and vice versa. This genetic predisposition is linked to variation in genes that encode targets of alcohol or their downstream effectors involved in intoxication. Genetic screens in C. elegans previously determined that the gene slo-1 represents a central target for alcohol intoxication in the worm. slo-1 encodes the highly conserved large-conductance, calcium-activated potassium (BK) channel. The BK channel has emerged as a key mediator for intoxication and tolerance across species from worm, fly, mouse to human. At the physiological level, BK channel activity is modulated by clinically relevant concentrations of ethanol (~20mM). To reveal specific residues in the BK channel that are required for activation by alcohol, and thus may influence sensitivity to behavioral intoxication, we are performing multiple genetic screens. First, we are screening slo-1 mutants from the million mutation project. Second, we are performing site-directed mutagenesis on the worm and human BK channels. We are able to study the human BK channel in the worm because we rescued ethanol sensitivity in a slo-1 null worm by expression of hslo. Novel mutants that are resistant to intoxication will either contain 1) a null mutation in slo-1 that provides no new information, or 2) a novel non-null mutation that provides insight into how specific residues on the BK channel interact with alcohol, and how this interaction alters intoxication. To distinguish non-null candidates, the locomotor posture of mutants recovered from the genetic screens will be compared against wild-type and a known slo-1 null mutant. Candidate non-null mutants will display different locomotor posture than the known slo-1 null mutant. Currently, we have isolated multiple candidate non-null mutants with resistance to intoxication. Further analysis using in vivo patch-clamp recordings will assess how specific mutations alter basal BK channel function and the response to alcohol.

Montgomery, Scott et al. (2009) International Worm Meeting "Analysis of the C. elegans Transcription Factor, LIN-31, Using the Yeast Two-Hybrid."

Montgomery, Scott, Teixeira, Elico, Smith, Adam, Miller, Leilani

[International Worm Meeting, 2009]

Cell fate, or how a cell decides to differentiate into a certain cell type, depends largely on which genes are expressed. Transcription factors are proteins responsible for such regulation, although the biochemical characterization of such systems is incomplete. For example, very few gene targets have been identified. In addition, transcription factors often heterodimerize with other factors in order to regulate their activity. The winged-helix transcription factor LIN-31 is involved in cell fate decisions during development of the vulva, an egg laying structure, in the model organism Caenorhabditis elegans. This protein contains a DNA-binding domain and several other regions of unknown function. The current model for LIN-31 function is that it plays two roles in vulval development: 1) it heterodimerizes with another transcription factor, LIN-1, to promote non-vulval cell fates in some cells and 2) when the dimer is disrupted due to cell-cell signaling events, it promotes a vulval cell fate in other cells (Miller et al, 1993 and Tan et al, 1998). In C. elegans, the LIN-31 protein is expressed in very few cells, making direct biochemical analysis difficult. Attempts to express and purify this protein in bacterial and insect cells have been impeded by low expression levels and insolubility. To learn more about the role of this protein without purifying it, we are using a yeast-based system for two-hybrid (Y2H) analysis. Prior to conducting a full-scale screen, we are directly testing interactions between LIN-31 and its interaction partner, LIN-1. First, we used the Y2H system to confirm LIN-31''s interaction with LIN-1. Previous Y2H experiments in other labs had failed to see this interaction, but removal of LIN-31''s DNA binding domain allowed interaction with LIN-1 in our Y2H system. Second, we showed that LIN-31''s small acidic domain is required for this interaction. Elimination of this domain by non-conservative amino acid substitutions resulted in disruption of the LIN-31/LIN-1 interaction. This is consistent with previous site-directed mutagenesis experiments indicating that this acidic region is required for proper LIN-31 function in non-vulval cells (Morris et al, in prep).

Scott Kennedy et al. (2001) International C. elegans Meeting "Pheromone Regulation of daf-7expression"

Scott Kennedy, Gary Ruvkun

[International C. elegans Meeting, 2001]

A major regulator of the decision to enter the dauer stage is the level in the environment of the secreted dauer promoting pheromone. Dauer pheromone controls the expression of the TGF- b -like ligand DAF-7 in the ASI amphid neuron (Ren et al. 1996, Schackwitz et al. 1996). We are studying the molecular mechanism by which this extracellular pheromone signal regulates daf-7 transcription. We have identified daf-11 as an important regulator of daf-7 expression. daf-11 encodes a transmembrane guanylyl cyclase, an enzyme that converts GTP to the intracellular second messenger cGMP. In daf-11 animals daf-7pGFP levels are dramatically reduced. We have also found that loss of function mutations in the a subunit of a cyclic nucloetide gated ion channel tax-4 also cause a loss of DAF-7 expression. The dauer and low daf-7p::GFP phenotypes of daf-11 but not tax-4 mutants are reversed by exogenous addition of cGMP analogues. The cilia mutant osm-1(p808) , while capable of suppressing the DAF-c phenotype of daf-11, does not suppress the associated low daf-7p::GFP phenotype suggesting that cilia mutants do not suppress daf-11 by activating the signaling pathway to daf-7 . These results suggests that pheromone signaling in C. elegans, like mammalian photoreceptor signaling, involves the second messenger cGMP and a cGMP regulated ion channel. To identify additional signaling molecules functioning between dauer pheromone and daf-7, we have undertaken two genetic screens. The first screen seeks to identify mutations that both suppress the daf-11 Daf-c phenotype and lead to a reversion in daf-7p::GFP expression. From an initial screen of 250,000 haploid genomes we have identified two mutations: p heromone s ignaling i nhibitor: psi-1( mg296) and psi-2( mg297) that satisfy these criteria. psi-1( mg296) is a recessive mutant that blocks dauer pheromone downregulation of daf-7p::GFP. We are now mapping psi-1 and psi-2 for molecular analysis. Our second genetic screen seeks to identify mutations that both suppress the tax-4 Daf-c phenotype and also lead to daf-7p::GFP expression. From an initial screen of 10,000 haploid genomes we have identified two mutations: psi-3( mg318) and psi-4( mg319 ) that satisfy these criteria. Progress on the characterization, mapping, and identification of these mutations will be reported.

Scott Alper et al. (2003) International Worm Meeting "Regulation of epidermal morphogenesis in C. elegans"

Cynthia Kenyon, Scott Alper

[International Worm Meeting, 2003]

The C. elegans epidermis is composed of several large multinucleate syncytia that are generated by the fusion of mononucleate cells throughout development. The largest such syncytium is hyp7, which spans most of the length of the worm and which contains more than 130 nuclei. hyp7 is initially generated during embryogenesis by the fusion of cells primarily on the dorsal surface of the worm. Subsequently during larval development, cells on the lateral and ventral surfaces of the worm fuse with hyp7 as well.The lateral epidermis of the worm is composed of a single row of blast cells known as seam cells that proliferate throughtout larval development, generating most of the cells that fuse with hyp7. During each larval stage, the seam cells undergo a repeated cycle of cell proliferation, cell fusion, and cell membrane rearrangement. For example, midway through the first larval stage, the seam cells divide. One of the daughter cells from each division then fuses with the growing hyp7 syncytium. The remaining unfused daughter cell then undergoes a cell shape change; it elongates along the A/P body axis until it is in contact with neighboring unfused cells. This cycle of cell proliferation, cell fusion, and cell membrane rearrangement is repeated in subsequent larval stages. Finally, in the fourth larval stage, the unfused cells all fuse with each other, generating their own smaller syncytium distinct from hyp7.We have generated a membrane-localized gfp fusion expressed in lateral epidermal cells throughout development. We have used this fusion to characterize lateral epidermal morphogenesis in more detail. We are also using this gfp strain to carry out several genetic and genomic RNAi screens. In preliminary screens, we have identified a variety of genes responsible for various aspects of epidermal morphogenesis.

Scott, Luisa et al. (2013) International Worm Meeting "Identifying Novel BK Channel Modulators."

Scott, Luisa, Davis, Scott, Shen, Angela, Nordquist, Sarah, Philpo, Ashley, Iyer, Sangeetha, Pierce-Shimomura, Jon

[International Worm Meeting, 2013]

The large-conductance calcium-activated potassium (BK) channel is an evolutionarily conserved target of ethanol. Physiologically relevant concentrations of ethanol increase BK channel opening. Additionally, genetic manipulation of BK channel function affects behavioral sensitivity to ethanol in multiple animal models. These findings suggest that the BK channel is a viable therapeutic target for alcohol intoxication and addiction. To begin to test this, we have developed a screen to identify peptides that alter BK channel function. First, we screened 30 million 9 amino acid peptide sequences using a monovalent phagemid display technique. Twenty-seven unique peptides remained after panning for sequences that bind to the human BKa channel but not the rat SK2 channel or human glycine a1 receptor. Sequences with clusters of positively charged amino acids were enriched 100-300 fold. Three motifs were enriched 3000-6000 fold. Next, select peptides were rapidly screened for functional effects using the nematode, C. elegans. Several peptides with highly enriched motifs affected an ethanol- and BK-channel dependent behavior in this animal. The behavioral assay suggests that peptide pskan4 enhances the ethanol mediated increase in BK channel opening, but does not alter channel activity in the absence of ethanol. Peptide pskan1 had BK channel- but not ethanol-dependent effects. In contrast, a peptide without a highly enriched motif had non-specific effects, and a 9 amino acid peptide not selected in the panning procedure did not alter locomotion. Finally, we have begun to confirm the function of the peptides electrophysiologically. Preliminary recordings support the putative action of peptides pskan1 and pskan4 as BK channel openers. Overall, these findings show that we have developed and successfully employed a screen for identifying and characterizing novel peptides that alter BK channel-dependent behavior in the presence of physiologically relevant concentrations of ethanol. This screening technique can be applied to identify modulators of other ion channels.