KA Dickinson (1999) International C. elegans Meeting "Nicotine Adaptation is PKC Dependent"

KA Dickinson

[International C. elegans Meeting, 1999]

We are interested in studying nicotine adaptation in C. elegans. When initially exposed to nicotine, worms become paralyzed but gradually adapt to the drug and regain their ability to move. Another consequence of nicotine adaptation is a change in egg-laying behavior. Wild type worms display a dose dependent stimulation of egg-laying under inhibitory conditions (M9 solution) by the nicotinic acetylcholine receptor agonist levamisole. However, long term nicotine treatment results in adapted worms that are able to move around but unable to be stimulated to lay eggs by levamisole in M9. We found that worms begin to lose their responsiveness to levamisole after one hour on nicotine and completely fail to respond to levamisole after three hours on nicotine. We also found that the effects of nicotine adaptation are long-lasting, with adapted worms failing to respond to levamisole after a 24 hour incubation on drug free medium following exposure to nicotine. To identify molecules involved in the nicotine adaptation process, we performed the egg- laying assay for nicotine adaptation on C. elegans mutants with known defects in neuronal signaling molecules. This assay produced interesting results when we tested a strain of worms with a mutation in the tpa-1 gene, a gene that encodes protein kinase C. Naïve tpa-1 worms respond to levamisole with a similar dose response to wild type. However, nicotine treated tpa-1 worms display no reduction in levamisole-induced egg-laying even after overnight exposure to nicotine. This suggests that the long-term effects of nicotine on egg-laying behavior are dependent on PKC. To identify more molecules involved in nicotine adaptation, we are screening for adaptation defective mutants. Ultimately, such studies may elucidate the molecular mechanisms behind nicotine adaptation in worms and eventually lead to an understanding of nicotine addiction in humans

Waggoner LE et al. (2000) West Coast Worm Meeting "Genetic Analysis of Nicotine Adaptation in C. elegans."

Poole DS, Dickinson KA, Schafer WR, Kim JS, Waggoner LE

[West Coast Worm Meeting, 2000]

In many organisms, prolonged exposure to nicotine causes long-lasting changes in the abundance and functional activity of nicotinic receptors, processes thought to underlie nicotine addiction in humans. At present, the molecular basis for nicotine adaptation is poorly understood. We have begun using a genetic approach to identify molecules required for nicotine adaptation in the egg-laying circuitry of C. elegans. Acute exposure to nicotine or the nicotinic agonist levamisole have dramatic effects on behavior, including stimulation of egg-laying. These acute effects of nicotine on egg-laying require the activity of a nicotinic receptor containing subunits unc-29, unc-38, and lev-1, as mutations in these genes confer resistance to nicotine and levamisole. This receptor appears to function in the vulval muscle, since expression of an unc-29 transgene under a vulval muscle-specific promoter in unc-29 mutant animals restored egg-laying sensitivity to levamisole. Upon long-term nicotine treatment, wildtype worms undergo adaptation, and acquire a long-lasting loss of egg-laying sensitivity. This effect is at least partially mediated at the level of receptor abundance, as the expression of an UNC-29:GFP chimera was drastically reduced upon overnight treatment with nicotine. In order to identify molecules required for the long-term effects of nicotine, we examined known mutants and screened for new mutants with defects in adaptation. From the known mutants, tpa-1, a PKC homologue, was shown to be necessary for the control of UNC-29 receptor abundance, since tpa-1 mutants remained sensitive to levamisole and retained high UNC-29 receptor levels in the vulval muscles even after long-term nicotine treatment. We also identified a new gene, nic-1, in a screen for adaptation defective mutants. These mutants displayed hypersensitivity to nicotine and a defect in adaptation, as well as an unusual locomotive phenotype and a deficiency in male mating ability, all of which are consistent with a defect in cholinergic function in the neuromusculature. Currently, we are in the process of mapping nic-1 in order to clone it, and hope to discover the identity of the molecule encoded by this gene.

Nathalie Strutz et al. (2001) International C. elegans Meeting "Identification of functional ion channel domains of C. elegans glutamate receptor subunits"

Nathalie Strutz, David Madsen, Michael Hollmann, Andres V Maricq

[International C. elegans Meeting, 2001]

Glutamate receptors are not only abundant in the nervous system of vertebrates but also of invertebrates. In Caenorhabditis elegans , 10 putative ionotropic glutamate receptor subunits have been identified so far (Brockie et al. 2001, J. Neurosci. 21, 1510). Two of these, GLR-1 and GLR-2, have been expressed in Xenopus oocytes and failed to show ligand-activated currents. Therefore, we set out to investigate if the pore-forming domains of these two subunits are intrinsically functional ion conducting domains. We have previously shown that domain transplantation between different subtypes of glutamate receptors can generate functional chimeras that allow the characterization of those domains. To test for pore function, we transplanted the pore-forming region of GLR-1 or GLR-2 into either rat GluR1, as a representative of the AMPA receptor family, or rat GluR6, as a representative of the KA receptor family. The resulting chimeras GluR1-PGLR1, GluR1-PGLR2, GluR6-PGLR1, and GluR6-PGLR2, as well as the respective wildtypes GluR1, GluR6, GLR-1, and GLR-2 were expressed and characterized in Xenopus oocytes. Surprisingly, GluR1-PGLR1, GluR1-PGLR2, and GluR6-PGLR2 produced ligand-activated currents, despite the GLR-1 and GLR-2 wildtype receptor's apparent lack of ion channel function. The maximal current amplitudes of GluR6-PGLR2 were comparable to wildtype GluR6. GluR1-PGLR1 and GluR1-PGLR2 showed reduced current amplitudes in comparison to wildtype GluR1. The ion channel properties of the chimeras classify GLR-1 and GLR-2 into the group of non-NMDA receptor subunits, consistent with conclusions derived from sequence homology data.

Calvi, I. et al. (2019) International Worm Meeting "Cell polarity regulation by the mitotic kinase PLK-1 in C. elegans embryos."

Gotta, M., Calvi, I.

[International Worm Meeting, 2019]

Cell polarity is a fundamental property of cells and it is crucial for many biological processes at the cellular and organismal level. The one-cell C. elegans embryo is polarized along the anterior-posterior axis and this mechanism is essential for asymmetric cell division, a specialized division that results in two cells with a different fate. PAR proteins, a network of highly dynamic proteins with scaffold and/or enzyme function, mediate and control cell polarization in the one-cell C. elegans embryos. They form two distinct cortical domains, the anterior and the posterior PAR domains. The formation of these domains takes place in two distinct phases: the polarity establishment and maintenance phases (reviewed in Rose and Gonczy, 2014). Just after the fertilization, the anterior PAR proteins are uniformly distributed at the cortex. During polarity establishment, PAR-3, an anterior PAR protein, oligomerizes and forms big clusters (Dickinson et al., 2017). Formation of these clusters is required for polarity establishment as it couples PAR-3 itself and the other anterior PAR proteins, PAR-6 and aPKC, to the cortical flows, therefore enabling movement to the anterior. After pronuclear meeting, PAR-3 clusters dissolve and the maintenance phase begins. We found that phosphorylation of PAR-3 by PLK-1 inhibits cluster formation, identifying PLK-1 as a regulator of this process. Phosphorylation by PLK-1 on PAR-3 must be relieved to allow cluster formation and polarity establishment. We hypothesized that a phosphatase counteracts PLK-1 phosphorylation during polarity establishment. To test this hypothesis we treated the embryos with phosphatases inhibitors and found that inhibits polarity establishment. To identify the phosphatases, we are currently performing RNAi interference and biochemical approaches. Our data will help to better understand how PLK-1 regulates cell polarity establishment.

Puri, Dharmendra et al. (2017) International Worm Meeting "Regulation of neuronal microtubule cytoskeleton."

Jin, Yishi, Puri, Dharmendra, Chisholm, Andrew, Ghosh-Roy, Anindya, Thyagarajan, Pankajam

[International Worm Meeting, 2017]

In the nervous system, directional flow of information depends on the polarized architecture of neuron. Neuron has a unique structure - many dendrites and one axon. Microtubule polymers are the building blocks of the polarized architecture of neuronal cell. Although MT protofilaments give stability in axon, they themselves are dynamic - undergoing polymerization and depolymerization. The dynamics of the microtubule network needs to be regulated in a context-dependent manner during polarization, maintenance or other remodeling processes. Loss of kinesin-13 family microtubule depolymerizing enzyme klp-7 (kinesin-like protein) leads to excess stabilization of microtubules, leading to a multipolar neuronal phenotype in C. elegans mechanosensory (1) and other neurons. We found that this phenotype in klp-7(lf) can be reversed by a microtubule-destabilizing drug Colchicine or in backgrounds lacking either alpha or beta tubulin. We hypothesized that a genetic screen for the suppressors of klp-7(lf) will help us identify regulators of microtubule cytoskeleton. We conducted a genetic screen saturating the mutagenized genome. By combining meiotic recombination mapping and whole genome sequencing, we have identified candidates such as tubulin genes, post-translation modification factors and other novel factors. We mapped one of the suppressors to a gene, muscleblind-1/mbl-1. We found that mbl-1 is necessary and sufficient for touch neuron axon extension in a cell-autonomous manner. Gentle touch response in posterior and anterior side is compromised in mbl-1 mutant. Mbl-1 function is mostly known in muscles as a splicing regulator. Recent study in worm suggested that MBL-1 is required for synapse stabilization in DA9 motor neuron (2). We hypothesize that MBL-1 is regulating splicing of genes required for microtubule stability. References: 1. Ghosh-Roy A, Goncharov A, Jin Y, & Chisholm AD (2012) Kinesin-13 and tubulin posttranslational modifications regulate microtubule growth in axon regeneration. Dev Cell 23(4):716-728. 2. Spilker KA, Wang GJ, Tugizova MS, & Shen K (2012) Caenorhabditis elegans Muscleblind homolog mbl-1 functions in neurons to regulate synapse formation. Neural Dev 7:7.regulation

Andersen*, Kristine et al. (2015) International Worm Meeting "An Investigation into the Affect of Neuronal Activity on Proper Neural Connectivity in C. elegans."

Andersen*, Kristine, Varshney, Aruna, Duong, Alex, Miller, Kristine, Park, Joori, Pyle, Jacqueline, Barsi-Rhyne*, Benjamin, VanHoven, Miri, Vargas, Christopher, Holdrich, Emma, Tsujimoto, Bryan, Tran, Alan

[International Worm Meeting, 2015]

Neuronal activity has been implicated in the establishment and maintenance of appropriate synaptic connections in vertebrate and invertebrate systems. However, the molecular mechanisms by which neuronal activity affects connectivity are poorly understood. To understand this process, we have focused our studies on the PHB phasmid sensory neurons. We have begun to elucidate the pathway by which sodium dodecyl sulfate (SDS) is sensed by the phasmid neurons using a high throughput assay adapted from the method developed by Hilliard and colleagues (Current Biology, 2002). Briefly, SDS is sensed by the PHB neurons, which terminate backward movement via their connections with AVA interneurons. Using this assay, we find that odr-3/Galphaolf and tax-2/CNG-channel beta subunit, in addition to previously discovered tax-4/CNG-channel alpha subunit (Hilliard et al., Current Biology, 2002), are required for SDS chemosensation. To determine if defects in sensory signaling affect sensory synapses, we utilized the split-GFP-based trans-synaptic marker NLG-1 GRASP (Neuroligin-1 GFP Reconstitution Across Synaptic Partners) to visualize synapses between PHBs and AVA interneurons in live animals. Interestingly, we find that neuronal activity is required for maintaining these sensory synapses. Time course experiments indicate that odr-3/Galphaolf mutant L1s have normal synapses, but synapses are significantly reduced in later larval stages, although phasmid neuron morphology appears to be unaffected. Cell-specific rescue experiments indicate that odr-3/Galphaolf likely functions in PHB neurons. Subcellular localization of odr-3/Galphaolf shows localization to the PHB cilia, consistent with a role in sensory signaling being required to maintain synaptic integrity. These results indicate that C. elegans may be a powerful model organism for elucidating the molecular mechanisms by which sensory activity affects synaptic connectivity. Our future goal is to further characterize the mechanism by which sensory activity maintains synapses using molecular genetic and physiological approaches. Funded by NIH (1R01NS087544 to MV at SJSU and NL at UCSF, 5T34GM008253 MARC undergraduate fellowship to CV, 2R25GM071381 RISE undergraduate fellowships to CV and JP), HHMI (SCRIBE 52006312 undergraduate fellowship to BB and KM), and NSF (RUMBA REU 1004350 fellowship to KA and EH).

Vidal-Gadea, Andres G et al. (2011) International Worm Meeting "C. elegans selects distinct crawling and swimming gaits via dopamine and serotonin."

Elbel, Erin, Axelrod, Abram, Erbguth, Karen, Topper, Stephen, Brauner, Martin, Vidal-Gadea, Andres G, Crisp, Ashley, Pierce-Shimomura, Jonathan, Young, Layla, Siegel, Dionicio, Maples, Thomas, Kressin, Leah, Gottschalk, Alexander

[International Worm Meeting, 2011]

The ability to select and switch between alternate locomotory gaits is shared among many animals that are required to transition between different environments. Failure to achieve motor transitions is often devastating to the organism as exemplified by patients with advanced Parkinson's disease. The nematode C. elegans is well adapted for locomotion on land and in water and is likely required to transition between these sub-niches in nature. Despite recent advances, it remains controversial if swimming and crawling are distinct gaits in C. elegans, and if so, how transitions between them are accomplished (1-4). Answering these questions could enable the use of this powerful model system to study the mechanisms underlying locomotory transitions. We used a multifaceted approach spanning in depth behavioral assays, mechanic and optogenetic stimulation, laser ablations, mutant analysis, and in vivo photolysis of caged amines to test whether the worm uses distinct gaits and to determine how they transition between them. We show through a series of behavioral assays that in C. elegans crawling and swimming are distinct gaits. Dopamine released by ADE and PDE neurons is necessary and sufficient to initiate and maintain crawling by a pathway activating D1-like receptors. Conversely, serotonin is necessary and sufficient to initiate and maintain transition from crawling to swimming and to inhibit a set of crawl-specific behaviors. We found these transitions to be modulated by the balance between dopamine and serotonin. These amines have been found to play crucial roles in transition between alternate locomotory forms in diverse species stressing the importance locomotor transitions have for survival. Further study of locomotory switching in C. elegans and its dependence on dopamine may provide insight into comparable motor deficits observed in Parkinson's disease. 1) Boyle H et al. N. Front. Behav. Neurosci. 2011 5:10 doi: 10.3389/fnbeh.2011.00010. 2) Mesce KA, Pierce-Shimomura JT. Front. Behav. Neurosci. 2010 4:49 doi: 10.3389/fnbeh.2010.0004 3) Vidal-Gadea AG et al. 2009 Neuroscience 366:17 Calabrese R: Faculty of 1000 Biology, 11 November, 2009. Conference 2009 October 16-21. Available at: http://f1000biology.com/ article/id/1182956/evaluation 4) Fang-Yen C et al. 2010 PNAS 107, 20323-20328.

Stevenson, Zachary et al. (2021) International Worm Meeting "Self-Selecting Clone-Free Transgene Integration in Caenorhabditis elegans- Expanding the Toolkit"

Phillips, Patrick, Petruccione, Erin, Moerdyk-Schauwecker, Megan, Stevenson, Zachary, Jamison, Brennen

[International Worm Meeting, 2021]

The integration of transgenes in Caenorhabditis elegans has traditionally utilized transposable elements or bombardment, with current protocols adopting CRISPR-based integration. CRISPR also allows for the creation of custom-designed integration loci. Here, we take advantage of this experimental freedom to design such loci, or 'landing pads,' to facilitate transgene integration. These landing pads are designed for integration-specific selection, thereby avoiding the selective benefit of array formation and consequent false positives, eliminating the need for anti-array selection or co-CRISPR methodologies. Within the genome, landing pads are built with a partial Hygromycin B resistance gene missing the promoter and the 5'coding sequence. In addition, a synthetic unique guide RNA target sequence is present upstream for targeting by Cas9. Six synthetic guides were pre-tested to choose an optimal guide target, and four were identified with similar integration efficiencies. Donor homology plasmids are constructed with the transgenic cargo plus the rps-0p::HYGR 5' coding sequence. As the Hygromycin B resistance gene has been split, there is no selectable advantage in the array. Only upon integration is the Hygromycin B resistance restored. A landing pad locus was initially constructed on Chr. II, and we recently expanded the toolkit, creating landing pad strains and plasmid donors for Chr. I and III. For each landing pad/donor pair, the Hygromycin B resistance gene is flanked by unique Lox sites, allowing for optional removal of the resistance gene without inducing interchromosomal rearrangements. Additional donor plasmids have been constructed to allow for self-excision of the resistance gene, similar to Dickinson et al. 2015. In addition to streamlining the integration process, we bypassed the requirement for pre-constructed plasmids. It is known that C. elegans can stitch together linear dsDNA fragments with homology-directed repair (HDR), which has been used for clone-free transgenesis. We tested two and six-part transgenic insertions for sqt-1(e1350) and found functionally integrated transgenes for both. We couple this strategy with our synthetic landing pads to streamline the transgenesis process from PCR to confirmed single copy integration in as little as five days. Overall, the combined approach provides an economical and rapid method to generating single-copy transgenes for C. elegans.

Torres, Edwin et al. (2019) International Worm Meeting "Characterizing the gonad-enriched transcripts in Caenorhabditis elegans ."

Torres, Edwin, Escobar, Giulianna, Kroetz, Mary B.

[International Worm Meeting, 2019]

Previous forward and reverse genetic screens have identified a handful of genes that when their expression is abrogated lead to defects in the development of the somatic gonad in C. elegans. Many genes that are important for gonadal development could be genetically redundant; alternatively, they could be essential for the viability of the organism. Either case would cause the genes not to have been easily isolated in previous screens. To help identify additional genes responsible for promoting gonadal development, a cell-specific transcriptional approach was conducted that identified transcripts enriched for expression in the developing somatic gonad compared to the whole animal in each sex. We are now working to further study how the gonad-enriched transcripts are driving the development of the somatic gonad in either sex. We are using CRISPR-Cas to N-terminally tag the endogenous loci of gonad-enriched genes of interest with GFP. To generate the GFP-tagged strains, we are using a self-excising cassette (Dickinson et al. 2015). Prior to the eviction of the self-excising cassette, the self-excising cassette is inserted between the promoter and open reading, disrupting the gene expression, and allowing for us to test the loss-of-function phenotype. After excision, the GFP-tagged protein is generated for expression studies. Of the gonad-enriched transcripts, approximately 15% are encoded by essential genes, and therefore the loss-of-function gonadal phenotype has not been possible to study. To determine the role that these essential genes are playing within the gonad, we are modifying a tool that has been shown to lead to tissue-specific degradation of genes (Wang et al. 2017). We are constructing a strain that allows us to degrade GFP-tagged proteins exclusively in the gonad, allowing us to test the gonadal function of essential genes. Finally, as part of a Course-based Undergraduate Research Experience, we are using CRISPR-Cas to create serial deletions in the upstream regulatory promoter of the fkh-6 gene, which is important for gonadal development of both sexes. We have identified a region in the regulatory promoter that leads to feminization of male gonads but does not drastically change the expression pattern of FKH-6 in either sex.