Brueggemann, Chantal et al. (2011) International Worm Meeting "Perception of bacteria by the AWC neuron."

O'Halloran, Damien, Brueggemann, Chantal, L'Etoile, Noelle

[International Worm Meeting, 2011]

Odor sensation and chemotaxis are essential cues that allow an organism to localize and tax towards food. However, the organism must also be able to ignore profitless odors. Thus, prolonged exposure of the olfactory sensory AWC neuron pair to benzaldehyde in the absence of food leads to desensitization of these sensory neurons, allowing the worm to ignore this odor [1, 2]. This adaptation process has to be modulated by the worm that the odor contained satisfying food remains attractive to the animal. Indeed, it is shown that in combination with food, adaptation to odor is blocked [3, 4]. In order to examine the molecular basis by which bacteria are able to block adaptation, we investigate bacterial molecules on cell surface or released into medium, which could allow the worm to distinguish between odor alone and odor with a nutritional benefit. One major molecule on the outer membrane of gram-negative bacteria is the strain specific lipopolysaccharide (LPS) structure, which acts as endotoxin and is able to induce strong immunological response in mammals and C. elegans [5-7]. We use E. coli strains with different LPS structures to ask how the LPS affect AWC during adaptation. Our results point out that altering LPS structure affects adaptation block. Interestingly, the ability of the bacteria to block adaptation depends on which strain the animals were grown on, indicating that there may be an LPS memory'. In order to understand how growth on specific LPS containing bacteria creates this LPS memory' we will determine which neurons within the animal are responsible for the memory. We are utilizing a combination of genetics and fluorescence based imaging to examine the calcium responses of the AWC neuron and its circuit to on-set and removal of bacteria or bacterial molecules. 1.Colbert, H.A. and C.I. Bargmann. Neuron, 1995. 14(4): p. 803-12. 2.Nuttley, W.M., S. Harbinder, and D. van der Kooy. Learn Mem, 2001. 8(3): p. 170-81. 3.Colbert, H.A. and C.I. Bargmann. Learn Mem, 1997. 4(2): p. 179-91. 4.Lin, C.H., et al. J Neurosci, 2010. 30(23): p. 8001-11. 5.O'Quinn, A.L., E.M. Wiegand, and J.A. Jeddeloh. Cell Microbiol, 2001. 3(6): p. 381-93. 6.Aballay, A., et al. Curr Biol, 2003. 13(1): p. 47-52. 7.Tenor, J.L. and A. Aballay. EMBO reports, 2007. 9(1): p. 103-109.

Brueggemann, Chantal et al. (2013) International Worm Meeting "Acute odor recognition in AWC neuron of C. elegans after adaptation."

Brueggemann, Chantal, L'Etoile, Noelle, O'Halloran, Damien

[International Worm Meeting, 2013]

Odor sensation and chemotaxis are essential responses that allow an organism to locate and move towards food. The paired AWCs neurons sense innately attractive odors such as benzaldehyde, butanone or isoamyl alcohol. However, the organism must also be able to ignore profitless odors. Thus, prolonged exposure of the AWC neurons to benzaldehyde (BA) in the absence of food leads to adaptation of the odor-seeking response. This allows the worm to ignore odors that are not associated with nutrition. Continuous exposure to BA in the absence of food, leads to a relocalization of the cGMP-dependent protein kinase EGL-4 from the cytoplasm into the nucleus. Nuclear EGL-4 is necessary and sufficient to adapt the AWC-mediated response to the odor. We examine the effects of both short- and long-term odor exposure on the acute odor response in AWC and its downstream interneurons by using the GFP-based calcium reporter GCaMP3. We hope to correlate the changes in intercellular calcium flux in response to odor with the subcellular localization of EGL-4 and in parallel with the behavior of the worms.

Parmidge, Amelia et al. (2015) International Worm Meeting "Finding Neuro: Automating calcium image analysis."

L'Etoile, Noelle, Parmidge, Amelia, Young, Jared, Brueggemann, Chantal

[International Worm Meeting, 2015]

In recent years, fluorescent imaging has been used more and more to capture the state of biological systems at different moments in time. For many researchers, analysis of the fluorescent image data has become the limiting factor of this new technique. We have developed NEPIC, a semi-automated tool for finding and tracking the soma of a single neuron over an entire movie of grayscale calcium image data. When tested on calcium image movies of the AWC neuron in C. elegans under highly variant conditions, NEPIC correctly identified the neuronal soma in 95.48% of the movie frames, and successfully tracked this soma feature across 98.60% of the frame transitions. Although support for finding and tracking multiple fluorescing Regions of Interest (or fROIs) has yet to be implemented, NEPIC displays promise as a tool for assisting researchers in the bulk analysis of fluorescent imaging data.

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.

Ablaza, Adriel et al. (2015) International Worm Meeting "An Environmentally Responsive 22G RNA Sensor."

Munoz-Lobato, Fernando, Balakrishnan, Sanjeev, Ablaza, Adriel, Gallegos, Maria, L'Etoile, Noelle, Mellman, Katie, Brueggemann, Chantal, Juang, Bi-Tzen

[International Worm Meeting, 2015]

Caenorhabditis elegans forages for food by distinguishing between various odorants in a dynamic environment. Their sensory neurons have the ability to adapt to persistent attractive odors in the absence of food (Colbert and Bargmann, 1995). One instance is the adaptation to the odor butanone, which is regulated in the AWC olfactory sensory neuron (L'Etoile et al., 2002).Olfactory adaptation in the AWC leads the repression of the odr-1 gene by an activated endogenous 22G small interfering RNA silencing pathway (L'Etoile and Bargmann, 2000; Juang et al., 2013). We found that mut-7, a 3'-5' exonuclease which is involved in the small RNA biosynthetic pathway, is implicated in odr-1 22G RNA production and butanone adaptation while a ChIP analysis of HPL-2 was shown to load heterochromatin on the odr-1 locus (Juang et al., 2013). This indicates that odr-1 22G RNA is a part of the endogenous siRNA pathway acting to silence odr-1 as a result of olfactory adaptation. However, there are limitations to qRT-PCR and ChIP analysis when investigating the activity of odr-1 22G RNA. Neither technique offers a dynamic or cell-specific readout of odr-1 22G RNA function.Here, we discuss the development of a fluorescent reporter that may be capable of visualizing cell specific changes in odr-1 22G RNA in response to olfactory adaptation to the odor butanone. The creation of a single copy insertion strain that includes an odr-1 22G small RNA sensor is designed to determine whether odr-1 22G small RNA can silence mRNA production in the AWC neuron and germline and whether this silencing is altered in response to olfactory adaptation. We show that odor mediates silencing of the reporter and that this silencing is dependent on the dsRNA import channel SID-1. We will discuss how olfactory adaptation may mediate this silencing.

O'Halloran, Damien M. et al. (2011) International Worm Meeting "Contribution of cyclic nucleotide gated (CNG) channel subunits to behavioral plasticity in C. elegans."

Yu, Yawei, L'Etoile, Noelle D., Brueggemann, Chantal, Chen, Tsung-Yu, Zhang, Xiao-Dong, Altshuler-Keylin, Svetlana, O'Halloran, Damien M.

[International Worm Meeting, 2011]

The Caenorhabditis elegans AWC neurons are responsible for sensation of a range of attractive volatile odors (Bargmann et al., 1993). Prolonged odor exposure in the absence of food leads to reversible decreases (adaptation) in the animal's attraction to that odor (Colbert and Bargmann, 1995; L'Etoile et al., 2002; Nuttley et al., 2002; Kaye et al., 2009; O'Halloran et al., 2009). It has been shown previously that the odor specificity of adaptation is determined by the feeding status of the animal (Colbert and Bargmann, 1997). That is, if a well-fed worm is exposed to benzaldehyde for a sustained period, it will adapt to both benzaldehyde and isoamyl alcohol (both sensed by AWC), this process is termed cross adaptation. In contrast, an unfed (starved) worm will adapt to benzaldehyde and its response to isoamyl alcohol will remain intact. The TAX-4 and TAX-2 cyclic nucleotide-gated (CNG) channel subunits are required for AWC-mediated olfactory responses (Coburn and Bargmann, 1996; Komatsu et al., 1999). TAX-4 is an a subunit that can form homomeric channels while TAX-2 is a b subunit that requires TAX-4 to form a functional channel (Coburn and Bargmann, 1996; Komatsu et al., 1999). C. elegans encodes two additional predicted a subunits, CNG-1 and CNG-3 (Cho et al., 2004a; 2004b; Coburn thesis, 1996). Here we report that CNG-1 is required in AWC to promote short-term (30-mins) cross adaptation between benzaldehyde and isoamyl alcohol. The ability of food to induce this cross adaptation is also dependent on the ASI sensory neurons. We also demonstrate that CNG-3 is required in AWC for adaptation to short-term (30-mins) exposures of odor. By using FRET, Bio-molecular Fluorescent Complementation assays, and genetically encoded calcium imaging we find that TAX-2::TAX-4::CNG-3 channels may promote short-term adaptation in AWC. Our data suggests the TAX-2::TAX-4::CNG-3 channel adopts a 3a:1b stoichiometry. By examining the electrophysiology of the CNG subunits in AWC, we also demonstrate that fast closing dynamics appear critical for proper short-term adaptation responses. Taken together our findings provide more understanding into the mechanisms and circuitry of how CNG channels shape olfactory plasticity.

Nagpal, Jatin et al. (2015) International Worm Meeting "Optogenetic control of cGMP mediated signal transduction ."

Woldemariam, Sarah, Schneider, Martin, Gottschalk, Alexander, Nagel, Georg, Nagpal, Jatin, Gao, Shiqiang, L'Etoile, Noelle, Bethke, Mary, Brueggemann, Chantal, Steuer Costa, Wagner

[International Worm Meeting, 2015]

Cyclic guanosine monophosphate (cGMP) is a widely used 2nd messenger in cellular signaling, acting via protein kinase G (PKG), or cyclic nucleotide gated (CNG) channels. In sensory neurons, cGMP allows for signal modulation and amplification, before depolarization. Manipulating cGMP levels is required to access this signalling and provide insights into signal encoding. We achieve this by implementing two photo-activatable guanylyl cyclases - 1) guanylyl cyclase rhodopsin from Blastocladiella emersonii (BeCyclOp) and 2) a mutated version of Beggiatoa sp. bacterial light-activated adenylyl cyclase (BlaC), with specificity for GTP, termed BlgC or bPGC (Beggiatoa photoactivated guanylyl cyclase). BeCyclOp enabled rapid and precise light-triggered cGMP increases in heterologous cells (Xenopus oocytes) and in C. elegans. BeCyclOp exhibits an unusual 8 transmembrane topology and cytosolic N-terminus. Oocyte experiments revealed light/dark activity ratio of ~5,000 and no cAMP production. Via co-expression of the TAX-2/-4 CNG channel in C. elegans body wall muscle, BeCyclOp photoactivation induced rapid light-driven depolarization and contraction of muscle cells. In C. elegans O2/CO2 sensory BAG neurons, BeCyclOp activation rapidly triggered slowing of locomotion, consistent with the normal sensory function of BAG, and in agreement with previous BAG activation by channelrhodopsin (ChR2). Interestingly, a quick 'recovery' of the slowing response was observed both in ChR2 and BeCyclOp stimulation, despite ongoing photostimulation, arguing that this apparent desensitization is neither mediated at the level of cGMP nor the CNG channel, but at the output synapses or in downstream networks.Light activation of bPGC expressed in muscle cells along with TAX-2 and TAX-4 caused a relatively slower and less pronounced contraction as compared to BeCyclOp. We could validate these differences in cGMP production from the two cyclases by directly imaging the cGMP rise, using a genetically encoded cGMP sensor, WincG2. WincG2 (or worm indicator of cGMP) is based on FlincG3, a circularly permutated EGFP fused to cGMP binding domain of PKG. Currently, we are expressing the cyclases in a variety of C. elegans sensory neurons that use cGMP as the 2nd messenger and are performing behavioural experiments that recapitulate cGMP mediated signal transduction in these sensory neurons using optogenetic activation of the cyclases.

Nathan, Sara et al. (2015) International Worm Meeting "Identifying and characterizing odor receptors in C. elegans."

Cox-Harris, Alesha, Brueggemann, Chantal, Mosqueda, Berenice, Young, Jared, Newman, Elizabeth, Nathan, Sara, Morton, Riva, Dalton, Cassidy, L'Etoile, Noelle, Apostol, Sherrlyne

[International Worm Meeting, 2015]

Although scientists have been studying olfaction in C. elegans for decades, olfactory receptor proteins remain largely uncharacterized. To address this knowledge gap, we are pursuing localization of candidate odor receptor proteins, and studying odor responses in genetically altered worms. We are producing lines with GFP-tagged putative odor receptors using constructs obtain from the Transgeneome project, and looking for proteins that localize to sensory cilia. We are testing chemotaxis to a variety of odors of worms in which expression of putative odor receptors have been reduced using RNA interference. We are also generating knockout worms for putative odor receptors using the CRISPR technique, to be tested in chemotaxis assays. .

Krzyzanowski, Michelle C et al. (2013) International Worm Meeting "The C. elegans cGMP-dependent Protein Kinase EGL-4 Regulates Nociceptive Behavioral Sensitivity."

Yu, Michael C, Ferkey, Denise M, Wood, Jordan F, Michaels, Kerry L, Ezak, Meredith J, Collins, Kimberly D, Brueggemann, Chantal, Jackson, Christopher A, Juang, Bi-Tzen, L'Etoile, Noelle D, Krzyzanowski, Michelle C

[International Worm Meeting, 2013]

The ability of an animal to detect and avoid noxious compounds in the environment is critical to its survival, and signaling levels within sensory neurons must be tightly regulated to allow cells to integrate information from multiple signaling inputs and to respond to new stimuli. G protein-coupled signaling pathways play a major role in mediating C. elegans avoidance of several ASH-detected nociceptive stimuli, including the bitter tastant quinine. Herein, we report a new role for the cGMP-dependent protein kinase EGL-4 in the negative regulation of G protein-coupled nociceptive chemosensory signaling. We have found that C. elegans lacking the cGMP-dependent protein kinase EGL-4 function are hypersensitive in their behavioral response to low concentrations of quinine and exhibit an elevated calcium flux in the ASH sensory neurons in response to quinine. We provide the first direct evidence for cGMP/PKG function in ASH and our data suggest that activated EGL-4 dampens quinine sensitivity via phosphorylation and activation of the regulator of G protein signaling (RGS) proteins RGS-2 and RGS-3, which in turn downregulate Ga signaling in ASH and, as a result, behavioral sensitivity. Moreover, animals lacking the function of the transmembrane quanylyl cyclases ODR-1 and GCY-27, or the soluble guanylyl cyclases GCY-33 and GCY-34, are also hypersensitive in their response to dilute quinine. However, these GCYs do not appear to function directly in the ASHs, suggesting that they act in a non-cell-autonomous manner to regulate ASH function, and that other neurons in the circuit may provide the cGMP that regulates EGL-4 and ASH function.

K Brunschwig et al. (2004) European Worm Meeting "GERMLINE DEVELOPMENT REQUIRES LET-418 AND A PUTATIVE INTERACTING PARTNER"

K Brunschwig, M Passannante, F Mueller, C Wicky, C Marti

[European Worm Meeting, 2004]

let-418, and chd-3, are two C. elegans Mi-2 orthologs (von Zelewsky et al., 2000). In vertebrates, Mi-2 is a component of the NuRD (nucleosome remodeling and histone deacetylase) complex. Among a variety of defects, gonad extension and germ cells proliferation are defective in let-418 mutants suggesting that let-418 plays an essential role in germline development. Recently, we also observed a presumed role of LET-418 in antagonizing the RAS/MAPK signaling pathway in the germline (see Chantal Wicky's abstract). Using a yeast two-hybrid screen, a transcriptional co-repressor and tumor suppressor homolog was identified to specifically interact with the C-term of LET-418. Similarly to let-418 mutants, RNAi interference as well as deletion mutations of this interaction partner exhibited an improper germline development. In both cases, the phenotype is enhanced in a chd-3(eh4) mutant background. In addition, the expression patterns of gfp fusion constructs and immunofluorescence straining of LET-418 and its putative interacting protein matched perfectly in most of the somatic gonad and germ cells. This suggests a common role of these two putative transcriptional repressors in the regulation of the development of the C. elegans reproductive system. Genetic and biochemical analyses will give us further insights into the function of these proteins in germline development.