Shui, Y. et al. (2017) International Worm Meeting "Molecular basis of antidromic rectification of gap junctions between AVA interneurons and motor neurons in C. elegans escape circuit."

Shui, Y., Liu, P., Wang, Z. W., Chen, B., Zhan, H.

[International Worm Meeting, 2017]

C. elegans AVA command interneurons play important roles in escape behavior, and contact A-type cholinergic motor neurons (A-MNs) through both electrical and chemical synapses. Our recent study shows that the gap junctions (GJs) between AVA and A-MNs only allow antidromic currents (from A-MNs into AVA), and that the function of these GJs depends on UNC-7 innexin in AVA and UNC-9 innexin in A-MNs (Liu et al., Nat Commun 2017). However, molecular basis of the antidromic rectification is unknown. To address this question, we began by expressing UNC-7 and UNC-9 in Xenopus oocytes, and analyzing biophysical properties of homotypic and heterotypic GJs formed by them. While UNC-9 has only one isoform, UNC-7 has at least three different isoforms (UNC-7a, UNC-7b and UNC-7c), which differ in the length of the amino terminal. UNC-7c has a short amino terminal (24 residues before the 1st membrane-spanning domain/TM1) like UNC-9 (26 residues before TM1) whereas UNC-7a and UNC-7c have additional 120 and 52 residues, respectively, before the first amino acid of UNC-7c. We recorded junctional currents (Ij) from paired oocytes by holding one oocyte at a constant voltage (-30 mV) while applying voltage steps of -150 mV to +50 mV (at 10-mV intervals) to the other oocyte. The voltage difference between the two oocytes is the junctional voltage (Vj). We measured the steady-state Ij at all the Vj steps (-120 to +120 mV), plotted the Gj - Vj relationship, and fitted the Gj - Vj relationship to a Boltzmann function. We found that homotypic GJs of UNC-9 and UNC-7c are similar in the Gj - Vj relationship but are very different from those of UNC-7a and UNC-7b. Although all the UNC-7 isoforms may form heterotypic GJs with UNC-9, only one of them can form heterotypic GJs that allow unidirectional current flow (from UNC-9 oocyte to UNC-7 oocyte). Expression of this specific UNC-7 isoform in AVA interneurons in an unc-7 mutant significantly restored the antidromic junctional currents between AVA and A-MNs whereas the other two UNC-7 isoforms had either no effect or a much weaker effect. Taken together, our results suggest that 1) the amino terminal domain of UNC-7 plays important roles in GJ gating; 2) GJs between AVA and A-MNs probably consist of UNC-9 in A-MNs and a specific UNC-7 isoform in AVA; and 3) interactions between UNC-7 and UNC-9 hemichannels can reciprocally influence their gating properties.

Bergstrom AR et al. (1991) International C. elegans Meeting "IDENTIFICATION OF A Gj-LlKE PROTEIN IN C. ELEGANS MEMBRANES: POSSIBLE ROLE IN POLYAMINE METABOLISM."

Schaeffer JM, Bergstrom AR

[International C. elegans Meeting, 1991]

Guanine nucleotide-binding regulatory proteins (G-proteins) represent a family of trimeric proteins which mediate communication between a variety of cell surface receptors and effector molecules such as adenylate cyclase, phosphoinositol-specific phospholipase C and ion channels. When an appropriate agonist binds to a G protein- linked receptor, the alpha subunit of the G protein is activated and binds GTP resulting in a dissociation of the three G protein subunits. The intrinsic GTPase activity of the alpha subunit hydrolyzes GTP to GDP and the subsequent GDP-alpha subunit recombines with the other two G protein subunits ending the activation cycle. There are both stimulatory- and inhibitory-G proteins which are specifically ribosylated by bacterial toxins. We have identified and characterized a 41,000 dalton protein from C. elegans membranes with properties similar to previously described Gj-like proteins. Our laboratory previously presented evidence that specific dibenzoguanidines (and structural analogs including robenidine) are potent inhibitors of C. elegans ornithine decarboxylase, possibly acting via a G-protein. Consequently, the effect of robenidine on pertussis toxin stimulated ADP-ribosylation and GTPase activity in C. elegans has been evaluated. As shown in this poster, robenidine is a potent inhibitor of pertussis toxin-mediated ADP-ribosylation and stimulator of GTPase activity. Further work is required to determine whether there is a direct link between this Gj-like protein and ODC activity.

Ge, Qian et al. (2009) International Worm Meeting "INX-18 and UNC-9 mediate electrical coupling of C. elegans body-wall muscle cells."

Wang, Zhao-Wen, Ge, Qian, Hall, David H., Altun, Zeynep F., Xu, X Z Shawn, Chen, Bojun, Rauthan, Manish

[International Worm Meeting, 2009]

Two or more gap junction (GJ) proteins (connexins, pannexins, and innexins) are often coexpressed in the same cells. A poorly resolved question is how different GJ proteins contribute to electrical coupling of the same cells. Analyses of C. elegans body-wall muscle might shed light on this question because contributions of different GJ proteins may be evaluated by a combination of electrophysiological and genetic approaches. We previously showed that electrical coupling of the muscle cells is deficient but not absent in a null mutant of the innexin unc-9, suggesting that UNC-9 and at least one additional innexin play roles in the coupling. To identify the remaining innexin(s) contributing to the coupling, we analyzed expression patterns for the remaining 24 members of the worm innexin family by expressing promoter::GFP transcriptional fusions. This analysis led to the identification of five more innexins expressed in body-wall muscle cells, including INX-8, INX-9, INX-11, INX-14 and INX-18. Comparisons of junctional currents (Ij), which are through intercellular channels, between wild-type and mutant worms suggest that INX-8, INX-9 and INX-11 do not contribute to the coupling. INX-14 also did not appear to play a role in the coupling because it was not localized to intercellular junctions. In contrast, Ij was significantly reduced in inx-18 mutant and completely absent in inx-18;unc-9 double mutant, suggesting that INX-18 is an important contributor to the coupling. Immunohistochemistry with worms expressing epitope-tagged INX-18 and UNC-9 in muscle cells showed that INX-18 colocalized with UNC-9 as puncta at intercellular junctions. Nevertheless, INX-18 and UNC-9 localizations were independent, as determined by analyzing localization of one innexin in mutant of the other. inx-18 mutant exhibited a significant increase in the bending amplitude of locomotion. Because inx-18 expression was not detected in any neurons important to locomotion, this mutant phenotype suggests that the function of INX-18 in muscle cells is important to generating the normal locomotion waveform. Collectively, our observations suggest that INX-18 and UNC-9 are the most important contributors to electrical coupling of body-wall muscle cells.

Starich TA et al. (2000) Midwest Worm Meeting "Gap junction proteins in C. elegans"

Shaw JE, Starich TA, Miller A, Hall DH

[Midwest Worm Meeting, 2000]

The innexins (invertebrate connexins) represent gap junction (GJ) proteins identified thus far only in invertebrates, includingC. elegans, Drosophila, and Schistocerca. The innexins encoded by the Drosophila shaking-B(lethal) gene (Phelan et al. 1998) and the C. elegans inx-3 gene (Landesman et al. 1999) are able to mediate electrical coupling when expressed in paired Xenopus oocytes. To verify that INX-3 is associated with GJs, we raised polyclonal antibodies to the carboxyl terminus of INX-3 for use in immuno-EM localization studies. Affinity-purified antibodies specifically labelled GJs in the pharynx in adult, larval, and late embryonic stages. In earlier embryos, very small GJ plaques showed labelling in cells which were not highly differentiated. To investigate the function of GJs during embryogenesis and establishment of neural wiring, we are using GFP fusions and specific antibodies to determine expression patterns of different innexin proteins. By these criteria at least three innexins, including INX-3, are expressed in 2-cell embryos. Two innexins, including UNC-7, are strongly expressed in the nerve ring, and UNC-7 is also detected in the ventral and dorsal nerve cords, pharyngeal nervous system and other process bundles. In the hypodermis, expression of at least four innexins has been observed. RNA interference experiments and mutant screens are ongoing for some of these innexin genes. A mutation in inx-3 has been isolated; inx-3 mutant embryos die during embryogenesis with defects evident during mid to late stages of embryonic morphogenesis. The mutant phenotype suggests a role for INX-3 in maintaining hypodermis integrity during embryogenesis and for attachment of the anterior pharynx to the nose.

TA Starich et al. (1999) International C. elegans Meeting "Localization of Gap Junction Proteins"

JE Shaw, DH Hall, TA Starich, A Miller

[International C. elegans Meeting, 1999]

The innexins ( in vertebrate con nexins ) represent gap junction (GJ) proteins in C. elegans and Drosophila. The strongest evidence for this is borne out of experiments showing that the Drosophila Shaking-B vit protein is able to mediate electrical coupling when expressed in paired Xenopus oocytes (Phelan et al., Nature 391:181, 1998). Similar experiments have now shown that the C. elegans INX-3 protein is also capable of mediating electrical coupling in paired Xenopus oocytes (Landesman, White, Paul and Goodenough, submitted). To investigate the function of GJs during embryogenesis and establishment of neural wiring we are using GFP fusions and specific affinity-purified antibodies to determine which innexins are expressed early in development and in the nervous system. By these criteria at least three innexins, including INX-3, are expressed in 2-cell embryos, although one of these does not appear to localize to plaques at this stage. Two innexins, including UNC-7, are strongly expressed in the nerve ring, and UNC-7 is also detected in the ventral and dorsal nerve cords, pharyngeal nervous system and other process bundles. In the hypodermis, expression of at least three innexins has also been observed. RNAi experiments and mutant screens are ongoing for these innexin genes; the RNAi phenotype for INX-3 suggests a role in maintaining hypodermis integrity during embryogenesis and attachment of the anterior pharynx in the head. To verify that innexins are associated with GJs, polyclonal antibodies raised to the carboxyl terminus of INX-3 were used in immuno-EM localization studies. A post-embedding immunogold technique (Miller and Hall, WBG 1998; Hall, Methods in Cell Biology, 1995) was employed after animals were immersed in fixative in a microwave oven to promote rapid fixation and to improve penetration of the embedding resin. Virtually all GJs could be labelled in the pharynx in adult, larval or late embryonic stages. This binding was specific since pre-mixing of the antibody with the fusion peptide reduced or eliminated labelling. In early embryos, very small GJ plaques showed labelling in cells which were not highly differentiated (i.e. prior to expression of pharyngeal markers).

Schwarz EM et al. (1996) East Coast Worm Meeting "Cellular and molecular analysis of thermosensation"

Chalfie M, Schwarz EM

[East Coast Worm Meeting, 1996]

While senses like sight, hearing, and mechanosensation are becoming well understood, thermosensation remains obscure. Work by Hedgecock (1) and Mori (2) has shown that C. elegans is a promising model system for metazoan thermosensation, but neither the cells nor the genes required for thermosensation in C. elegans are fully known. Previous work in this laboratory (3) has shown that at least three new deg mutations cause cells in the head to degenerate while partially crippling thermosensation; none of these are allelic to previously described ttx mutations . We have therefore begun to determine which cells are defective in these deg mutations. We have also begun genetic mapping experiments aimed at positional cloning of the wild-type deg loci. References: 1. Hedgecock, E.M. and Russell, R.L. (1975). Proc. Natl. Acad. Sci. U.S.A. 72, 4061-4065. 2. Mori, I. and Ohshima, Y. (1995). Nature 376, 344-348. 3. Treinin, M. and Chalfie, M. (1993). International C. elegans meeting abstracts, p. 446.

Ephraim Tsalik et al. (2002) West Coast Worm Meeting "AIY-Mediated Temperature Modulation of Behavior"

Ephraim Tsalik, Oliver Hobert

[West Coast Worm Meeting, 2002]

Thermotaxis has served as one of the key behavioral paradigms in C. elegans since its initial characterization in 1975 (Hedgecock and Russell, PNAS, 1975. 72(10):4061-5). With the neuronal pathway mediating thermosensation established, there has been comparatively little advance in understanding how temperature input might relate to other known C. elegans behaviors (Mori and Ohshima, Nature. 1995. 376:344-348). We have begun a preliminary analysis of how various behaviors are modulated by temperature as well as what role the AIY interneuron class plays in this modulation. By testing various mutant backgrounds as well as laser ablation studies, we have looked at known behaviors such as defecation, chemotaxis and radial locomotion. We have also analyzed a new behavior, which we call thermokinesis and define as the temperature and experience modulated movement of an animal with no specified body-axis orientation. This behavior is based on the previously described thrashing of worms in liquid media (Miller et al., PNAS, 1996. 93(22):12593-8), (Tsalik and Hobert, 2001 International C. elegans Meeting).

Arpagaus M et al. (1991) International C. elegans Meeting "ACETYLCHOLINESTERASES IN STEINERNEMA CARPOCAPSAE AND CAENORHABDITIS ELEGANS."

Arpagaus M, Thierry-Mieg D, Toutant J-P, Berge J-B

[International C. elegans Meeting, 1991]

We are interested in the structure of the different molecular forms of acetylcholinesterase (AChE) with a particular interest for the association of AChE to membranes, and in cloning the three ace genes of C.elegans. Preliminary studies have been conducted on the nematode Steinernema carpocapsae since it shows a much higher AChE activity than C. elegans. Sucrose gradient centrifugation analysis of crude extracts shows four AChE peaks sedimenting at 4S, 6S, 12S and 14S, a situation similar to that found in C. elegans wild type strains ( Johnson and Russell, 1983). Further analysis of gradient fractions by non denaturing electrophoresis reveals the existence of both hydrophilic and hydrophobic components in 6S and 14S forms. The hydrophobic 6S form is completely converted into its hydrophilic counterpart by phosphatidylinositol-specific-phospholipase C (PIPLC), indicating that this molecule possesses a glycolipid anchor close to the type already described in insect and vertebrate dimeric AChEs. The hydrophobic 14S component however is not susceptible to PIPLC, and the nature of the hydrophobic domain is still under investigation, as well as the molecular organization of all forms. We started the cloning of C. elegans ace 3 gene. For this purpose we improved the genetic map at the tip of chromosome II. ace 3 maps 0.4 map unit from unc 52, which has been cloned by D. Moerman, and lies in the center of a 600 kb contig. We are trying to clone ace 3 by transformation rescue.

Gal Haspel et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Stretch Receptors and Pattern Generation in C. elegans Locomotion Circuit"

Anne Hart, Gal Haspel

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

The anatomy of the nervous system of the nematode C. elegans has been comprehensively described compared to other animal species; the location and connectivity of each neuron are known. This affords us the opportunity to address the neural circuitry underlying various behaviors such as locomotion. Only five pairs of interneurons and six groups of motoneurons have been are central to locomotion based on ablation studies. Yet how, these neurons orchestrate their activity during locomotion is unclear. The sinusoid motion locomotive pattern might rise from a sensory feedback loop or involve a central pattern generator. Even in the latter case, sensory feedback might serve to adjust the pattern generator to environmental cues. Taking advantage of C. elegans optical transparency and the relative ease of genetic manipulation, we have used cameleon (a genetically encoded calcium indicator) to record the activity in specific motoneurons during fictive locomotion. Two initial findings stand out. First, mechanically moving the nematode activates motoneuron activity- suggesting the presence of stretch receptors. This supports a hypothesis first suggested by R.L. Russell based on motoneuron anatomy 30 years ago. Second, by presenting an aversive stimulus, we were able to trigger rhythmic activity in the motoneurons of immobilized animals, indicating the presence of a central pattern generator, active even in the absence of sensory feedback. These findings begin to unravel the characteristics of the nematode locomotion circuit. Understanding the orchestrated activity of neurons within this circuit will provide us with general insights into how neurons produce rhythmic movements in animals and into nematode behaviors that involve locomotion.

Collet J et al. (1996) Midwest Worm Meeting "ANALYSIS OF osm-6, A GENE THAT AFFECTS CHEMOSENSATION AND SENSORY CILIA STRUCTURE IN C. ELEGANS"

Herman RK, Shaw JE, Collet J, Lundquist EA, Spike CA

[Midwest Worm Meeting, 1996]

Many sensory neurons in C. elegans have ciliated dendritic endings. Mutations in several genes disrupt the structure of the sensory cilia and lead to defects in chemosensory behaviors. One of these genes is osm-6, which was first defined by a mutation that resulted in abnormal avoidance of high osmolarity (Culotti and Russell 1978). An ultrastructural analysis has shown that the tips of osm-6 mutant sensory neurons have greatly shortened axonemes and ectopic membrane-attached microtubules assembled proximal to the cilia (Perkins et al. 1986). No defects in the microtubular structures were detected. The osm-6 gene may thus play a role in the assembly of sensory cilia. A total of five osm-6 mutations are now available (Starich et al. 1995). All result in severe defects in the updake of fluorescent dyes such as FITC and DiO by amphid and phasmid neurons in living animals (Perkins et al. 1986; Starich et al. 1995). We have cloned osm-6 by transposon tagging. Two osm-6 spontaneous mutations identified in the mutator strain RW7097 were found to be associated with Tc1 insertions, at sites about 0.5 kb apart. Spontaneous revertants of these mutations were correlated with excision of the Tc1 elements. In germline transformation experiments, a 3.8-kb genomic clone that includes the two Tc1 insertion sites was able to rescue fully the dye filling defect of an osm-6 mutant. We have identified DNA alterations in this region for all five osm-6 mutations. One of the mutations was shown by sequence analysis to be an amber mutation, and it is suppressed by the amber suppressor sup-5. On northern blots of poly(A)-enriched RNA, we detected a single osm-6 transcript, which is about 1,500 nucleotides long and most abundant in embryos and L1 larvae. We have sequenced the 3.8-kb rescuing genomic clone and a 1.5-kb cDNA clone. Comparison of the sequences indicates that the cDNA clone has 11 exons derived from a 3-kb sequence within the 3.8-kb rescuing genomic sequence. The cDNA sequence has an open reading frame that can encode a polypeptide (OSM-6) of 472 amino acid residues. The OSM-6 amino acid sequence suggests that it is cytoplasmic. OSM-6 is similar over nearly its full length (40% identical, overall) to the putative translation product of a mammalian mRNA that is down regulated in a neuroblastoma-glioma hybrid cell line after opioid treatment (Wick et al. 1995). Sequences nearly identical to segments of the latter message have been identified as expressed sequence tags from rat and human. We have conducted a genetic mosaic analysis of osm-6 function, using an extrachromosomal array containing wild-type copies of osm-6, ncl-1 and unc-36. Animals in which different neurons within a single amphid have different genotypes, as judged by their Ncl phenotypes, indicate that osm-6 affects DiO filling cell autonomously. References: Culotti, J. G., and R. L. Russell (1978).Genetics 90, 243-256. Perkins, L. A., Hedgecock, E. M., Thomson, J. N., and Culotti, J. G. (1986). Dev. Biol. 117, 456-487. Starich, T. A., et al. (1995) Genetics 139, 171-188. Wick, M. J., D. K. Ann, and H. H. Loh (1995) Mol. Brain Res. 32, 171-175.