Shin Takagi et al. (2001) International C. elegans Meeting "Involvment of PlexinA in vulval morphogenesis"

Shin Takagi, Hajime Fujisawa, Akira Nukazuka, Go Shioi, Yukimasa Shibata, Takashi Fujii

[International C. elegans Meeting, 2001]

Involvment of PlexinA in vulval morphogenesis Shin Takagi, Takashi Fujii, Akira Nukazuka, Yukimasa Shibata, Go Shioi, Hajime Fujisawa Laboratory of Developmental Neurobiology, Division of Biological Science, Graduate School of Science, Nagoya University, Japan, CREST, JST The plexin family transmembrane proteins are putative receptors for semaphorins, which are implicated in the morphogenesis of animal embryos including axonal guidance. We have generated and characterized putative null mutants of the plx-1 ( pka. cep-2 ) gene encoding C. elegans plexinA. plx-1 mutants exhibited morphological defects in several organs of epidermal origin, such as Ray1 in the male tail (see Fujii et al . in this Meeting) and alae. Here we report on the defects in vulvae of plx-1 mutants. plx-1 adult hermaphrodites sometimes had a deformed vulva or extra vulva-like structures. plx-1::gfp transgene was expressed in the vulval primordial cells. Since the epidermal cells composing the vulval primordium are known to undergo dynamic changes in shape and position during the vulval morphogenesis, we analyzed the vulval primordium with MH27::gfp . The epidermal cells of the vulval primordium were aberrantly arranged in plx-1 mutants: the rotation-symmetric configuration of the primordum was often disrupted, and some cells were not in contact with each other. Some cells failed to participate in the formation of the main vulva and formed an extra vulva-like depression. The results indicate that plx-1 regulates the arrangement and/or migration of epidermal cells in this system.

Pin-An Chen et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Identification of an activated allele of Go alpha"

Pin-An Chen, Erik Jorgensen

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

Neurotransmission can be regulated by G protein pathways. In C. elegans, Gq signaling is the main pathway that stimulates neurotransmitter release. Go plays an inhibitory role in neurotransmission, possibly by negative regulating Gq . However, the downstream effectors of Go and the linkage between Go and Gq are still unclear. We cloned the gene mutated in a dominant paralyzed mutant, unc-109(n499). We found that unc-109(n499) has a R179C mutation in Go alpha;, encoded by goa-1. This arginine residue has been shown to be important for GTPase activity of G alpha; suggesting that unc-109(n499) is an activated allele of goa-1. The phenotype of unc-109(n499) is what we would predict for an activated Go alpha;. First, the phenotype of n499 worms is remarkably similar to null alleles of Gq alpha;: worms are paralyzed and have a straight body posture. Second, activated alleles of Gq alpha;, encoded by egl-30 (tg26), can suppress the lethality of n499 homozygotes and the uncoordinated movement of the n499 heterozygote. Third, mutations in a potential downstream target of Go, DAG kinase, also suppresses n499. Thus, unc-109(n499) is a great tool to dissect the Go pathway by looking for suppressors. We have screened 24,000 haploid genomes and obtained 18 candidates. We are now characterizing and mapping these suppressor candidates.

Mendel JE et al. (1995) International C. elegans Meeting "The Heterotrimeric G protein, Go, regulates several C. elegans behaviors"

Mendel JE, Plasterk RHA, Korswagen HC, Liu KK, Simon MI, Sternberg PW, Hajdu-Cronin YM

[International C. elegans Meeting, 1995]

The C. elegans gene encoding the alpha subunit of the heterotrimeric G protein Go, goa-1, is expressed in most neurons, the pharynx, and in muscles involved in defecation, egg-laying, and male mating. The reduction of function mutations, goa-1(pk62) and goa-1(sy192), cause a variety of behavioral defects including hyperactive movement, premature egg-laying, and male impotence. Expression of activated Go alpha subunit in transgenic nematodes results in lethargic movement, slow pharyngeal pumping in the presence of food, irregular defecation, delayed egg-laying, and reduced mating efficiency. Induction of activated Go alpha subunit by heat-shock at any developmental stage results in rapid arrest of movement, pumping, and in adults, egg production and egg-laying. Go is thus involved in neuron and muscle function throughout C. elegans life. We are investigating the role of Go alpha subunit in regulating behavior by analysis of presumed dominant negative mutations, epistatic interactions between goa-1 and other G protein subunit genes, and the isolation and analysis of extragenic suppressor mutations.

Kaplan JM et al. (2000) West Coast Worm Meeting "Is the DAG kinase DGK-1 an effector of Go alpha (GOA-1)?"

Dybbs M, Kaplan JM, Nurrish SJ

[West Coast Worm Meeting, 2000]

We (and others) have previously described two competing G-protein pathways acting in motor neurons to either facilitate or inhibit synaptic transmission at neuromuscular junctions (NMJs) [1-4]. Facilitation of release occurs via a pathway composed of a Gq alpha (EGL-30), a phospholipase C beta (EGL-8), and the diacylglycerol (DAG) binding protein UNC-13 [2,4]. The Gq alpha pathway can be activated by muscarinic agonists leading to the formation of the membrane bound second messenger DAG by EGL-8 and the subsequent recruitment of UNC-13 to sites of Acetylcholine release [2]. UNC-13 is essential for neurotransmitter release and it's enrichment at release sites correlates with an increase in neurotransmitter release. Addition of serotonin agonists inhibits acetylcholine release at NMJs. Inhibition of synaptic transmission by serotonin requires Go alpha (GOA-1) and a diacylglycerol kinase (DGK-1) which phosphorylates DAG to Phosphatididic Acid which is unable to bind UNC-13. Serotonin antagonists and goa-1 mutations result in the recruitment of UNC-13 to NMJs suggesting that the Gq and Go pathways converge to regulate UNC-13 localization, most likely by regulating levels of DAG [1,2]. As yet the Go signaling pathway is not well defined. Go may reduce DAG levels by activating DGK-1, by negatively regulating the Gq signaling pathway, or by an as yet undefined pathway. We are currently testing the first of these models. Co-expression of DGK-1 and activated Go alpha in human tissue culture cells (HEK cells) has no effect on the kinase activity of DGK-1. This suggests that there is no direct interaction between Go alpha and DGK-1. However, it is possible that Go alpha indirectly regulates DGK-1 via intermediates that are not conserved in HEK cells. Therefore we have generated a rescuing MYC tagged GFP::DGK-1 construct. The myc-DGK-1 protein can be immunoprecipitated from adult animals and possesses DAG kinase activity. We are currently testing whether the DGK-1 DAG kinase activity changes in response to levels of Go alpha signaling. We are also testing whether Go regulates the subcellular localization of DGK-1. Nurrish et al.,(1999) Neuron:24 p231-242 Lackner et al.,(1999) Neuron:24 p335-346 Hajdu-Cronin et al.,(1999) Genes Dev 13: 1780-93 Miller et al.,(1999) Neuron 24: 323-33

Daniel L Chase et al. (2003) International Worm Meeting "Dopamine-resistant mutants of C. elegans identify components of Go and Gq signaling pathways"

Daniel L Chase, Judy S Pepper, Michael R Koelle

[International Worm Meeting, 2003]

Dopamine, a major neurotransmitter in the mammalian central nervous system, acts through two classes of G protein-coupled receptors known as D1-like and D2-like receptors. These two classes of receptor signal to antagonistically regulate neural activity. Despite intensive research, the signaling mechanisms responsible for the antagonistic effects of dopamine in the brain remain debated. We describe the first genetic screen designed to identify proteins required for dopamine signaling. C. elegans uses endogenous dopamine to inhibit locomotion behavior1, and treatment with excess exogenous dopamine paralyzes worms2. We isolated mutants unresponsive to exogenous dopamine and found that they are also defective for endogenous dopamine signaling. Eleven mutations identified five different genes. Four of these genes encode components of the Go and Gq signaling pathways, including the G protein Go itself and subunits of the RGS complex that inhibits Gq signaling. The Go and Gq signaling pathways act antagonistically in C. elegans to control behavior and their components are conserved in humans. Mutations in these signaling components that decrease Go signaling or increase Gq signaling cause dopamine resistance, while mutations that have opposite effects on these two signaling pathways cause dopamine hypersensitivity. Thus the physiological effects of dopamine are mediated by Go in C. elegans and are antagonized by Gq signaling. Such a dopamine signaling mechanism in mammals could explain many of the opposing effects of D1-like and D2-like receptor activation. Our screen also identified one gene that appears to encode a new Go/Gq signaling component. 1. Sawin, E.R., Ranganathan, R., and Horvitz, H.R. (2000). Neuron 26, 619-631; 2. Schafer, W.R., and Kenyon, C.J. (1995). Nature 375, 73-78.

Sarah Straud Anselmo et al. (2002) West Coast Worm Meeting "Developing a screen for HERG blocking drugs using the C. elegans pharynx"

Sarah Straud Anselmo, Leon Avery

[West Coast Worm Meeting, 2002]

The HERG (human ether-a-go-go related gene) potassium channel is expressed in the human heart and is responsible for proper repolarization of cardiac muscle at the end of the action potential. Compromising HERG function gives rise to Long QT Syndrome, a disorder in which one is predisposed to ventricular fibrillation and sudden death. Many drugs, including Seldane (an anti-histamine) and Propulsid (an acid reflux inhibitor), block HERG as an unwanted side effect and consequently have been taken off of the market.

Nurrish SJ et al. (1996) East Coast Worm Meeting "Downstream of goa-1; characterization of a serotonin signaling pathway."

Elkes DA, Nurrish SJ, Segalat LS, Kaplan JM

[East Coast Worm Meeting, 1996]

Addition of the neurotransmitter serotonin to C.elegans results in a reduction of locomotion and foraging behaviors; in contrast, mutations which reduce levels of endogenous serotonin cause animals to locomote and forage hyperactively. To identify genes required for serotonin signaling we have identified mutants displaying hyperactive locomotion and foraging behaviors which could not be corrected by the addition of exogenously added serotonin. These mutants are likely to define genes encoding proteins required at or downstream of the serotonin receptor; 17 such serotonin resistant mutants have been identified. Two of the genes represented by these mutants have been cloned, the first of which, the goa-1 gene encodes the alpha subunit of the G-protein Go, demonstrating that serotonin modulates behavior via a G-protein coupled receptor. Goa-1 mutants are hyperactive for locomotion, whereas transgenic animals that overexpress Go alpha mimic some of the effects of adding exogenous serotonin, i.e. display a severely reduced rate of locomotion . These results demonstrate that the levels of Go alpha are critical for the control of at least some serotonin modulated C.elegans behaviors. A second serotonin resistant mutant has identified the dgk-1 gene which encodes a diacylglycerol kinase(DGK) . In Drosophila DGK has been shown to be required for signaling downstream of light activated rhodopsin in a pathway that also requires the Gq alpha subunit and Phospholipase C (PLC). These results suggest a model in which at least some of the behavioral effects of serotonin are mediated by activation of PLC, perhaps via Go alpha. We will test this model by identifying further components of the serotonin signaling pathway. As a first step we have determined which serotonin resistant mutants are able to suppress the reduced locomotion phenotype associated with overexpression of goa-1. We are also testing the ability of the serotonin resistant mutants to suppress the reduced locomotion defect of animals expressing a constitutively active Go alpha mutant expressed from a heat shock promoter (kindly provided by P.Sternberg). Those mutants which suppress the defects resulting from overexpression of both wild type and constitutively active Go alpha are likely to define effectors downstream of Go alpha and will be further characterized.

Elkes DA et al. (1995) International C. elegans Meeting "SEROTONIN SIGNALING AND G-ALPHA O"

Kaplan JM, Segalat LS, Elkes DA

[International C. elegans Meeting, 1995]

We are interested in identifying the molecules required for serotonin (5-HT)signaling. Mutants that have low levels of 5-HT(e.g.cat-4) locomote and forage hyperactively. Therefore, to identify effectors of 5-HT signaling, we screened for hyperactive mutants whose hyperactivity is resistant to exogenous 5-HT. 28 mutants corresponding to at least 13 different genes fall into this category. In addition to the mutants we isolated, the following pre-existing mutations were also shown to disrupt 5-HT signaling: egl-21 IV, phm-2 I, and the allelic mutations n1134 and n363 I, which were isolated by Ambros and Horvitz. We have cloned the gene defined by n1134 andn363 and determined that it corresponds to goa-1, which encodes the alpha subunit of the heterotrimeric G-protein Go. Several lines of evidence suggest that Go mediates signaling by 5-HT. First, null mutations and overexpression of goa-1 cause reciprocal defects in three 5-HT controlled behaviors: locomotion, egg laying, and feeding. This indicates that the level of Go alpha activity is a critical determinant of these behaviors. Second, goa-1 mutants are resistant to the effects of 5- HT on locomotion and defecation but not feeding. Thus for feeding, 5-HT may act through other G proteins as well as Go for signaling. Third, goa-1 mutants are fully sensitive to other metabotropic agonists. Interestingly, Go appears to act differently in locomotion and egg laying. For locomotion, goa-1 animals behave like animals that lack 5-HT whereas for egg laying these mutants behave like worms in exogenous 5-HT. We propose that 5-HT inhibits locomotion and defecation via G-alpha o (hence goa-1 mutants lack signaling), whereas 5-HT stimulates egg laying through Go beta gamma (hence goa-1 mutants constitutively signal). Studies in mammalian neurons indicate that G- alpha o may play a role in axon outgrowth or guidance. We found that nearly all axons are normal in goa-1 mutants. However, a small but significant portion of HSN neurons have defects in cell and axon migration. Thus, in C. elegans, Go has a relatively modest role in axon guidance.

Pin-An Chen et al. (2007) International Worm Meeting "Identifying suppressors of an activated Go mutant."

Pin-An Chen, Erik Jorgensen

[International Worm Meeting, 2007]

Neurotransmission is modulated by G protein pathways. In C. elegans, Gq signaling is the major stimulatory pathway regulating neurotransmitter release. Conversely, Go plays an inhibitory role in neurotransmission, possibly by negatively regulating the Gq signaling. However, the downstream effectors of Go<font face=symbol>a</font> remain unclear. To characterize the Go pathway, we screened for suppressors of the activated Go<font face=symbol>a</font> mutant unc-109(n499ox304). unc-109(n499ox304) mutant worms are paralyzed and have a straight body posture. We screened approximately 24,000 haploid genomes for mutants that suppress the paralyzed movement or the straight body posture phenotype of unc-109(n499ox304). 17 candidate mutants were identified including two eat-16 mutants. EAT-16 is the RGS protein specific for Gq<font face=symbol>a</font>, and mutations in eat-16 were previously shown to suppress the paralyzed phenotype of animals overexpressing goa-1 (Hajdu-Cronin et al., 1999). In addition to eat-16, we also identified a dominant suppressor which is a gain-of-function allele of nca-1. nca-1 encodes a novel conserved channel, which we believe may be a target of G protein signaling. I am now in the process of mapping and characterizing the other candidate suppressors.

Segalat LS et al. (1995) International C. elegans Meeting "Signal transduction by G-proteins in C. elegans"

Kaplan JM, Segalat LS, Elkes DA

[International C. elegans Meeting, 1995]

Serotonin is a neuromodulator which acts mainly through seven transmembrane receptors (7-TMRs) coupled to heterotrimeric G proteins. Upon ligand binding, this class of receptors activates G proteins whose activity modulates the level of cytoplasmic second messengers, which in turn alter the activity of ion channels. The ion channels whose activities are regulated by serotonin are poorly understood. We have taken advantage of the fact that serotonin modulates locomotion and foraging, 2 behaviors of C. elegans easy to follow, to screen for mutants with altered behaviors. Twenty eight of the mutations we have characterized appear to be post-synaptic, because these mutants do not respond to the serotonergic agonist CGS 12066B. The genes corresponding to these mutations are candidates for being involved in the reception and/or the transduction of the signal, or in the ion channels modulated by serotonin. These mutations represent at least 12 genes. We have cloned one of them (n363), originally described by V. Ambros and M. Finney, which turned out to be the goa-1 gene, encoding the alpha subunit of the C. elegans Go protein and previously cloned by homology in the Sternberg lab. We have shown that the absence or the overexpression of Go alpha leads to opposite phenotypes for several serotonin-controlled behaviors (locomotion, foraging, feeding, egg-laying), suggesting that the level of Go alpha has a direct influence on several of the animal's behaviors. The site of action of Go seems to be the neurons for locomotion, and the vulva muscle for egg-laying. In mammals, heterotrimeric G proteins can signal either by the alpha or the beta-gamma subunit. We are currently testing which subunit is signaling in the different behaviors modulated by Go.