Cheryl Copelman et al. (2001) International C. elegans Meeting "A screen to identify regulators of DGK-1"

Stephen Nurrish, Cheryl Copelman

[International C. elegans Meeting, 2001]

Diacylglycerol (DAG) is a critical regulator of the release of acetylcholine (ACh) by motorneurons onto the body wall muscles [1-4]. Increases in DAG levels result in the recruitment of the DAG binding protein UNC-13, which is essential for neurotransmitter release [5], to release sites and this correlates with increases in ACh release. ACh release is increased by activation of Gq alpha (EGL-30) which in turn activates a Phospholipase C beta (EGL-8) which hydrolyzes PIP2 to produce DAG. Such Gq alpha/ PLC beta signaling pathways have been extensively characterized in many systems. Levels of DAG, and thus ACh release, are lowered by the action of the DAG kinase DGK-1 which phosphorylates DAG to phosphatidic acid to which UNC-13 cannot bind. In contrast to the activation of PLC beta little is known of how DAG kinases are regulated, although our previous work has suggested that DGK-1 may be a component of a Go alpha (GOA-1) signaling pathway. We aim to identify regulators of DGK-1 using a genetic approach. Animals overexpressing DGK-1 [dgk-1(xs)] have reduced rates of locomotion and to identify DGK-1 regulators we have performed an EMS screen for suppressors of the dgk-1(xs) reduced locomotion phenotype. We have screened 3906 haploid genomes and have identified 6 suppressors. We are currently mapping these suppressors using SNIP-SNPs. 1. Nurrish et al.,(1999) Neuron:24 231-242 2. Lackner et al.,(1999) Neuron:24 335-346 3. Hajdu-Cronin et al.,(1999) Genes Dev 13: 1780-1793 4. Miller et al.,(1999) Neuron 24: 323-333 5. Richmond et al., (1999) Nat.Neurosci 2: 959-964

Cheryl Copelman et al. (2002) European Worm Meeting "Screens to identify positive and negative regulators of neurotransmission"

Sarah Woolner, Cheryl Copelman, Stephen Nurrish

[European Worm Meeting, 2002]

The drug Aldicarb inhibits acetylcholine-esterases preventing the removal of the neurotransmitter Acetylcholine (ACh) from the synaptic cleft. The build up of ACh results in hypercontraction of muscles, paralysis and eventual death of animals. Mutants unable to release ACh are resistant to Aldicarb and screens for Aldicarb resistance, performed by many groups, have greatly increased our understanding of the essential components of the synaptic release cycle. However, less is known about the signalling pathways that impinge upon the synaptic release cycle to alter rates of ACh release. We are interested in defining the regulators of the synaptic release cycle using 2 genetic approaches based on our (and others) previous work demonstrating that serotonin inhibits ACh release in a diacylglycerol kinase DGK-1 dependent manner[1-4]. Activation of DGK-1 causes the secondary messenger diacylglycerol to be phosphorylated to form phosphatidic acid and this is believed to cause the loss of the DAG binding protein UNC-13 from release sites. UNC-13 is essential for neurotransmitter release [5] and it's enrichment at release sites correlates with an increase in neurotransmitter release. In the first screen we have slowed ACh release by over-expressing DGK-1 [dgk-1(xs)] resulting in reduced rates of locomotion. We hope to identify either positive regulators of DGK-1 or negative regulators of the synaptic release cycle as a whole by identifying suppressors of the reduced locomotion phenotype. We have presently screened 3906 haploid genomes and have identified 8 candidates of which 4 are strong suppressors of DGK-1 over-expression. We are currently mapping these suppressors using single nucleotide polymorphisms (SNIP-SNPs) and have associated 2 of these suppressors to the left arm of chromosome 1. In the second screen we have increased the rates of ACh release by the addition of the serotonin antagonist methiothepin which results in hypersensitivity to Aldicarb. We are identifying animals that are unable to sustain high levels of ACh release caused by methiothepin but which are normal for ACh release in the absence of methiothepin. We have screened 1330 haploid genomes and have identified 9 suppressors one of which has been mapped to the left arm of X by SNIP-SNPs. These mutants are candidates for positive regulators of ACh release.

Paul Morrison et al. (2003) International Worm Meeting "Identifying regulators of DGK-1: genetic and biochemical approaches."

Stephen Nurrish, Cheryl Copelman, Paul Morrison

[International Worm Meeting, 2003]

Addition of the neuromodulator serotonin to C.elegans causes a reduction in locomotion. Mutations in the G alpha subunit GOA-1 or the DAG kinase DGK-1 substantially blocks the ability of serotonin to reduce locomotion. Very little is known about DAG kinases. DGK-1 possesses a number of protein:protein and protein:lipid interaction domains in addition to its lipid kinase domain, suggesting that either its lipid kinase activity or localisation can be regulated. The purpose of this study is to utilise two complementary approaches to identify potential interacting partners of DGK-1 with the aim of delineating its regulatory mechanisms. Firstly we have adopted a genetic approach screening worms over-expressing DGK-1 [dgk-1(xs)] which reduces ACh release resulting in reduced rates of locomotion. We hope to identify either positive regulators of DGK-1 or negative regulators of the synaptic release cycle as a whole by identifying suppressors of the reduced locomotion phenotype. We have presently screened 3906 haploid genomes and have identified 8 candidates of which 4 strong suppressors of DGK-1 over-expression proved to be alleles of goa-1. We are currently mapping the remaining suppressors. Secondly we have taken a biochemical approach to identify protein binding partners of DGK-1. We have carried out a yeast two-hybrid screen using full length DGK-1 as bait and identified a number of positive clones which are currently under analysis. In addition we have generated a rescuing MYC tagged GFP::DGK-1 construct and a range of GST::DGK-1 fusion proteins. We are using these constructs to test whether other proteins are co-associated with DGK-1.

Stephen Nurrish et al. (2002) European Worm Meeting "Genetic and biochemical approaches to study modulation of neurotransmitter release."

Iwan Evans, Paul Morrison, Stephen Nurrish, Sarah Woolner, Cheryl Copelman

[European Worm Meeting, 2002]

My lab is investigating the mechanisms by which certain neuromodulators cause widespread changes in synaptic strength which are believed to underlie changes in global behavioral states. For example, serotonin has been implicated in several aspects of mood and behavior, including depression, eating disorders, alcohol consumption, and aggression. To investigate neuromodulation we have carried out a genetic analysis of inhibition and facilitation of neurotransmission in an in vivo system, the C.elegans neuromuscular junction (NMJ). C.elegans changes it's rate of locomotion in response to a variety of stimuli, such as nutritional state and response to touch. In addition locomotion rates can be altered by the addition of exogenous neuromodulators, for example, addition of exogenous serotonin causes a pronounced decrease in locomotion. A screen for mutants defective in their response to serotonin has defined two competing G protein pathways that facilitate or inhibit release of acetylcholine (ACh) at the NMJ and thus alter rates of locomotion [1-4]. These two pathways, containing the trimeric G-protein alpha subunits goa-1 Go and egl-30 Gq, antagonistically control levels of the second messenger DAG and this in turn regulates the abundance of the DAG binding protein UNC-13 at synaptic release sites. Since UNC-13 interacts with syntaxin, an essential component of the synaptic vesicle release machinery, these experiments define a genetic pathway that leads from G-protein coupled receptors to the general synaptic release machinery. We are analyzing the biochemical pathways controlled by Go which lead to a reduction in DAG levels, in particular the role of a DAG kinase, DGK-1, which was identified in the screen for serotonin resistant mutants. Current projects that will be discussed are a screen for suppressors of a dgk-1(gf) phenotype, suppressors of constitutively high levels of ACh release caused by the serotonin antagonist methiothepin, identification of DGK-1 interactors by yeast 2 hybrid and co- immunoprecipitation, and analysis of another 4 DAG kinases in the C.elegans genome.

Rachel McMullan et al. (2003) International Worm Meeting "Activated RHO-1 stimulates neurotransmitter release."

Emma Hiley, Stephen Nurrish, Rachel McMullan

[International Worm Meeting, 2003]

Mutations in the diacylglycerol kinase DGK-1 or the Go alpha subunit GOA-1 substantially block the ability of the neuromodulator serotonin to reduce C.elegans locomotion. The simplest model for serotonin signalling would be that serotonin binding to its receptor causes activation of GOA-1 and that activated GOA-1 subsequently stimulates DGK-1 activity. However, we have been unable to show any regulation of DGK-1 by GOA-1. DGK-1 contains many putative protein:protein and protein:lipid interaction domains, in addition to its lipid kinase domain, suggesting that DGK-1 can be regulated, possibly in a serotonin dependent manner. We are following 3 strategies to identify DGK-1 regulators. Genetic and biochemical approaches are described in a poster (Morrison, Copelman, and Nurrish). Here we describe a candidate regulator approach.The human DGK-1 homolog DGK theta co-IPs with, and is inactivated by, activated RhoA. Initial attempts to test whether DGK-1 can bind directly to C.elegans RHO-1 by a yeast 2 hybrid approach have been unsuccessful and we are now testing whether they co-IP in worm extracts. Expression of activated RHO-1 from a heat shock promoter in adult animals causes the animals to become hypersensitive to the cholinesterase inhibitor aldicarb and to move in a loopy hyperactive manner, suggesting an increase in release of the neurotransmitter acetylcholine (ACh), a phenotype very similar to dgk-1 mutants. Prolonged expression of RHO-1 causes sterility and death over 3 days but we are unsure if this is related to the effects on ACh release. These results are consistent with activated RHO-1 down regulating the activity of DGK-1. We have performed a genetic screen for mutant animals which fail to become hypersensitive to aldicarb upon expression of activated RHO-1. In a screen of 4488 haploid genomes we identified over 100 suppressors. In the absence of activated RHO-1 at least 5 of these suppressors have a wild type response to aldicarb; thus these animals do not have a general synaptic release defect. These animals are likely to contain either a mutation in a heat-shock response gene or in a RHO-1 effector that allows RHO-1 to regulate synaptic vesicle release. Each of these suppressors becomes sterile and dies after multiple heat-shocks (as is true for non-suppressed animals) indicating that the heat shock response remains normal. Thus at least some of the suppressors are likely to represent mutations in at least one RHO-1 effector which can modulate synaptic vesicle release.