G Punkosdy et al. (1999) International C. elegans Meeting "CUL-2 is required for mitotic chromatin condensation"

G Punkosdy, H Feng, ET Kipreos, W Zhong

[International C. elegans Meeting, 1999]

cul-2 is a member of the cullin gene family in which two members, cul-1 and CDC53 , function as E3s in the ubiquitin-mediated proteolysis of cell cycle regulators. cul-2(ek1) homozygous progeny of heterozygous parents have normal embryonic and postembryonic somatic cell lineages. cul-2 germ cells undergo a late onset G1 phase arrest that correlates with increased level of CKI-1 protein. The late onset of germ cell arrest as well as the observation that cul-2 dsRNA induces an immediate embryonic arrest, suggests that cul-2 is supplied as maternal product that perdures through the larval stages. cul-2 homozygotes lay only a small number of eggs, which all arrest with approximately 24 cells. These embryos have defective cytoskeletal movements with numerous cytoplasmic projections. The length of mitosis, and prometaphase in particular, is greatly increased. cul-2 embryos have a seemingly normal mitotic spindle, however, the chromosomal DNA in cul-2 mutants is not condensed. This uncondensed DNA appears to contribute to other embryonic cul-2 phenotypes: unequal DNA segregation, chromosome bridging, and multinuclei formation. cul-2 mutants have defective meiosis II in approximately 2/3 of cases, however, we observe uncondensed chromosomes at the first mitotic division even when the correct 4n DNA content is present upon pronuclei fusion. Other mutants that have defective meiosis also have multinuclei formation, the classic example is loss-of-function mei-1 alleles. We found that mei-1(b284) embryos with multinuclei have normal chromosome condensation although they have 30 or more chromosomes, suggesting that, in contrast to cul-2 , mei-1 multinuclei form due to excessive chromosome numbers. In cul-2 oocytes in diakinesis, chromosomes have normal size and shape, suggesting that cul-2 is not required for meiotic chromosome condensation. However, in late cul-2 homozygote adults (4-5 days post-larvae), the 6 bivalents separate to produce 12 univalents. This effect is not a result of the old age of the cul-2 adults as young adult cul-2 homozygotes from germline mosaic parents also have univalents. The observed chromatin condensation defect as well as the separation of bivalents suggests that cul-2 has role(s) promoting chromosome condensation/integrity.

Daniel L Chase et al. (2001) International C. elegans Meeting "Two RGS proteins that inhibit Galphao and Galphaq signaling in C. elegans neurons require a Gbeta5-like subunit for function"

Michael R Koelle, Daniel L Chase, Georgia A Patikoglou

[International C. elegans Meeting, 2001]

Gbeta proteins have traditionally been thought to complex with G g proteins to function as subunits of G protein heterotrimers. The divergent Gbeta 5 protein, however, can bind either G g proteins or r egulator of G protein s ignaling (RGS) proteins that contain a G - g amma- l ike (GGL) domain. RGS proteins are inhibitors of G protein signaling that act as G a GTPase activators. While Gbeta 5 appears to bind RGS proteins in vivo , its association with G g proteins in vivo has not been clearly demonstrated. It is unclear how Gbeta 5 might influence RGS activity. In C. elegans there are exactly two GGL-containing RGS proteins, EGL-10 and EAT-16, and they inhibit G a o and G a q signaling, respectively. We knocked out the gene encoding the C. elegans Gbeta 5 ortholog, GPB-2, to determine its physiological roles in G protein signaling. The gpb-2 mutation reduces the functions of EGL-10 and EAT-16 to levels comparable to those found in egl-10 and eat-16 null mutants. gpb-2 knockout animals are viable and exhibit no obvious defects beyond those that can be attributed to a reduction of EGL-10 or EAT-16 function. GPB-2 protein is nearly absent in eat-16; egl-10 double mutants, and EGL-10 protein is severely diminished in gpb-2 mutants. These results indicate that Gbeta 5 functions in vivo complexed with GGL-containing RGS proteins. In the absence of Gbeta 5 , these RGS proteins have little or no function. The formation of RGS-Gbeta 5 complexes is required for the expression or stability of both the RGS and Gbeta 5 proteins. Appropriate RGS-Gbeta 5 complexes regulate both G a o and G a q proteins in vivo .

Nicole K Reynolds et al. (2004) West Coast Worm Meeting "The Major Function of UNC-31 (CAPS) - Mediated Secretion is to Activate the GSA-1 (Gs) Pathway of the Synaptic Signaling Network"

Kenneth G Miller, Michael A Schade, Nicole K Reynolds

[West Coast Worm Meeting, 2004]

The C. elegans Synaptic Signaling Network is composed of at least three interacting G a signaling pathways: G a o (GOA-1), G a q (EGL-30), and G a s (GSA-1). Genetic epistasis studies of the G a q and G a s pathways suggest that these two major pathways converge downstream of the messenger molecule diacylglycerol (DAG), which is produced by the G a q pathway. 1 Knocking out either the G a q or the G a s pathway results in strong paralysis that is the result of functional (non-developmental) neuronal defects. Curiously, however, only the G a q pathway, which is the core, obligatory synaptic vesicle priming pathway, appears required for neurotransmitter release: knocking out the neuronal G a s pathway, which is known to be required for learning and memory in other organisms, does not significantly alter overall levels of neurotransmitter release. 1 Our observations of neuronal G a s pathway nulls in different genetic backgrounds, combined with our finding that the G a q and G a s pathways converge, led us to hypothesize that the neuronal G a s pathway may transduce positional information onto the downstream part of the G a q pathway to activate optimal synapses for specific behaviors. To investigate the source(s) of this hypothesized positional information, we sought mutants with phenotypes similar to mutants that lack a neuronal G a s pathway. Our search led us to UNC-31, the C. elegans homolog of CAPS, which is required for the fusion of one or more classes of secretory vesicles. Like G a s pathway nulls, unc-31 mutants are strongly paralyzed, and yet they exhibit only slightly reduced levels of neurotransmitter release. Using a heat-shock inducible promoter, we found that the paralysis of unc-31 nulls is the result of functional (non-developmental) defects and that expression of an unc-31 cDNA in the ventral cord cholinergic neurons of an unc-31 null restores locomotion rate to wild type levels. To investigate whether UNC-31 provides input for the G a q pathway, the G a s pathway, or both, we undertook an extensive genetic epistasis analysis, in which we analyzed double mutants containing an unc-31 null mutation in combination with gain- and loss-of-function mutations in the G a q and G a s pathways. Our results suggest that the major function of UNC-31 (CAPS) - mediated secretion is to activate the neuronal G a s pathway. We propose that one or more classes of molecules released by UNC-31 activates the neuronal G a s pathway, which in turn acts on downstream components of the core G a q priming pathway to activate optimal synapses for locomotion. 1 Reynolds, N.K., Schade, M. A., and K. G. Miller, submitted.

Edith M Myers et al. (2004) East Coast Worm Meeting "Characterization of the identity and specificty of RGS protein targets in C. elegans"

Michael R Koelle, Edith M Myers

[East Coast Worm Meeting, 2004]

R egulator of G protein s ignaling (RGS) proteins shorten the duration of signaling through heterotrimeric G proteins by stimulating the intrinsic GTPase activity of G a proteins. In mammals, there are more than 25 RGS proteins and more than 20 G a proteins. Each RGS protein contains an RGS domain sufficient to activate GTPase activity, and most contain other domains that may confer divergent functions to each of the RGS proteins. The role of the other domains remains unclear and, despite extensive in vitro and cell culture studies, it is still unclear whether full-length RGS proteins stimulate the GTPase activity of specific G a proteins in vivo . In addition, little is known about proteins that may modify RGS protein activity, stability, or sub-cellular localization. We will purify and analyze RGS protein complexes from C. elegans . By characterizing the identity of RGS targets and the specificity of such interactions, our goal is to convert current genetic models for RGS function into concrete molecular mechanisms. We first plan to examine biochemically whether RGS proteins specifically bind their genetically defined G a targets. Extensive genetic analysis of two antagonistic G protein signaling pathways in C. elegans demonstrates that the RGS protein EAT-16 inhibits signaling through the G a q EGL-30, while the RGS protein EGL-10 inhibits signaling through the G a o GOA-1 . We have expressed functional tagged EAT-16 and EGL-10 RGS proteins in C. elegans . After stabilizing the normally transient RGS . G a interactions by treating C. elegans homogenates with GDP-AlF 4 - , we will affinity purify RGS . G protein complexes using the tandem affinity purification (TAP) tag. By Western blot analysis of the complexes, we will determine specifically whether EAT-16 and EGL-10 bind, and presumably activate, their genetically identified G a targets, EGL-30 and GOA-1, respectively. Using this same experimental methodology, we also plan to test a model in which the RGS protein acts as a subunit in a new type of G protein heterotrimer. EAT-16 and EGL-10 are members of a family of RGS proteins that contain G g -like domains, through which they are constitutively bound to a G b 5 subunit. Such G b 5 . RGS dimers suggest that G a . G b 5 . RGS heterotrimers may form in addition to the analogous classical G a . G b . G g heterotrimers. The existence of G a . G b 5 . RGS heterotrimers would provide an elegant mechanism for a switch between the antagonistic EGL-30 and GOA-1 pathways. In this proposed mechanism, the inactive heterotrimers would be released from the G a subunit upon GTP binding. The released G b 5 . RGS dimers would activate the GTPase activity of the opposing G a protein, thus turning off signaling through the antagonistic pathway. For instance, upon GTP binding, the G b 5 . EGL-10 dimer would be released from GDP-bound EGL-30, and would activate the GTPase activity of activated GOA-1. We will affinity purify RGS complexes, and look for the EGL-30 . G b 5 . EGL-10 or GOA-1 . G b 5 . EAT-16 heterotrimers with Western blot analysis. These assays will be performed in the absence of GDP-AlF 4 - , since our aim is to identify proteins interacting with the GDP-bound G a subunit. Finally, we plan to identify other proteins in the purified RGS complexes and determine their role in G protein signaling. Components of such complexes will be identified by mass spectrometry, and their role in signaling will be determined after phenotypic analysis of animals carrying knockouts of the corresponding genes.

Oppenheimer AJ et al. (1995) International C. elegans Meeting "G-PROTEIN SIGNALING IN C. ELEGANS"

Kaplan JM, Oppenheimer AJ

[International C. elegans Meeting, 1995]

We are interested in determining how G-proteins modulate the activity of ion channels in the C. elegans nervous system. To generate behavioral phenotypes dependent on G-protein signaling, we targeted expression of mammalian G-proteins to a set of neurons including those controlling foraging and locomotion (RMD, AVA, AVB, AVD, and PVC). Ectopic expression of several G-proteins under the control of the not-3 promoter results in behavioral defects. Expression of constitutively active rat G-alpha-i1 causes uncoordinated forward and backward locomotion. Expression of wild-type rat G-alpha-s impairs backward locomotion, while constitutively active rat G-alpha-s causes defective backward locomotion and lethargy. In order to identify components of the G-alpha-s signaling pathway, we have initiated a screen for mutations that suppress the behavioral effects of activated G-alpha-s expression. In a screen of approximately 7000 haploid genomes, we have identified 11 independent mutations that improve backward locomotion. Several of these mutations result in a hyperactive phenotype in the absence of activated G-alpha-s expression. Mapping and complementation tests will determine whether the suppressors are allelic to other hyperactive mutants isolated by D. Elkes and J. Kaplan. By cloning the suppressor genes, we hope to identify ion channels regulated by G-alpha-s as well as other components of the G-alpha-s signaling pathway. We also plan to determine whether the behavioral effects of G-alpha-s expression are mediated by known second messenger systems. We are first testing the role of cAMP-dependent protein kinase (PKA), because it is activated by G-alpha-s in other systems and has been shown to phosphorylate ion channels. If G-alpha-s signals through PKA, increasing the level of PKA activity should mimic the effect of activated G-alpha-s expression, and inhibiting PKA should suppress the effect of activated G- alpha-s expression.

Kevin Collins et al. (2008) C.elegans Neuronal Development Meeting "A Hyperactive Egg Laying Mutant May Define a New Regulator of Neurotransmitter Release in C. elegans"

I Amy Bany, Kevin Collins, Michael Koelle, Antony Jose, Andrew Bellemer

[C.elegans Neuronal Development Meeting, 2008]

Two heterotrimeric G proteins, G(alpha)o and G(alpha)q, antagonistically modulate neurotransmitter release. In C. elegans these G proteins regulate specific behaviors that can be conveniently scored and quantitatively measured, allowing signaling to be studied genetically. Egg laying is one such behavior. Mutations in G(alpha)q and its downstream signaling components UNC-73/Trio or EGL-8/PLC-beta decrease egg laying frequency, leading to the accumulation of unlaid eggs. Conversely, mutations in G(alpha)o cause eggs to be laid almost immediately after their fertilization after having undergone only one or two cell divisions. The specific downstream effector(s) that mediate the effects of G(alpha)o remain unknown, but genetic experiments suggest that G(alpha)o antagonizes G(alpha)q signaling. The one- to four-cell eggs laid by G(alpha)o mutants can be easily distinguished from those laid by wild type animals, which have typically advanced through several more cell divisions. A genetic screen for mutants that lay early-stage eggs has yielded mutations in several genes that act in G protein signaling to inhibit neurotransmitter release. These include mutations in DGK-1, a diacylglycerol kinase which terminates DAG signaling, and in the G protein regulator EAT-16, a conserved RGS protein which terminates G(alpha)q signaling by activating G(alpha)q GTPase activity. By looking for additional mutants that share the phenotype of G(alpha)o mutants, I hope to define those factors through which G(alpha)o mediates its inhibition of neurotransmitter release. I am presently mapping a new mutant, vs39, that displays the hyperactive egg-laying phenotype as well as the locomotion defects characteristic of animals with impaired G(alpha)o signaling. Two additional mutant alleles have been isolated in a separate screen for suppressors of egl-47(gf), a candidate G protein coupled receptor which appears to activate G(alpha)o signaling in the egg laying cells. Like G(alpha)o mutants, these mutants lay mostly early-stage eggs. Genetic mapping experiments have placed these mutants to a 1 Mb region of chromosome III. High resolution mapping using the polymorphic Hawaiian strain of C. elegans is underway and should reduce the mutant interval to the point where transformation rescue can be attempted. Progress toward the identification of the affected gene and preliminary phenotypic analyses will be presented.

E Cuppen et al. (1999) International C. elegans Meeting "Heterotrimeric G proteins of C. elegans: cDNA sequence analysis and interaction studies"

G Jansen, RH Plasterk, E Cuppen

[International C. elegans Meeting, 1999]

Heterotrimeric G proteins are signal transduction molecules situated at the interface between extracellular signals received via G protein-coupled receptors (over 600 predicted in the C.elegans genome) and intracellular second messenger systems. We set out to study the function of all heterotrimeric G protein genes in C.elegans . Thus far, two G b subunit genes ( gpb-1 and 2 ), two G g genes ( gpc-1 and 2 ) and 20 G a genes have been identified in the genomic sequence (Jansen et al., Nature Gen. in press ). Besides one member ( gsa-1 , goa-1 , egl-30 and gpa-12 ) of each of the mammalian G a classes (Gs, Go/Gi, Gq, and G12) there are 16 new C.elegans specific genes ( gpa-1 to 11 , 13 to 16 and odr-3 ). We have cloned the cDNA's for all the G protein subunits. RT-PCR and sequence analysis gave no indications for alternative splicing, but we found that about 10% of the exons of this highly conserved protein family were predicted incorrectly by Genefinder. We will employ all cDNA's in yeast two- and three-hybrid interaction studies to test for interaction specificity between the different subunits. Furthermore, we will screen cDNA libraries for interacting proteins. The interaction specificity of novel proteins can easily be tested using mating assays, because all C.elegans heterotrimeric G protein subunits are available. Since we know that multiple G a subunits are expressed in particular cells (Jansen et al., Nature Gen. in press ), specificity in interactions mediated by these subunits may provide an efficient mechanism regulating signaling via G protein coupled receptors.

Oppenheimer AJ et al. (1996) East Coast Worm Meeting "G-alpha-s signaling in C. elegans"

Kaplan JM, Oppenheimer AJ

[East Coast Worm Meeting, 1996]

We are interested in determining how G-proteins modulate the activity of ion channels in the C. elegans nervous system. To generate phenotypes dependent on G-protein signaling, we expressed mammalian G-proteins under the control of the glr-1 promoter, which drives expression in a set of neurons including those controlling foraging and locomotion (RMD, AVA, AVB, AVD, and PVC). Expression of wild-type rat G-alpha-s impairs backward locomotion, while consitutively active rat G-alpha-s causes paralysis and neuronal degeneration in a subset of G-alpha-s-expressing cells, primarily the PVC and AVD neurons. Our model for the neurodegeneration is that activated G-alpha-s is misregulating an ion channel in AVD and PVC, causing osmotic imbalance and consequent swelling. Degenerating neurons appear enlarged and vacuolated, similar to those in worms carrying gain-of-function mutations in deg-1 and other degenerins. We tested whether activated G-alpha-s is killing cells by misregulating known ion channels. Loss-of-function mutations in deg-1, mec-6, unc-36, unc-2, and egl-19 were unable to suppress the degenerations, suggesting that G-alpha-s is not acting through these degenerins or calcium channels. Mutations in ced-3 and ced-4 do not prevent G-alpha-s-induced degenerations, indicating that G-alpha-s is not killing through apoptosis. To identify the components of the signaling pathway downstream of G-alpha-s, we screened for mutations that suppress the paralysis caused by activated G-alpha-s. In a screen of 7,760 haploid genomes, we identified 8 mutations that suppress paralysis and neurodegeneration and 8 mutations that suppress only paralysis. Five of the degeneration suppressors map to chromosome III and may be allelic. These mutations are semidominant, and several cause a hyperactive phenotype in the absence of activated G-alpha-s. Because hyperactivity also results from mutations in the Go signaling pathway, our suppressor mutations may identify a gene invovled in both the Gs and Go signaling pathways. We are continuing to map and characterize the suppressor genes.

Hofler C et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "The GPR Domain Protein AGS-3 May Be an Activator of Gαo Signaling"

Koelle M, Hofler C

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Many neurotransmitters signal via G protein coupled receptors that activate the heterotrimeric G protein Gαo to inhibit the subsequent release of neurotransmitters. The G protein Regulator (GPR) domain is a ~25 amino acid motif that can bind Gαo. The in vitro interactions of GPR proteins with Gαo might allow them to either activate or inhibit signaling, and thus the physiological functions of GPR proteins remain unclear. We are using a combination of genetic and biochemical studies of the C. elegans GPR protein AGS-3 to try to resolve what the in vivo effects of this protein are on Gαo signaling. C. elegans AGS-3 and its human ortholog AGS3 each have seven tetratricopeptide repeats followed by four GPR domains. We found that, in vitro, AGS-3 binds C. elegans Gαo in both its GDP- and GTP-bound states, specifically through its GPR domains. We are investigating whether different GPR domains in AGS-3 bind the different states of Gαo. We have also engineered a GPR insensitive mutation into Gαo that eliminates binding to AGS-3. We created a polyclonal antibody against AGS-3 and have found that, like Gαo, AGS-3 is predominantly membrane-associated in C. elegans lysates. Membrane localization of each protein persists in the absence of the other, but overexpression of GFP::Gαo, which is predominantly cytosolic, moves AGS-3 into the cytosolic fraction. In addition, GFP::Gαo can co-immunoprecipitate with AGS-3 from C. elegans lysates. We have knocked out the AGS-3 gene to examine its in vivo functions. The HSN motor neurons that release serotonin to stimulate egg laying also feedback-inhibit themselves via Gαo-coupled serotonin auto-receptors on the HSNs. Our analysis suggests that AGS-3 may function with the non-receptor nucleotide exchange factor RIC-8 in this feedback signaling. Mutants for the serotonin receptor SER-4, Gαo, AGS-3, and RIC-8 all share a common defect in which animals fail to stop laying eggs in the absence of food, and in which exogenous serotonin appears to fail to inhibit HSN function. We have shown that in vitro RIC-8 can act as a guanine nucleotide exchange factor on a preformed complex of AGS-3 and Gαo. We hypothesize that SER-4 initially activates Gαo, separating it from Gβγ subunits, and that AGS-3 and RIC-8 subsequently act on Gαo to maintain it in the active GTP-bound state. We plan to test this hypothesis through further in vivo and in vitro studies.

Jessica E. Tanis et al. (2007) International Worm Meeting "Serotonin signaling in C. elegans is regulated through transcriptional control of serotonin biosynthesis by the G proteins Gao and Gaq."

Michael R. Koelle, James J. Moresco, Robert A. Lindquist, Jessica E. Tanis

[International Worm Meeting, 2007]

Changes in signaling by the neurotransmitter serotonin may underlie depression in humans. To determine how serotonin signaling is modulated, we analyzed how C. elegans regulates function of the serotonergic HSN motor neurons that stimulate egg-laying behavior. Egg laying is inhibited by the G protein G<font face=symbol>a</font>_o (known as GOA-1 in C. elegans) and activated by the G protein G<font face=symbol>a</font>_q (known as EGL-30 in C. elegans). We found that G<font face=symbol>a</font>_o and G<font face=symbol>a</font>_q act directly in the HSN motor neurons to control egg laying. We used quantitative confocal microscopy to examine expression and localization of a variety of fluorescent reporter proteins in the HSNs to look for effects of the G proteins on HSN structure and function. The G protein mutants showed no detectable changes in the structure of the HSN, its synapses, or in localization of the synaptic vesicle priming protein UNC-13S, all of which have been suggested to be affected by the G proteins in other neurons. However, we found that the G proteins do act in the HSNs to have opposing effects on transcription of the tryptophan hydroxylase gene tph-1, which encodes the rate-limiting enzyme for serotonin biosynthesis. Anti-serotonin staining confirmed that G<font face=symbol>a</font>_o and G<font face=symbol>a</font>_q antagonistically affect serotonin levels in the HSNs. Altering tph-1 gene dosage showed that small changes in tph-1 expression were sufficient to affect egg-laying behavior. Our results suggest that serotonin signaling is regulated at the level of serotonin biosynthesis. G<font face=symbol>a</font>_o and G<font face=symbol>a</font>_q are expresed in all neurons and have opposing effects on many behaviors besides egg laying. These two G proteins may generally have their opposing effects by antagonizing each other directly in the same neurons, as they do in the HSNs. Thus they may integrate information from multiple signals and set levels of neurotransmitter release, in part through antagonistic effects on transcription of neural genes.