Ketting RF et al. (1997) International C. elegans Meeting "HOW DO THE Tc1 AND Tc3 TRANSPOSASES FIND THEIR TARGETS?"

Fischer SE, Sixma TK, Ketting RF, Plasterk RHA, van Pouderoyen G

[International C. elegans Meeting, 1997]

From previous studies it is clear that the Tc1 and Tc3 transposons do not integrate their DNA randomly into the genome. These transposons always integrate into a TA dinucleotide, but the sequences flanking that dinucleotide are also of importance. Both in vivo and in vitro studies show a distinct, and similar, Tc1 insertion pattern into the gpa-2 gene (Nucleic Acids Res.(1994) 22, 262-269, Genes & Dev.(1996) 10, 755-761), indicating that an interaction between the Tc1 transposase and naked target DNA is sufficient to generate the observed micro-distribution. From the analysis of 500 endogenous Tc1 flanks, a consensus sequence was isolated (Proc. Natl. Acad. Sci. USA (1996) 93, 14680- 14685). Hot-sites found in the gpa-2 gene indeed fit this consensus sequence. We now took one such hot-site from the gpa-2 gene, subcloned it into the lacZ gene, and studied it using the in vitro transposition system. The results show that four basepairs flanking the TA dinucleotide are sufficient to make a hot-site hot, and that purified Tc1A transposase is capable of making the right choices. Mutagenesis of the four basepair flanks shows that the consensus sequence nicely reflects the interaction between transposase and target DNA. We also noticed a surprising effect of supercoiling on Tc1 integration. To perform transposition, the N-terminal part of the transposase protein binds to the inverted repeat ends of the transposable element. We will present the crystal structure of the N-terminal 65 amino acids of the Tc3 transposase in complex with its DNA binding site. The protein shows a helix-turn-helix fold, with high similarity to paired box proteins. The supermolecular structure of the complex shows a dimer, in which the two ends of the transposon are brought together.

Wolf FW et al. (1998) West Coast Worm Meeting "Yes! you can suppress vab-8"

Wolf FW, Garriga G

[West Coast Worm Meeting, 1998]

vab-8 acts in posteriorly directed cell and axon growth cone migrations in a cell autonomous manner. vab-8 functions in posterior ALM cell migration but not in later anterior ALM axon migration, and continuous vab-8 expression in the ALM can reorient anterior ALM axon outgrowth posteriorly. This finding suggests that the direction of migration can be regulated at the level of vab-8 activity. vab-8 encodes two isoforms of a novel intracellular protein, and the opal null allele e1017 is predicted to remove both activities. Our analysis of vab-8 has led us to propose that there exists at least one mechanism for the global guidance of posterior migrations. To identify other genes that function in posterior guidance, we suppressed the highly penetrant CAN cell migration defect of vab-8(e1017) mutants. Extragenic suppressors could identify genes that act downstream of vab-8 as well as genes that act in parallel pathways. From screening over 20,000 genomes we isolated four partially penetrant van (variably abnormal normal) suppressors, each defining a distinct gene. van-1(gm128) is a semi-dominant suppressor of vab-8 cell and axon outgrowth defects that is allele non-specific but at least so far is gene specific. Alas, van-1 has no phenotype on its own. In dosage studies van-1(gm128) appears to alter gene function. We have mapped van-1 to LGI between stu-4 and unc-11 and transformation rescue experiments are underway.

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.

Monique van der Voet et al. (2006) European Worm Meeting "The Function of LIN-5 in Chromosome Segregation and Asymmetric Division"

Matilde Galli, Christian Berends, Monique van der Voet, Justin Peters, Sander Van Den Heuvel, Mike Boxem, Marjolein Wildwater, Adri Thomas, Inge The

[European Worm Meeting, 2006]

Monique van der Voet1, Marjolein Wildwater1, Mike Boxem, Justin Peters1, Christian Berends1, Matilde Galli1, Inge The1, Adri Thomas1 and Sander van den Heuvel. The position of the spindle apparatus determines the plane of cell division. During asymmetric division, proper positioning of the spindle is needed in the generation of daughter cells that differ in size, cytoplasmic determinants and developmental fate. We have focused our attention on the C. elegans gene lin-5 to identify novel regulatory mechanisms and molecules that control spindle-mediated functions.. Our previous studies demonstrated that lin-5 is generally required for chromosome segregation and spindle positioning during meiotic and mitotic M phase. lin-5 encodes a coiled-coil protein that localizes to the cell cortex as well as spindle asters, and recruits the G protein regulators GPR-1/GPR-2 to the same locations. Results from our lab as well as others support that LIN-5 and GPR act in concert with the GOA-1/GPA-16 G?i/o subunits of heterotrimeric G proteins in an evolutionarily conserved pathway for spindle positioning. Many aspects of this process remain poorly understood. In particular, it is unclear how polarity determinants at the cortex affect LIN-5/GPR/G?i/o function to create asymmetry in the pulling forces that act on the spindle asters. In addition, the downstream targets of the LIN-5/GPR/G?i/o pathway remain unknown. To better understand the molecular mechanisms of these processes, we continue to use a combined genetic and biochemical approach. We have identified novel candidate partners of LIN-5 through immunoprecipitation from embryonic lysates followed by mass spectrometry. In addition, we identified multiple residues of LIN-5 and GPR that are phosphorylated in vivo. We are currently testing the in vivo relevance of these interactions and modifications and hope to present our results at the meeting.

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.

Merrilee Robatzek et al. (2001) International C. elegans Meeting "eat-11 encodes GPB-2, a Gbeta5 ortholog that interacts with Goalpha and Gqalpha to regulate C. elegans behavior"

Kate Steger, Merrilee Robatzek, Leon Avery, Timothy Niacaris, James H Thomas

[International C. elegans Meeting, 2001]

In C. elegans , the GOA-1 G o a /EGL-30 G q a signaling network regulates locomotion and egg laying. Genetic analysis shows that the locomotion and egg-laying defects of activated Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) are suppressed by perturbations of this network, which include loss of the GOA-1 G o a , DGK-1 diacylglycerol kinase, EAT-16 G-protein g subunit-like (GGL)-containing RGS, or the unidentified protein encoded by the gene eat-11 [1]. To define the role of eat-11 in G o /G q signaling, we cloned eat-11 and report that it encodes GPB-2, an ortholog of mammalian G b 5 . Unlike other G b proteins, G b 5 has been shown to bind specifically to GGL-containing RGS proteins, and the G b 5 /RGS complex can promote the GTP-hydrolyzing activity of G a subunits. In addition to EAT-16, the GGL-containing RGS protein EGL-10 also participates in the GOA-1/EGL-30 signaling network. We find that several gpb-2 ( lf ) alleles, including a putative null allele, confer locomotory and egg-laying phenotypes intermediate between eat-16 ( lf ) and egl-10 ( lf ). This indicates that GPB-2 may regulate the opposing activities of G o a and G q a via an interaction with both the EAT-16 and EGL-10 RGS proteins. Similar models have been proposed based on independent studies of gpb-2 deletion mutants [2, 3]. Two other gpb-2 ( lf ) mutations, C266Y and D307N, confer different behavioral phenotypes which suggest that these mutations specifically disrupt the activity of the EAT-16 RGS without affecting EGL-10. We also analyzed the role of gpb-2 in defecation and feeding. gpb-2 and eat-16 regulate enteric muscle activity similarly, and this function appears to be independent of G o signaling. In this behavior, the C266Y and D307N mutants behave like the other gpb-2 ( lf ) mutants, including the putative null, indicating that GPB-2 normally interacts with only the EAT-16 RGS in the enteric muscle. For feeding behavior, we find that loss of GPB-2 or EAT-16 function decreases the activity of the M3 pharyngeal motor neuron. M3 is important for generating coordinated pharyngeal motions that allow efficient feeding. Thus, the misregulation of M3 in gpb-2 ( lf ) mutants may explain their starved appearance, which was first described by L. Avery. We also show that some aspects of GPB-2 function may occur downstream of a pharyngeal muscarinic receptor, and that these functions are required for normal feeding and growth. 1. Robatzek, M. and Thomas, J.H. Genetics 156:1069-1082 (2000) 2. Chase, D.L. et al. Curr Biol 11:222-231 (2001) 3. van der Linden, A.M. et al. Genetics, in press (2001) * These authors contributed equally

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.