RIC-8 (Synembryn): a novel conserved protein that is required for G(q)alpha signaling in the C. elegans nervous system

Recent studies describe a network of signaling proteins centered around G(o)alpha and G(q)alpha that regulates neurotransmitter secretion in C. elegans by controlling the production and consumption of diacylglycerol (DAG). We sought other components of the Goalpha-G(q)alpha signaling network by screening for aldicarb-resistant mutants with phenotypes similar to egl-30 (G(q)alpha) mutants. In so doing, we identified ric-8, which encodes a novel protein named RIC-8 (synembryn). Through cDNA analysis, we show that RIC-8 is conserved in vertebrates. Through immunostaining, we show that RIC-8 is concentrated in the cytoplasm of neurons. Exogenous application of phorbol esters or loss of DGK-1 (diacylglycerol kinase) rescues ric-8 mutant phenotypes. A genetic analysis suggests that RIC-8 functions upstream of, or in conjunction with, EGL-30 (G(q)alpha).     

Serotonin promotes G(o)-dependent neuronal migration in Caenorhabditis elegans

BACKGROUND: The directed migration of neurons during development requires attractive and repulsive cues that control the direction of migration as well as permissive cues that potentiate cell motility and responsiveness to guidance molecules. RESULTS: Here, we show that the neurotransmitter serotonin functions as a permissive signal for embryonic and postembryonic neuronal migration in the nematode C. elegans. In serotonin-deficient mutants, the migrations of the ALM, BDU, SDQR, and AVM neurons were often foreshortened or misdirected, indicating a serotonin requirement for normal migration. Moreover, exogenous serotonin could restore motility to AVM neurons in serotonin-deficient mutants as well as induce AVM-like migrations in the normally nonmotile neuron PVM; this indicates that serotonin was functioning as a permissive cue to enable neuronal motility. The migration defects of serotonin-deficient mutants were mimicked by ablations of serotonergic neuroendocrine cells, implicating humoral release of serotonin in these processes. Mutants defective in G(q) and G(o) signaling, or in N-type voltage-gated calcium channels, showed migration phenotypes similar to serotonin-deficient mutants, and these molecules appeared to genetically function downstream of serotonin in the control of neuronal migration. CONCLUSIONS: Thus, serotonin is important for promoting directed neuronal migration in the developing C. elegans nervous system. We hypothesize that serotonin may promote cell motility through G protein-dependent modulation of voltage-gated calcium channels in the migrating cell.     

Presynaptic Gαo (GOA-1) signals to depress command neuron excitability and allow stretch-dependent modulation of egg laying in Caenorhabditis elegans

Egg laying in the nematode worm Caenorhabditis elegans is a two-state behavior modulated by internal and external sensory input. We have previously shown that homeostatic feedback of embryo accumulation in the uterus regulates bursting activity of the serotonergic HSN command neurons that sustains the egg-laying active state. How sensory feedback of egg release signals to terminate the egg-laying active state is less understood. We find that Gα_o, a conserved Pertussis Toxin-sensitive G protein, signals within HSN to inhibit egg-laying circuit activity and prevent entry into the active state. Gα_o signaling hyperpolarizes HSN, reducing HSN Ca²⁺ activity and input onto the postsynaptic vulval muscles. Loss of inhibitory Gα_o signaling uncouples presynaptic HSN activity from a postsynaptic, stretch-dependent homeostat, causing precocious entry into the egg-laying active state when only a few eggs are present in the uterus. Feedback of vulval opening and egg release activates the uv1 neuroendocrine cells which release NLP-7 neuropeptides which signal to inhibit egg laying through Gα_o-independent mechanisms in the HSNs and Gα_o-dependent mechanisms in cells other than the HSNs. Thus, neuropeptide and inhibitory Gα_o signaling maintain a bi-stable state of electrical excitability that dynamically controls circuit activity in response to both external and internal sensory input to drive a two-state behavior output.     

An N-terminal region of Caenorhabditis elegans RGS proteins EGL-10 and EAT-16 directs inhibition of G(alpha)o versus G(alpha)q signaling

Regulator of G protein signaling (RGS) proteins contain an RGS domain that inhibits G(alpha) signaling by activating G(alpha) GTPase activity. Certain RGS proteins also contain a Ggamma-like (GGL) domain and a poorly characterized but conserved N-terminal region. We assessed the functions of these subregions in the Caenorhabditis elegans RGS proteins EGL-10 and EAT-16, which selectively inhibit GOA-1 (G(alpha)(o)) and EGL-30 (G(alpha)(q)), respectively. Using transgenes in C. elegans, we expressed EGL-10, EAT-16, their subregions, or EGL-10/EAT-16 chimeras. The chimeras showed that the GGL/RGS region of either protein can act on either GOA-1 or EGL-30 and that a key factor determining G(alpha) target selectivity is the manner in which the N-terminal and GGL/RGS regions are linked. We also found that coexpressing N-terminal and GGL/RGS fragments of EGL-10 gave full EGL-10 activity, whereas either fragment alone gave little activity. Biochemical analysis showed that coexpressing the two fragments caused both to increase in abundance and also caused the GGL/RGS fragment to move to the membrane, where the N-terminal fragment is localized. By coimmunoprecipitation, we found that the N-terminal fragment complexes with the C-terminal fragment and its associated Gbeta subunit, GPB-2. We conclude that the N-terminal region directs inhibition of G(alpha) signaling by forming a complex with the GGL/RGS region and affecting its stability, membrane localization, and G(alpha) target specificity.     

Distribution of U3 small nucleolar RNA and fibrillarin during early embryogenesis in Caenorhabditis elegans

U3 small nucleolar RNA (snoRNA) is one of the members of the box C/D class of snoRNA and is essential for ribosomal RNA (rRNA) processing to generate 18S rRNA in the nucleolus. Although U3 snoRNA is abundant, and is well conserved from yeast to mammals, the genes encoding U3 snoRNA in C. elegans have long remained unidentified. A recent RNomics study in C. elegans predicted five distinct U3 snoRNA genes. However, characterization of these candidates for U3 snoRNA has yet to be performed. In this study, we isolated and characterized four candidate RNAs for U3 snoRNA from the immunoprecipitated RNAs of C. elegans using an antibody against the 2,2,7-trimethylguanosine (TMG) cap. The sequences were identical to the predicted U3 sequences in the RNomics study. Here, we show the several lines of evidence that the isolated RNAs are the true U3 snoRNAs of C. elegans. Moreover, we report the novel expression pattern of U3 snoRNA and fibrillarin, which is an essential component of U3 small nucleolar ribonucleoprotein complex, during early embryo development of C. elegans. To our knowledge, this is the first observation of the inconsistent localization U3 snoRNA and fibrillarin during early embryogenesis, providing novel insight into the mechanisms of nucleologenesis and ribosome production during early embryogenesis.     

Light-sensitive coupling of rhodopsin and melanopsin to G(i/o) and G(q) signal transduction in Caenorhabditis elegans

Activation of G-protein-coupled receptors (GPCRs) initiates signal transduction cascades that affect many physiological responses. The worm Caenorhabditis elegans expresses >1000 of these receptors along with their cognate heterotrimeric G proteins. Here, we report properties of 9-cis-retinal regenerated bovine opsin [(b)isoRho] and human melanopsin [(h)Mo], two light-activated, heterologously expressed GPCRs in the nervous system of C. elegans with various genetically engineered alterations. Profound transient photoactivation of G(i/o) signaling by (b)isoRho led to a sudden and transient loss of worm motility dependent on cyclic adenosine monophosphate, whereas transient photoactivation of G(q) signaling by (h)Mo enhanced worm locomotion dependent on phospholipase Cβ. These transgenic C. elegans models provide a unique way to study the consequences of G(i/o) and G(q) signaling in vivo with temporal and spatial precision and, by analogy, their relationship to human neuromotor function.     

Calcium/calmodulin-dependent protein kinase II regulates Caenorhabditis elegans locomotion in concert with a G(o)/G(q) signaling network

Caenorhabditis elegans locomotion is a complex behavior generated by a defined set of motor neurons and interneurons. Genetic analysis shows that UNC-43, the C. elegans Ca(2+)/calmodulin protein kinase II (CaMKII), controls locomotion rate. Elevated UNC-43 activity, from a gain-of-function mutation, causes severely lethargic locomotion, presumably by inappropriate phosphorylation of targets. In a genetic screen for suppressors of this phenotype, we identified multiple alleles of four genes in a G(o)/G(q) G-protein signaling network, which has been shown to regulate synaptic activity via diacylglycerol. Mutations in goa-1, dgk-1, eat-16, or eat-11 strongly or completely suppressed unc-43(gf) lethargy, but affected other mutants with reduced locomotion only weakly. We conclude that CaMKII and G(o)/G(q) pathways act in concert to regulate synaptic activity, perhaps through a direct interaction between CaMKII and G(o).     

Suppressors of zyg-1 define regulators of centrosome duplication and nuclear association in Caenorhabditis elegans

In Caenorhabditis elegans, the kinase ZYG-1 is required for centrosome duplication. To identify factors that interact with ZYG-1, we used a classical genetic approach and identified 21 szy (suppressor of zyg-1) genes that when mutated restore partial viability to a zyg-1 mutant. None of the suppressors render animals completely independent of zyg-1 activity and analysis of a subset of the suppressors indicates that all restore the normal process of centrosome duplication to zyg-1 mutants. Thirteen of these suppressor mutations confer phenotypes of their own and cytological examination reveals that these genes function in a variety of cellular processes including cell cycle timing, microtubule organization, cytokinesis, chromosome segregation, and centrosome morphology. Interestingly, several of the szy genes play a role in attaching the centrosome to the nuclear envelope. We have found that one such szy gene is sun-1, a gene encoding a nuclear envelope component. We further show that the role of SUN-1 in centrosome duplication is distinct from its role in attachment. Our approach has thus identified numerous candidate regulators of centrosome duplication and uncovered an unanticipated regulatory mechanism involving factors that tether the centrosome to the nucleus.     

eat-11 encodes GPB-2, a Gbeta(5) ortholog that interacts with G(o)alpha and G(q)alpha to regulate C. elegans behavior

In C. elegans, a G(o)/G(q) signaling network regulates locomotion and egg laying [1-8]. Genetic analysis shows that activated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is suppressed by perturbations of this network, which include loss of the GOA-1 G(o)alpha, DGK-1 diacylglycerol kinase, EAT-16 G protein gamma subunit-like (GGL)-containing RGS protein, or an unidentified protein encoded by the gene eat-11 [9]. We cloned eat-11 and report that it encodes the Gbeta(5) ortholog GPB-2. Gbeta(5) binds specifically to GGL-containing RGS proteins, and the Gbeta(5)/RGS complex can promote the GTP-hydrolyzing activity of Galpha subunits [10, 11]. However, little is known about how this interaction affects G protein signaling in vivo. In addition to EAT-16, the GGL-containing RGS protein EGL-10 participates in G(o)/G(q) signaling; EGL-10 appears to act as an RGS for the GOA-1 G(o)alpha, while EAT-16 appears to act as an RGS for the EGL-30 G(q)alpha [4, 5]. We have combined behavioral, electrophysiological, and pharmacological approaches to show that GPB-2 is a central member of the G(o)/G(q) network and that GPB-2 may interact with both the EGL-10 and EAT-16 RGS proteins to mediate the opposing activities of G(o)alpha and G(q)alpha. These interactions provide a mechanism for the modulation of behavior by antagonistic G protein networks.     

The Dunce cAMP phosphodiesterase PDE-4 negatively regulates G alpha(s)-dependent and G alpha(s)-independent cAMP pools in the Caenorhabditis elegans synaptic signaling network

Forward genetic screens for mutations that rescue the paralysis of ric-8 (Synembryn) reduction-of-function mutations frequently reveal mutations that cause hyperactivation of one or more components of the G alpha(s) pathway. Here, we report that one of these mutations strongly reduces the function of the Dunce cAMP phosphodiesterase PDE-4 by disrupting a conserved active site residue. Loss of function and neural overexpression of PDE-4 have profound and opposite effects on locomotion rate, but drug-response assays suggest that loss of PDE-4 function does not affect steady-state acetylcholine release or reception. Our genetic analysis suggests that PDE-4 regulates both G alpha(s)-dependent and G alpha(s)-independent cAMP pools in the neurons controlling locomotion rate. By immunostaining, PDE-4 is strongly expressed throughout the nervous system, where it localizes to small regions at the outside boundaries of synaptic vesicle clusters as well as intersynaptic regions. The synaptic subregions containing PDE-4 are distinct from those containing active zones, as indicated by costaining with an antibody against the long form of UNC-13. This highly focal subsynaptic localization suggests that PDE-4 may exert its effects by spatially regulating intrasynaptic cAMP pools.     

The NED-8 conjugating system in Caenorhabditis elegans is required for embryogenesis and terminal differentiation of the hypodermis

This work has identified the enzymes involved in the activation and conjugation of the ubiquitin-like protein NED-8 in Caenorhabditis elegans. A C. elegans conjugating enzyme, UBC-12, is highly specific in its ability to utilize NED-8 as a substrate. Immunostaining shows that NED-8 is conjugated in vivo to a major target protein with a conjugate size of 90 kDa. While the amount of this conjugate is developmentally regulated with reduced levels in the larval stages, the mRNA encoding C. elegans UBC-12 is constitutively produced throughout development, as is NED-8 itself. The importance of the NED-8 conjugating system in C. elegans was determined by RNA interference (RNAi) assays using double-stranded RNA encoding NED-8, UBC-12, or the NED-8 activating enzyme component ULA-1. The progeny of both ned-8 and ubc-12 RNAi-treated hermaphrodites either arrested during embryonic development or underwent abnormal postembryonic development. The effect on postembryonic development was pleiotropic, the most frequent gross abnormality being vulval eversion during the L4 stage. Individuals with an everted vulva either burst at the L4 to adult molt or gave rise to adults incapable of egg laying. Additionally, both ned-8 and ubc-12 RNAi induced a striking abnormality in the alae, structures produced by the lateral hypodermal seam cells in the adult nematode. Affected alae were patchy and frequently diverged around a central space. Vulval defects were also produced by RNAi directed at C. elegans ula-1. This is the first demonstration of a requirement for NED-8 conjugation in metazoan development.     

RIC-8 is required for GPR-1/2-dependent Galpha function during asymmetric division of C. elegans embryos

Heterotrimeric G proteins are crucial for asymmetric cell division, but the mechanisms of signal activation remain poorly understood. Here, we establish that the evolutionarily conserved protein RIC-8 is required for proper asymmetric division of one-cell stage C. elegans embryos. Spindle severing experiments demonstrate that RIC-8 is required for generation of substantial pulling forces on astral microtubules. RIC-8 physically interacts with GOA-1 and GPA-16, two Galpha subunits that act in a partially redundant manner in one-cell stage embryos. RIC-8 preferentially binds to GDP bound GOA-1 and is a guanine nucleotide exchange factor (GEF) for GOA-1. Our analysis suggests that RIC-8 acts before the GoLoco protein GPR-1/2 in the sequence of events leading to Galpha activation. Furthermore, coimmunoprecipitation and in vivo epistasis demonstrate that inactivation of the Gbeta subunit GPB-1 alleviates the need for RIC-8 in one-cell stage embryos. Our findings suggest a mechanism in which RIC-8 favors generation of Galpha free from Gbetagamma and enables GPR-1/2 to mediate asymmetric cell division.     

Molecular cloning and characterization of a novel alpha 1,2-fucosyltransferase (CE2FT-1) from Caenorhabditis elegans

Here we report the discovery of a unique fucosyltransferase (FT) in Caenorhabditis elegans. In studying the activities of FTs in extracts of adult C. elegans, we detected activity toward the unusual disaccharide acceptors Galbeta1-4Xyl-R and Galbeta1-6GlcNAc-R to generate products with the general structure Fucalpha1-2Galbeta1-R. We identified a gene encoding a unique alpha1,2FT (designated CE2FT-1), which contains an open reading frame encoding a predicted protein of 355 amino acids with the type 2 topology and domain structure typical of other glycosyltransferases. The predicted cDNA for CE2FT-1 has very low identity (5-10%) at the amino acid level to alpha1,2FT sequences in humans, rabbits, and mice. Recombinant CE2FT-1 expressed in human 293T cells has high alpha1,2FT activity toward the simple acceptor Galbeta-O-phenyl acceptor to generate Fucalpha1-2Galbeta-R, which in this respect resembles mammalian alpha1,2FTs. However, CE2FT-1 is otherwise completely different from known alpha1,2FTs in its acceptor specificity, since it is unable to fucosylate either Galbeta1-4Glcbeta-R or free lactose and prefers the unusual acceptors Galbeta1-4Xylbeta-R and Galbeta1-6GlcNAc-R. Promoter analysis of the CE2FT-1 gene using green fluorescent protein reporter constructs demonstrates that CE2FT-1 is expressed in single cells of early stage embryos and exclusively in the 20 intestinal cells of L(1)-L(4) and adult worms. These and other results suggest that multiple fucosyltransferase genes in C. elegans may encode enzymes with unique activities, expression, and developmental roles.     

Caenorhabditis elegans inhibitor of apoptosis protein (IAP) homologue BIR-1 plays a conserved role in cytokinesis

BACKGROUND: Inhibitor of apoptosis proteins (IAPs) suppress apoptotic cell death in several model systems and are highly conserved between insects and mammals. All IAPs contain at least one copy of the approximately 70 amino-acid baculovirus IAP repeat (BIR), and this domain is essential for the anti-apoptotic activity of the IAPs. Both the marked structural diversity of IAPs and the identification of BIR-containing proteins (BIRPs) in yeast, however, have led to the suggestion that BIRPs might play roles in other, as yet unidentified, cellular processes besides apoptosis. Survivin, a human BIRP, is upregulated 40-fold at G2-M phase and binds to mitotic spindles, although its role at the spindle is still unclear. RESULTS: We have identified and characterised two Caenorhabditis elegans BIRPs,BIR-1 and BIR-2; these proteins are the only BIRPs in C. elegans. The bir-1 gene is highly expressed during embryogenesis with detectable expression throughout other stages of development; bir-2 expression is detectable only in adults and embryos. Overexpression of bir-1 was unable to inhibit developmentally occurring cell death in C. elegans and inhibition of bir-1 expression did not increase cell death. Instead, embryos lacking bir-1 were unable to complete cytokinesis and they became multinucleate. This cytokinesis defect could be partially suppressed by transgenic expression of survivin, the mammalian BIRP most structurally related to BIR-1, suggesting a conserved role for BIRPs in the regulation of cytokinesis. CONCLUSIONS: BIR-1, a C. elegans BIRP, is probably not involved in the general regulation of apoptosis but is required for embryonic cytokinesis. We suggest that BIRPs may regulate cytoskeletal changes in diverse biological processes including cytokinesis and apoptosis.     

A potential role for fatty acid biosynthesis genes during molting and cuticle formation in Caenorhabditis elegans

Caenorhabditis elegans undergoes a developmental molting process that involves a coordinated interplay among diverse intracellular pathways. Here, we investigated the functions of two fatty acid biosynthesis genes; pod-2, encoding acetyl-CoA carboxylase, and fasn-1, encoding fatty acid synthase, in the C. elegans molting process. Although both the pod-2 and fasn-1 genes were expressed at constant levels throughout C. elegans development, knockdown of the proteins encoded by these genes using RNA interference produced severe defects in triglyceride production, molting, and reproduction that were coupled to suppression of NAS-37, a metalloprotease. An assessment of the structure and integrity of the cuticle using a COL-19::GFP marker and Hoechst 33258 staining showed that downregulation of either pod-2 or fasn-1 impaired cuticle formation and disrupted the integrity of the cuticle and the hypodermal membrane.     

Genetic evidence for a dystrophin-glycoprotein complex (DGC) in Caenorhabditis elegans

A novel alpha-tubulin gene (alpha6) was cloned from a genomic library of Naegleria gruberi strain NB-1 and characterized. The open reading frame of alpha6 contained 1359 nucleotides encoding a protein of 452 amino acids (aa) with a calculated molecular weight of 50.5 kDa. The nucleotide sequence of the open reading frame of alpha6 showed considerable divergence (68.4% identity) when compared with previously cloned N. gruberi alpha-tubulin genes, which share about 97% identity in DNA sequences. The deduced aa sequence of alpha6-tubulin was 61.9% identical to that of alpha13-tubulin, which was cloned from the same strain, and showed similar identities to those of alpha-tubulins from other species (54 approximately 62%). These data showed that alpha6-tubulin is one of the most divergent alpha-tubulins so far known. Alpha6-tubulin was found to be expressed in actively growing cells and repressed quickly when these cells were induced to differentiate. Immunostaining with an antibody against alpha6-tubulin showed that alpha6-tubulin is present in the nuclei and mitotic spindle-fibers but absent in flagellar axonemes or cytoskeletal microtubules. These data finally established the presence of an alpha-tubulin that is specifically utilized for spindle-fiber microtubules and distinct from the flagellar axonemal alpha-tubulins in N. gruberi, hence confirmed the multi-tubulin hypothesis in this organism.