http://repub.eur.nl/res/aut/11530/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/18023/ 2009-10-01T00:00:00Z The neuropeptides pigment dispersing factor (PDF) and vasoactive intestinal peptide (VIP) are known as key players in the circadian clock system of insects and mammals, respectively. In this study, we report the discovery and characterization of a widely conserved PDF-like neuropeptide precursor pathway in nematodes. Using a combinatorial approach of biochemistry and peptidomics, we have biochemically isolated, identified and characterized three PDF-like neuropeptides in the free-living nematode Caenorhabditis elegans. The two PDF encoding genes, which were designated pdf-1 and pdf-2, display a very strong conservation within the phylum of nematodes. Many of the PDF expressing cells in C. elegans play a role in the control of locomotion and the integration of environmental stimuli, among which light. Our real-time PCR analysis indicates that both PDF genes are consistently expressed during the day and do not affect each other's expression. The transcription of both PDF genes seems to be regulated by atf-2 and ces-2, which encode bZIP transcription factors homologous to Drosophila vrille and par domain protein 1 (Pdp1epsilon), respectively. Together, our data suggest that the PDF neuropeptide pathway, which seems to be conserved throughout the protostomian evolutionary lineage, might be more complex than previously assumed. http://repub.eur.nl/res/pub/17776/ 2009-07-01T00:00:00Z The lifespan of the nematode Caenorhabditis elegans is regulated by sensory signals detected by the amphid neurons. In these neurons, C. elegans expresses at least 14 Galpha subunits and a Ggamma subunit. We have identified seven sensory Galpha subunits that modulate lifespan. Genetic experiments suggest that multiple sensory signaling pathways exist that modulate lifespan and that some G proteins function in multiple pathways, most of which, but probably not all, involve insulin/IGF-1 like signaling. Interestingly, of the sensory G proteins involved in regulating lifespan, only one Galpha probably functions directly in the detection of sensory cues. The other G proteins seem to function in modulating the sensitivity of the sensory neurons. We hypothesize that in addition to the mere detection of sensory cues, regulation of the sensitivity of sensory neurons also plays a role in the regulation of lifespan. http://repub.eur.nl/res/pub/18014/ 2009-05-18T00:00:00Z Heterochromatin protein 1 (HP1) family members are chromatin-associated proteins involved in transcription, replication, and chromatin organization. We show that HP1 isoforms HP1-{alpha}, HP1-β, and HP1-{gamma} are recruited to ultraviolet (UV)-induced DNA damage and double-strand breaks (DSBs) in human cells. This response to DNA damage requires the chromo shadow domain of HP1 and is independent of H3K9 trimethylation and proteins that detect UV damage and DSBs. Loss of HP1 results in high sensitivity to UV light and ionizing radiation in the nematode Caenorhabditis elegans, indicating that HP1 proteins are essential components of DNA damage response (DDR) systems. Analysis of single and double HP1 mutants in nematodes suggests that HP1 homologues have both unique and overlapping functions in the DDR. Our results show that HP1 proteins are important for DNA repair and may function to reorganize chromatin in response to damage. http://repub.eur.nl/res/pub/17778/ 2009-01-01T00:00:00Z The lifespan of the nematode Caenorhabditis elegans is regulated by sensory signals detected by the amphid neurons. In these neurons, C. elegans expresses at least 14 Galpha subunits and a Ggamma subunit. We have identified seven sensory Galpha subunits that modulate lifespan. Genetic experiments suggest that multiple sensory signaling pathways exist that modulate lifespan and that some G proteins function in multiple pathways, most of which, but probably not all, involve insulin/IGF-1 like signaling. Interestingly, of the sensory G proteins involved in regulating lifespan, only one Galpha probably functions directly in the detection of sensory cues. The other G proteins seem to function in modulating the sensitivity of the sensory neurons. We hypothesize that in addition to the mere detection of sensory cues, regulation of the sensitivity of sensory neurons also plays a role in the regulation of lifespan. http://repub.eur.nl/res/pub/14274/ 2008-11-01T00:00:00Z While naïve Caenorhabditis elegans individuals are attracted to 0.1-200 mM NaCl, they become strongly repelled by these NaCl concentrations after prolonged exposure to 100 mM NaCl. We call this behavior gustatory plasticity. Here, we show that C. elegans displays avoidance of low NaCl concentrations only when pre-exposure to NaCl is combined with a negative stimulus, e.g., a repellent, or in the absence of food. By testing serotonin and/or dopamine signaling mutants and rescue by exogenously supplying these neurotransmitters, we found that serotonin and dopamine play a role during the plasticity response, while serotonin is also required during development. In addition, we also show that glutamate plays an important role in the response to NaCl, both in chemoattraction to NaCl and in gustatory plasticity. Thus, C. elegans can associate NaCl with negative stimuli using dopaminergic, serotonergic, and glutamatergic neurotransmission. Finally, we show that prolonged starvation enhances gustatory plasticity and can induce avoidance of NaCl in most gustatory plasticity mutants tested. Only mutation of the glutamate-gated Cl- channel gene avr-15 affected starvation-enhanced gustatory plasticity. These results suggest that starvation induces avoidance of NaCl largely independent of the normal gustatory plasticity mechanism. http://repub.eur.nl/res/pub/14554/ 2008-09-30T00:00:00Z G protein-coupled receptors (GPCRs) have a key role in many biological processes and are important drug targets for many human diseases. Therefore, understanding the molecular interactions between GPCRs and their ligands would improve drug design. Here, we describe an approach that allows the rapid identification of functional agonists expressed in bacteria. Transgenic Caenorhabditis elegans expressing the human chemokine receptor 5 (CCR5) in nociceptive neurons show avoidance behavior on encounter with the ligand MIP-1α and avoid feeding on Escherichia coli expressing MIP-1α compared with control bacteria. This system allows a simple activity screen, based on the distribution of transgenic worms in a binary food-choice assay, without a requirement for protein purification or tagging. By using this approach, a library of 68 MIP-1α variants was screened, and 13 critical agonist residues involved in CCR5 activation were identified, four of which (T8, A9, N22, and A25) have not been described previously, to our knowledge. Identified residues were subsequently validated in receptor binding assays and by calcium flux assays in mammalian cells. This approach serves not only for structure/function studies as demonstrated, but may be used to facilitate the discovery of agonists within bacterial libraries. http://repub.eur.nl/res/pub/18024/ 2008-05-01T00:00:00Z Here, we report the identification, cloning, and functional characterization of three Caenorhabditis elegans G protein-coupled pigment dispersing factor (PDF) receptors, which we designated as Ce_PDFR-1a, -b, and -c. They represent three splice isoforms of the same gene (C13B9.4), which share a high degree of similarity with the Drosophila PDF receptor and are distantly related to the mammalian vasoactive intestinal peptide receptors (VPAC2) and calcitonin receptors. In a reverse pharmacological screen, three bioactive C. elegans neuropeptides, which were recently identified as the Drosophila PDF orthologues, were able to activate these receptors in a dose-dependent manner with nanomolar potency (isoforms a and b). Integrated green fluorescent protein reporter constructs reveal the expression of these PDF receptors in all body wall muscle cells and many head and tail neurons involved in the integration of environmental stimuli and the control of locomotion. Using a custom data analysis system, we demonstrate the involvement of this newly discovered neuropeptide signaling system in the regulation of locomotor behavior. Overexpression of PDF-2 phenocopies the locomotor defects of a PDF-1 null mutant, suggesting that they elicit opposite effects on locomotion through the identified PDF receptors. Our findings strengthen the hypothesis that the PDF signaling system, which imposes the circadian clock rhythm on behavior in Drosophila, has been functionally conserved throughout the protostomian evolutionary lineage. http://repub.eur.nl/res/pub/18025/ 2007-04-01T00:00:00Z In the cilia of the nematode Caenorhabditis elegans, anterograde intraflagellar transport (IFT) is mediated by two kinesin-2 complexes, kinesin II and OSM-3 kinesin. These complexes function together in the cilia middle segments, whereas OSM-3 alone mediates transport in the distal segments. Not much is known about the mechanisms that compartmentalize the kinesin-2 complexes or how transport by both kinesins is coordinated. Here, we identify DYF-5, a conserved MAP kinase that plays a role in these processes. Fluorescence microscopy and EM revealed that the cilia of dyf-5 loss-of-function (lf) animals are elongated and are not properly aligned into the amphid channel. Some cilia do enter the amphid channel, but the distal ends of these cilia show accumulation of proteins. Consistent with these observations, we found that six IFT proteins accumulate in the cilia of dyf-5(lf) mutants. In addition, using genetic analyses and live imaging to measure the motility of IFT proteins, we show that dyf-5 is required to restrict kinesin II to the cilia middle segments. Finally, we show that, in dyf-5(lf) mutants, OSM-3 moves at a reduced speed and is not attached to IFT particles. We propose that DYF-5 plays a role in the undocking of kinesin II from IFT particles and in the docking of OSM-3 onto IFT particles. http://repub.eur.nl/res/pub/18026/ 2007-03-15T00:00:00Z The life span of the nematode Caenorhabditis elegans is under control of sensory signals detected by the amphid neurons. In these neurons, C. elegans expresses at least 13 Galpha subunits and a Ggamma subunit, which are involved in the transduction and modulation of sensory signals. Here, we show that loss-of-function mutations in the Galpha subunits odr-3, gpa-1 and gpa-9, in the Ggamma subunit gpc-1 and the introduction of extra copies of the Galpha subunit gpa-11 extend the life span of C. elegans. Loss-of-function of odr-3 and extra copies of gpa-11 act synergistically and can together extend life span more than two-fold, indicating that sensory signals play an important role in regulating life span. We show that gpa-1, gpa-11, odr-3 and gpc-1 all signal via the daf-16 FOXO family transcription factor. In addition, odr-3 and gpa-11 might suppress life span extension partially independent of the insulin/IGF-1 like receptor homologue daf-2. Our results suggest that the previously unanticipated nociceptive ASH and/or ADL neurons regulate longevity. We expect that the implication of specific G proteins will eventually contribute to the identification of the sensory cues that determine the rate of aging in C. elegans. http://repub.eur.nl/res/pub/18027/ 2007-01-01T00:00:00Z The life span of the nematode Caenorhabditis elegans is under control of sensory signals detected by the amphid neurons. In these neurons, C. elegans expresses at least 13 Galpha subunits and a Ggamma subunit, which are involved in the transduction and modulation of sensory signals. Here, we show that loss-of-function mutations in the Galpha subunits odr-3, gpa-1 and gpa-9, in the Ggamma subunit gpc-1 and the introduction of extra copies of the Galpha subunit gpa-11 extend the life span of C. elegans. Loss-of-function of odr-3 and extra copies of gpa-11 act synergistically and can together extend life span more than two-fold, indicating that sensory signals play an important role in regulating life span. We show that gpa-1, gpa-11, odr-3 and gpc-1 all signal via the daf-16 FOXO family transcription factor. In addition, odr-3 and gpa-11 might suppress life span extension partially independent of the insulin/IGF-1 like receptor homologue daf-2. Our results suggest that the previously unanticipated nociceptive ASH and/or ADL neurons regulate longevity. We expect that the implication of specific G proteins will eventually contribute to the identification of the sensory cues that determine the rate of aging in C. elegans. http://repub.eur.nl/res/pub/14025/ 2006-08-01T00:00:00Z In the sensory system of C. elegans, the candidate odorant receptor gene str-2 is strongly expressed in one of the two AWC neurons and weakly in both ASI neurons. Asymmetric AWC expression results from suppression of str-2 expression by a Ca2+/MAPK signaling pathway in one of the AWC neurons early in development. Here we show that the same Ca2+/MAPK pathway promotes str-2 expression in the AWC and ASI neurons together with multiple cell-autonomous and noncell-autonomous G-protein-signaling pathways. In first-stage larvae and adult animals, signals mediated by the Galpha subunits ODR-3, GPA-2, GPA-5, and GPA-6 and a Ca2+/MAPK pathway involving the Ca2+ channel subunit UNC-36, the CaMKII UNC-43, and the MAPKK kinase NSY-1 induce strong str-2 expression. Cell-specific rescue experiments suggest that ODR-3 and the Ca2+/MAPK genes function in the AWC neurons, but that GPA-5 and GPA-6 function in the AWA and ADL neurons, respectively. In Dauer larvae, the same network of genes promotes strong str-2 expression in the ASI neurons, but ODR-3 functions in AWB and ASH and GPA-6 in AWB. Our results reveal a complex signaling network, encompassing signals from multiple cells, that controls the level of receptor gene expression at different developmental stages. http://repub.eur.nl/res/pub/18062/ 2006-01-25T00:00:00Z Caenorhabditis elegans shows chemoattraction to 0.1-200 mM NaCl, avoidance of higher NaCl concentrations, and avoidance of otherwise attractive NaCl concentrations after prolonged exposure to NaCl (gustatory plasticity). Previous studies have shown that the ASE and ASH sensory neurons primarily mediate attraction and avoidance of NaCl, respectively. Here we show that balances between at least four sensory cell types, ASE, ASI, ASH, ADF and perhaps ADL, modulate the response to NaCl. Our results suggest that two NaCl-attraction signalling pathways exist, one of which uses Ca(2+)/cGMP signalling. In addition, we provide evidence that attraction to NaCl is antagonised by G-protein signalling in the ASH neurons, which is desensitised by the G-protein-coupled receptor kinase GRK-2. Finally, the response to NaCl is modulated by G-protein signalling in the ASI and ADF neurons, a second G-protein pathway in ASH and cGMP signalling in neurons exposed to the body fluid. http://repub.eur.nl/res/pub/13490/ 2004-08-01T00:00:00Z The two pairs of sensory neurons of C. elegans, AWA and AWC, that mediate odorant attraction, express six Galpha-subunits, suggesting that olfaction is regulated by a complex signaling network. Here, we describe the cellular localization and functions of the six olfactory Galpha-subunits: GPA-2, GPA-3, GPA-5, GPA-6, GPA-13, and ODR-3. All except GPA-6 localize to sensory cilia, suggesting a direct role in sensory transduction. GPA-2, GPA-3, GPA-5, and GPA-6 are also present in cell bodies and axons and GPA-5 specifically localizes to synaptic sites. Analysis of animals with single- to sixfold loss-of-function mutations shows that olfaction involves a balance between multiple stimulatory and inhibitory signals. ODR-3 constitutes the main stimulatory signal and is sufficient for the detection of odorants. GPA-3 forms a second stimulatory signal in the AWA and AWC neurons, also sufficient for odorant detection. In AWA, signaling is suppressed by GPA-5. In AWC, GPA-2 and GPA-13 negatively and positively regulate signaling, respectively. Finally, we show that only ODR-3 plays a role in cilia morphogenesis. Defects in this process are, however, independent of olfactory behavior. Our findings reveal the existence of a complex signaling network that controls odorant detection by C. elegans. http://repub.eur.nl/res/pub/13022/ 2002-03-01T00:00:00Z Caenorhabditis elegans has two heterotrimeric G-protein gamma subunits, gpc-1 and gpc-2. Although GPC-1 is specifically expressed in sensory neurons, it is not essential for the detection of odorants or salts. To test whether GPC-1 is involved in sensory plasticity, we developed a water soluble compound adaptation assay. The behaviour of wild-type animals in this assay confirms that prolonged exposure to salts can abolish chemo-attraction to these compounds. This process is time and concentration dependent, partly salt specific and reversible. In contrast, gpc-1 mutant animals show clear deficits in their ability to adapt to NaAc, NaCl and NH4Cl, but normal wild-type adaptation to odorants. Two other loci previously implicated in odorant adaptation, adp-1 and osm-9, are also involved in adaptation to salts. Our finding that G proteins, OSM-9 and ADP-1 are involved in taste adaptation offer the first molecular insight into this process.