Huitema K et al. (2004) EMBO J "Identification of a family of animal sphingomyelin synthases."

Brouwers JF, Huitema K, Holthuis JC, van den Dikkenberg J

[EMBO J, 2004]

Sphingomyelin (SM) is a major component of animal plasma membranes. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, yielding diacylglycerol as a side product. This reaction is catalysed by SM synthase, an enzyme whose biological potential can be judged from the roles of diacylglycerol and ceramide as anti- and proapoptotic stimuli, respectively. SM synthesis occurs in the lumen of the Golgi as well as on the cell surface. As no gene for SM synthase has been cloned so far, it is unclear whether different enzymes are present at these locations. Using a functional cloning strategy in yeast, we identified a novel family of integral membrane proteins exhibiting all enzymatic features previously attributed to animal SM synthase. Strikingly, human, mouse and Caenorhabditis elegans genomes each contain at least two different SM synthase (SMS) genes. Whereas human SMS1 is localised to the Golgi, SMS2 resides primarily at the plasma membrane. Collectively, these findings open up important new avenues for studying sphingolipid function in animals.

Steven J Husson et al. (2009) European Worm Neurobiology Meeting "Optogenetics tools to dissect a nociceptive neuronal network and neuropeptide signalling pathways in C. elegans"

Jana Liewald, Alexander Gottschalk, Liliane Schoofs, Steven J Husson, Martin Brauner, Wagner Steuer Costa

[European Worm Neurobiology Meeting, 2009]

Light-gated cation channels such as the blue light-activated depolarizing Channelrhodopsin-2 (ChR2), allow optical activation of individual neurons of live and behaving C. elegans at the millisecond time-scale in a non-invasive manner (Nagel et al., 2005, Zhang et al., 2007, Liewald et al., 2008). This optogenetics approach paves the way for further functional dissection of peptide signalling pathways or individual neuronal networks in a detail that is not possible in higher organisms. The huge advantage is that we can specifically stimulate the sensory input neurons, while other potentially contributing neurons are kept silent. The sensory PVD neurons that envelop the nematode with highly branched dendritic arbors are involved in harsh touch nociception. Expression and activation of ChR2 in PVD results in a forward escape movement and sometimes a reversal. These results are in line with the fact that the PVD neurons make synaptic contacts with the locomotory command interneurons PVC and AVA that regulate forward and backward movement, respectively. Using electrophysiology, we will assess the physiology of the PVD cells, as well as the downstream interneurons in response to photoactivation, while the involvement of different ion channels, receptors or neurotransmitters will be assessed by RNAi. We are also investigating the flp-15 and nlp-38 neuropeptidergic signalling pathways by optogenetics. Neuropeptide release can be triggered by photo-activating the respective neurons in an acute fashion while effects on behaviour can be observed at the same time. This way, we can correlate neuropeptide action with acute behavioural changes or effects, about which very limited knowledge is currently available in any system. References Liewald JF, Brauner M, Stephens GJ, Bouhours M, Schultheis C, Zhen M, and Gottschalk A (2008) Optogenetic analysis of synaptic function. Nat Methods, 5, 895-902. Nagel G, Brauner M, Liewald JF, Adeishvili N, Bamberg E, and Gottschalk A (2005) Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr Biol, 15, 2279-2284. Zhang F, Wang LP, Brauner M, Liewald JF, Kay K, Watzke N, Wood PG, Bamberg E, Nagel G, Gottschalk A, and Deisseroth K (2007) Multimodal fast optical interrogation of neural circuitry. Nature, 446, 633-639.

Husson, Steven J et al. (2009) International Worm Meeting "Optogenetics tools to dissect small neuronal networks and neuropeptide signalling systems."

Schedletzky, Thorsten, Brauner, Martin, Gottschalk, Alexander, Husson, Steven J, Schoofs, Liliane, Erbguth, Karen, Schultheis, Christian, Liewald, Jana

[International Worm Meeting, 2009]

Light-gated ion channels or pumps such as the blue light-activated depolarizing Channelrhodopsin (ChR2) and the yellow light-driven hyperpolarizing Halorhodopsin (HR) allow optical activation or inhibition in muscles and neurons of live and behaving C. elegans (Zhang et al., 2007). Furthermore, inward currents evoked by either ChR2 or HR, as well as muscle currents in response to activating ChR2 in motor neurons, can be directly measured by electrophysiology, while photo-evoked body contraction or elongation of the animal could be monitored at the behavioural level (Nagel et al., 2005; Liewald et al., 2008). These state-of-the-art technologies pave the way for further functional dissection of individual neuronal networks in a detail that is not possible in higher organisms. Doing so, we are investigating some defined neuropeptidergic signalling pathways. Neuropeptide release can be triggered by photo-activating the respective neurons in an acute fashion while effects on behaviour can be observed at the same time. This way, we can correlate neuropeptide action with acute behavioural changes or effects, about which very limited knowledge is currently available in any system. While higher organisms display millions of contributing neurons, only a handful of neurons take part in individual neuronal networks in C. elegans. This opens the possibility to study the contribution of each neuron to the function of a small network, for example involved in nociception. The huge advantage of our optogenetics tools is that we can specifically stimulate the sensory input neurons, while other potentially contributing neurons are kept silent. The involvement of different ion channels, receptors or neurotransmitters can be assessed by using different genetic backgrounds, while the physiological properties of each individual neuron will be monitored by electrophysiology. Liewald JF, Brauner M, Stephens GJ, Bouhours M, Schultheis C, Zhen M, and Gottschalk A (2008) Optogenetic analysis of synaptic function. Nat Methods, 5, 895-902. Nagel G, Brauner M, Liewald JF, Adeishvili N, Bamberg E, and Gottschalk A (2005) Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr Biol, 15, 2279-2284. Zhang F, Wang LP, Brauner M, Liewald JF, Kay K, Watzke N, Wood PG, Bamberg E, Nagel G, Gottschalk A, and Deisseroth K (2007) Multimodal fast optical interrogation of neural circuitry. Nature, 446, 633-639.

Segade F et al. (2006) Arch Otolaryngol Head Neck Surg "Association of the FBXO11 gene with chronic otitis media with effusion and recurrent otitis media: the Minnesota COME/ROM Family Study."

Daly KA, Brown M, Rich SS, Allred D, Bowden DW, Cox M, Brown SD, Segade F, Hardisty-Hughes RE, Hicks PJ

[Arch Otolaryngol Head Neck Surg, 2006]

OBJECTIVE: The FBXO11 gene is the human homologue of the gene mutated in the novel deaf mouse mutant jeff (Jf), a single gene model of otitis media. We have evaluated single nucleotide polymorphisms (SNPs) in the FBXO11 gene for association with chronic otitis media with effusion/recurrent otitis media (COME/ROM). DESIGN: A total of 13 SNPs were genotyped across the 98.7 kilobases of genomic DNA encompassing FBXO11. Data were analyzed for single SNP association using generalized estimating equations, and haplotypes were evaluated using Pedigree Disequilibrium Test methods. PATIENTS: The Minnesota COME/ROM Family Study, a group of 142 families (619 subjects) with multiple affected individuals with COME/ROM. MAIN OUTCOME MEASURES: Genetic association of COME/ROM with polymorphisms in FBXO11. RESULTS: The FBXO11 SNPs are contained in a single linkage disequilibrium haplotype block. Ten of the 13 SNPs were sufficiently polymorphic in the sample to permit analysis. In univariate genetic analysis, 1 reference SNP (hereinafter rs) (rs2134056) showed nominal evidence of association to COME/ROM (P = .02), and 2 SNPs approached significance (rs2020911, P = .06; rs3136367, P = .09). In multivariable analyses, including known risk factors for COME/ROM (sex, exposure to smoking, attending day care centers, no prior breastfeeding, and having allergies), the evidence of independent association was reduced for each SNP (eg, rs2134056, from P = .02 to P = .08). In subsequent analyses using the Pedigree Disequilibrium Test, the association of FBXO11 SNP rs2134056 (P = .06) with COME/ROM was confirmed. Incorporating multiple SNPs in 2- and 3-locus SNP haplotypes, those haplotypes containing rs2134056 also exhibited evidence of association of FBXO11 and COME/ROM (P values ranging from .03 to .10). CONCLUSION: We have observed evidence consistent with an association between polymorphisms in FBXO11, the human homologue of the Jeff mouse model gene, and COME/ROM.

Ellen Nollen et al. (2003) International Worm Meeting "Genome-wide RNAi screen for genes involved in polyglutamine aggregate formation"

Ellen Nollen, Susana Garcia, Richard I Morimoto, Gijs van Haaften, Ronald H Plasterk

[International Worm Meeting, 2003]

Accumulation of polyglutamine containing proteins into intracellular aggregates is associated with various CAG trinucleotide expansion disorders, including neurodegenerative diseases such as Huntingtons disease and spinocerebellar ataxias. The aggregation properties of polyglutamine proteins are directly related to the length of the polyglutamine stretch. With polyglutamine stretches above a threshold of approximately 30 glutamine residues, the aggregation rate increases with increasing numbers of glutamine residues. The length-dependent kinetics of aggregation recapitulates the length-dependent increase in cellular toxicity and age of onset of disease. Expression of polyglutamine stretches of 0, 24, 33, 35, 40, 44, and 82 glutamine residues as YFP-fusion proteins under control of the muscle specific unc-54 promoter in C. elegans reconstitutes the length and age dependence of aggregation. 1 Whereas worms expressing YFP-fusions with polyglutamine stretches up to 24 (Q24) show a diffuse YFP staining in all muscle cells, Q82 animals show a punctate staining in most of the cells. Interestingly, all Q lengths show variability in aggregation within individual animals, depending on the cell and the age of the worm, which is influenced by the genetic background of the worms. For example, aggregation of Q82-YFP is greatly delayed in the aging mutant age-11. This heterogeneity of aggregation suggests that genes exist that influence the formation of polyglutamine aggregates. We are using a genome-wide RNAi screen to identify genes involved in polyglutamine aggregation. In a candidate gene approach with RNAi against genes encoding molecular chaperones and molecules involved in proteins degradation, we have already identified genes that may play a role in aggregate formation. 1Morley JF, Brignull HR, Weyers JJ, Morimoto RI. Proc Natl Acad Sci U S A 2002 99(16):10417-22

Anusha Narayan et al. (2007) International Worm Meeting "Transfer at C. elegans synapses."

Tod Thiele, Gilles Laurent, Paul Sternberg, Anusha Narayan, Shawn Lockery

[International Worm Meeting, 2007]

Neural circuits in C. elegans have been studied using light and electron microscopic techniques, focal laser ablations and, more recently, calcium imaging techniques. For a clearer functional understanding of these circuits, however, some knowledge of the rules of synaptic information transfer is required. How is the dynamic range of the post-synaptic neuron set? What are the mechanisms for synaptic integration and gain control? Questions such as these can best be answered by monitoring or controlling connected pre- and post-synaptic neurons simultaneously. We chose to focus on the synapses between the AFD/ASER and AIY neurons, since the functional relevance of these neurons has been established and there is anatomical evidence for synapses between them. Channelrhodopsin-2 (chR2) is a light activated cation channel with fast kinetics (order of milliseconds1). We express chR2 under a neuron-specific promoter2 in the presynaptic neuron, and use whole-cell patch-clamp recording techniques to monitor membrane voltage or currents in the postsynaptic neuron. We are first calibrating the response to light of chR2-expressing neurons. Currently, we are calibrating this light response in worms expressing chR2 in ASER. We have observed depolarizations of 10-30 mV in response to light (450-490 nm) in current clamp, and inward currents of 5-10 pA in voltage-clamp. We have also seen evidence of spontaneous synaptic activity, in the form of discrete synaptic events (potentials or currents) with different reversal potentials (some depolarizing, others Cl-dependent). We are beginning to characterize the ASER-AIY synapse, and will then move on to the AFD-AIY synapse. References 1. Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K. Millisecond-timescale, genetically targeted optical control of neural activity, Nat. Neurosci. 8 (2005), pp. 1263-1268. 2. Nagel G, Brauner M, Liewald JF, Adeishvili N, Bamberg E, Gottschalk A. Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr. Biol. 15 (2005), pp. 2279-2284.

Hidehito KUROYANAGI et al. (2001) International C. elegans Meeting "C. elegans SR protein kinase, SPK-1, is required for germline proliferation and maturation."

Masatoshi Hagiwara, Hidehito KUROYANAGI

[International C. elegans Meeting, 2001]

SR-protein kinases (SRPKs) and their substrates, the serine/arginine-rich pre-mRNA splicing factors (SR proteins), are key components of splicing machinery and are well conserved across phyla. Despite extensive biochemical investigation, the physiological functions of SRPKs remain unclear. In C. elegans , one SRPK gene and six SR protein genes have been reported. Functional analyses by us and other labs utilizing RNAi technique revealed that one member of SR protein family, CeSF2/ASF, is essential for embryogenesis (1,2), while other SR proteins play redundant roles (2,3); spk-1 is required for early embryogenesis (1,2). However, we have also demonstrated that spk-1 is required in germline proliferation and gametogenesis utilizing various RNAi protocols (1). Here we report the isolation and analyses of a deletion mutant within the spk-1 gene. spk-1 mutants are comparable to Wild-Type in locomotion and body size. However, spk-1 mutants shows recessive fully penetrated phenotype of sterility in both hermaphrodites and males. In spk-1 hermaphrodites germlines are underproliferated and lack oogenesis. spk-1 males have comparable number of germ nuclei in a gonadal arm, suggesting deficiency in sperm maturation. These observations indicate that zygotic expression of spk-1 is required for germline proliferation in hermaphrodites and maturation in both hermaphrodites and males. In order to analyze the epistasis between germline proliferation deficiency in spk-1 mutant and ectopic or tumorous proliferation of germline in gld-2 (h292) and gld-1; gld-2 double mutant, we generated double and triple mutants. Certain population of gld-2 (h292); spk-1 showed ectopic proliferation of germ nuclei in the most proximal region of the gonad, indicating that gld-2 (h292) is epistatic. On the other hand, gld-2 (q497) gld-1 (q485); spk-1 showed Glp phenotype, indicating that spk-1 is epistatic. With these characteristics, spk-1 mutant may serve as a useful tool to analyze the molecular mechanisms in sex-determination, proliferation and maturation of C. elegans germline. 1: Kuroyanagi H, Kimura T, Wada K, Hisamoto N, Matsumoto K, Hagiwara M. SPK-1, a C. elegans SR protein kinase homologue, is essential for embryogenesis and required for germline development. Mech Dev. 2000; 99(1-2): 51-64. 2: Longman D, Johnstone IL, Caceres JF.@ Functional characterization of SR and SR-related genes in Caenorhabditis elegans . EMBO J. 2000; 19(7): 1625-37. 3: Kawano T, Fujita M, Sakamoto H. Unique and redundant functions of SR proteins, a conserved family of splicing factors, in Caenorhabditis elegans development. Mech Dev. 2000; 95(1-2): 67-76.

Squire MD et al. (1994) Worm Breeder's Gazette "Molecular Cloning and Functional Expression of a C. elegans Nicotinic Acetylcholine Receptor Subunit (acr-2)."

Baylis HA, Sattelle DB, Squire MD, Tornoe C, Fleming JT, Barnard EA

[Worm Breeder's Gazette, 1994]

MOLECULAR CLONING AND FUNCTIONAL EXPRESSION OF A C. ELEGANS NICOTINIC ACETYLCHOLINE RECEPTOR SUBUNIT (ACR-Z). Michael Squire, Camilla Torn0e, Howard Baylis, John Fleming1, Eric Barnard2 and David Sattelle. The Babraham Institute Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, UK. 1 MGH Cancer Centre, Harvard University, 2Molecular Neurobiology Unit, Royal Free Hospital School of Medicine, London, UK. As part of an ongoing program to study neurotransmiter receptors in C. elegans we set out to isolate nicotinic acetylcholine receptor (nAChR) subunit clones from C. elegans by screening a C. elegans genomic DNA library with a probe derived from the Drosophila ARD gene (a non-a nAChR subunit clone, kindly supplied by Dr I Hermans-Borgmeyer). A number of putative nAChR subunit clones were isolated. The resulting C elegans clones were mapped to 8 loci on the physical map of the genome (Fleming JT, PhD Thesis, University of Cambridge). We extended our studies on one of these loci (represented by the clone JF#WA56) now called acr-2 which is located between svp-7 and unc-6 on the X chromosome. Iev-9 is also located in this part of the chromosome but cosmids carrying acr-2 do not rescue lev-9. A full length acr-2 cDNA has been isolated and sequenced and the genomic structure is being determined. The cDNA encodes a putative non-a subunit of a nicotinic acetylcholine receptor which shows many of the conserved features of vertebrate and invertebrate non-alpha nAChR subunits, for instance the four putative transmembrane domains and the cysteine delimited extracellular loop. Amongst other nAChR subunits acr-2 is most similar to the Drosophila ARD subunit to which it shows 49% identity. In order to investigate the function of the subunit acr-2, cRNA was produced by in vitro transcription and micro-injected into Xenopus oocytes. When expressed alone acr-2 shows no levamisole gated channel activity. Unusually, such activity is observed when acr-2 is co-expressed with another C. elegans non-a subunit (lev-1, Fleming et al, in preparation). Such activity is normally only observed in the presence of an a-subunit. Whether this activity is the result of endogenous a subunit activity or reflects novel behaviour by the C. elegans subunits remains to be determined. When co expressed with a C. elegans a subunit (unc-38, which is itself unable to form functional homo-oligomers) acr-2 contributed to the formation of a functional (a, non-a) channel. In both of these cases the levamisole induced current was inhibited by mecamylamine.

Ederle S et al. (1999) Worm Breeder's Gazette "Searching the genome for DNA sites recognized by binding proteins."

Ederle S, La Volpe A, Pulitzer JF

[Worm Breeder's Gazette, 1999]

In the past two decades much effort in biology has gone into cloning and sequencing of genes. The accomplishment of genome sequencing projects, such as that of Caenorhabditis elegans, is leading to a new scenario, where all genomic information is available, and the remaining challenge is to interpret it in biological terms. It is becoming essential to develop strategies to link non coding sequence to function. We have set up a one hybrid system kit, using C. elegans as model, to search the DNA for sites of binding of structural and regulatory proteins. We have constructed yeast strains in which C. elegans non-coding DNA sequences (baits) are inserted, in place of the UAS, upstream the two test genes HIS-3 and LacZ. The recipient strain is a diploid ura- yeast strain carrying a deletion of the HIS-3 gene on one chromosome and a wild type copy on the other. We have chosen to use diploid strains because the most frequent cause of HIS spontaneous reversion is the transposition of a Ty element upstream the HIS coding region; in diploid strains the transposable element Ty is quiescent. Our bait sequences are cloned upstream the HIS-3 gene in an integrative plasmid and transformed in yeast after restriction digestion. The integrative plasmid had been constructed by inserting a polylinker into the partially deleted HIS-3 promoter for ease of manipulation and it contains homology cassettes appropriate for gene replacement and to facilitate the screening of the integrants. It contains, in fact, HIS-3 flanking regions for proper integration of the plasmid, and the selectable marker URA3 in such a position to be deleted, after integration, via homologous recombination together with the wild type copy of HIS-3. Such event can be selected by plating the recombinant strains on FOA (5-fluor-orotic acid), only the ura- cells will growth in such conditions. Most pop-outs occur by recombination in the extensive tracts of homology between the recombinant HIS-3 sequence and the wild type. The obtained yeast strains are auxotrophic for histidin in the presence of only 5mM aminotriazol unless a protein binds to the bait sequence and activates transcription by mean of an activation domain such as that of GAL-4. This is the selection tool for the screening of our cDNA library (described in Ederle et al. European C. elegans Meeting 1996). In all cases the second test gene (LacZ) is carried as an episome by the cell. We have tested our tools by cloning the GATA-box as DNA bait and successfully screening our library. We are now proceeding in using this kit for the analysis of gene promoter regions and other DNA sequences potentially target of binding proteins. 1) Ederle S, De Felice B, Pulitzer JF, La Volpe A (1996) A C. elegans cDNA library for one hybrid/two hybrid system screening in yeast. 2nd European C. elegans Meeting Mont Saint-Odile, France. p.23.