Tina Burton et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Multiple FLPs are Involved in Locomotion and Egg-laying Behaviors"

Tina Burton, Lawrence Ha, Jenny Chang, Chris Li, Diego Levy, Adewale Olajubelo

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Neuropeptides are involved in all complex behaviors in mammals. FMRFamide and related peptides (FaRPs) all share a common Arg-Phe-amide at their C-termini, and are found in organisms throughout the animal kingdom, where they are involved in a variety of behaviors, including cardioactivity, pain modulation, and reproduction. At least 29 flp genes encode over 70 FaRPs in C. elegans. Each flp gene encodes a unique set of peptides and is expressed in a distinct set of cells. Most, if not all, of the predicted FLP peptides are produced, and signal through G protein coupled receptors. To determine the role of the flp genes, we have been isolating animals carrying deletions in the different flp genes, and examining the mutants for locomotory and egg-laying defects. To date, we have isolated 11 flp mutants. Surprisingly, eight of the flp mutants have defects in locomotion and seven are egg-laying defective, suggesting that many FLP peptides modulate these behaviors. Furthermore, five flp mutants are resistant to serotonin-induced sluggishness. We have also been characterizing mutants carrying deletions in G protein coupled receptors, including some of the FLP receptors. These animals also have defects in locomotion and egg laying. The potential complexity with which the FLPs modulate certain behaviors is enormous and awaits further examination.

Christopher J Cronin et al. (2004) West Coast Worm Meeting "The Combined UC San Diego/Caltech Quantitative Behavior Analysis System"

William Schafer, Christopher J Cronin, Zhaoyang Feng, Paul W Sternberg

[West Coast Worm Meeting, 2004]

California Institute of Technology and University of California, San Diego have each developed practical, automated movement analysis systems to quantify aspects of worm behavior and morphology (see previous meeting abstracts). To better serve the Worm Research Community, we have combined our development efforts and merged our independent system designs to create a single, unified behavior analysis system. Our combined system provides the researcher with all of the quantification tools and database functionality of the individual systems, plus provides the framework for continued future development to satisfy the needs of the community. A list of the necessary hardware is available upon request.

Christopher J Cronin et al. (2005) International Worm Meeting "The Combined UC San Diego/Caltech Quantitative Behavior Analysis System"

William R Schafer, Zhaoyang Feng, Kuang-man Huang, Paul W Sternberg, Christopher J Cronin

[International Worm Meeting, 2005]

California Institute of Technology and University of California, San Diego have each developed practical, automated movement analysis systems to quantify aspects of worm behavior and morphology (see An imaging system for standardized quantitative analysis of C. elegans behavior, Zhaoyang Feng, et al, BMC Bioinformatics 2004, 5:115, An automated system for measuring parameters of nematode sinusoidal movement, Christopher J Cronin, et al, BMC Genetics 2005, 6:5, and previous meeting abstracts). We have consolidated our development efforts and merged our independent system designs to create a single, unified behavior analysis system. The combined system provides researchers with all of the quantification tools and database functionality of the individual UCSD and Caltech systems, but also establishes a common, open software and hardware foundation for continued development by Caltech, UCSD and the community. Current efforts include expanding system capabilities for the automated analysis of mating behavior with the challenge of tracking multiple moving individuals, both in contact with and separated from each other.

Siddiqui ZK et al. (1997) International C. elegans Meeting "SUPPRESSION OF THE TEMPERATURE SENSITIVE MUTANTS EMB-8 AND HIM-14 BY DPY-10"

Ali MY, Miwa J, Siddiqui ZK, Hori F, Nishikawa K, Hori Y, Siddiqui SS

[International C. elegans Meeting, 1997]

Earlier, some alleles of different genes glp-1 (Kimble. J.) and emb-5 (K. Nishiwaki) have been shown to be suppressed by mutants in dpy-10(e128). The dpy-10 gene encodes a collagen which is similar to the collagen encoded by dpy-2 (Levy et al., 1993). Here we have examined the suppression of two temperature sensitive maternal effect embryonic mutants by the dpy-10(e128) mutation. Mutants in emb-8 (hc69) have a number of growth defects, such as embryonic cell division arrest (early to mid cleavage), at 25 C, eggs are osmotically sensitive, and have slow cell division rate (Also see abst. by Khan et al.). The double mutants of emb-8(hc69); dpy-10(e128) showed remarkable improvement in hatching efficiency than the single emb-8 homozygous mutants at 25 C. Similarly, mutants in him-14(it44) that produce 2-40% males, 95% dead eggs (Kemphues et al., 1988), when combined with dpy-10(e128) show suppression of the non-disjunction defect, reducing the number of males, and increasing the brood size of the progeny at 25 C. Interestingly, a high level of the HSP-20 transcript is observed in dpy-10 mutants at 15 and 25 C (K. Nishiwaki, unpublished, suggesting a general mechanism for the suppression of ts mutnts through a possible role of the HSP-20 in dpy-10 mutants.

RV Aroian et al. (1999) International C. elegans Meeting "Using C. elegans to Teach Embryonic Development to Undergraduates"

G Wienhausen, RV Aroian, D Johnson

[International C. elegans Meeting, 1999]

We have begun to use C. elegans as a model system in our undergraduate embryology laboratory class at the University of California, San Diego. In the past, this 10 week class has used Sea Urchin, Xenopus, Chick, and other non-genetic systems to demonstrate to students embryonic development. In this poster we will outline the 6 new weeks of C. elegans modules. These modules allow the students to use advanced techniques to explore widely applicable principals of embryonic development. These techniques include wild-type lineage analysis, mutant lineage analysis, use of specific inhibitors to alter embryonic development, immunofuorescence analysis of wild type and mutants using antibodies to various cell biology and developmental markers, genome searching, and knocking out genes using RNA-mediated interference. Our goal is to one day publish an article on our results in the Worm Breeder's Gazette. The embryonic development of more advanced model organisms such as Xenopus and chick are later used to compare and contrast to C. elegans. This work is greatly enhanced by the cooperativity of the C. elegans community in the sharing of antibodies, mutants, and other reagents such as GFP strains. Last year we used protocols, stains, and antibodies from to Geraldine Seydoux, Craig Mello, Pete Okkema, Michel Labouesse, Bruce Bowerman and the CGC. Thanks to them and others that have generously shared their reagents.

Naomi Levy-Strumpf et al. (2005) International Worm Meeting "Directional decisions-The vab-8 unc-40 connection."

Joseph G Culotti, Naomi Levy-Strumpf

[International Worm Meeting, 2005]

C. elegans comprises 6 touch neurons responsible for mediating touch responses: ALML/R, PLML/R, AVM and PVM. The later two send an axon ventrally in response to the Netrin guidance cue. The ALMs on the other hand are refractory to the Netrin and send a process longitudinally. Ectopic expression of UNC-5 in the ALMs results in dorsal reorientation of their axons in an unc-6 dependent manner (1), suggesting that the ALMs contain the necessary machinery to respond to UNC-6 signal. Moreover, the ectopic UNC-5 induced axon rerouting, was shown to be highly dependent on unc-40 (2), demonstrating that not only is UNC-40 expressed in the ALMs (3), but that it can also be functional in those neurons. In order to get a better insight into the mechanisms that guide longitudinal versus circumferential migrations, we overexpressed an UNC-40GFP fusion protein in the touch neurons using the mec-7 promoter. The most common phenotype we observe is that the ALM process, which normally migrates longitudinally, reorients itself towards the ventral cord (4). Interestingly, we also found that the overexpression of unc-40(+) resulted in 180<sym05> reversal of the ALM process. While the ventral rerouting of the ALM is dependent on unc-6(+), the posterior reorientation of the ALM axon is independent of the UNC-6 netrin guidance cue. Similar to UNC-40, expression of mec-3::VAB-8L in the touch neurons also resulted in the rerouting of the ALM axons posteriorly (5). This prompted us to check whether vab-8 and unc-40 might work together to mediate the posterior rerouting of the ALM axon. We had found that although vab-8 mutations do not suppress the UNC-40 induced rerouting, unc-40 does suppress the VAB-8L induced posterior rerouting. This was later also found to be true for sax-3 (6), implying that VAB-8L might function by regulating guidance cue receptors. It has been shown that VAB-8L acts upstream of MIG-2/RAC and UNC-73/TRIO to mediate the ALM axon rerouting response (7). The ALM reorientation resulting from the mec-7::UNC-40GFP is independent of UNC-73 suggesting the intriguing possibility that mig-2 and unc-73 act downstream of vab-8L and upstream of unc-40 to mediate longitudinal posterior migration. We are currently looking directly to see whether VAB-8L affects the localization of UNC-40. 1.Hamelin et al., (1993) Nature 364, 327-30. 2. Colavita, A. and Culotti, J. G. (1998). Dev Biol 194, 72-85. 3. Chan et al., (1996). Cell 87, 187-95. 4. Levy-Strumpf, N., and Culotti , J. G. WBG 17(1): 37 (2001). 5. Wolf et al., (1998). Neuron 20, 655-66. 6. Watari-Goshima, N., Levy-Strumpf, N., Culotti , J. G. and Garriga, G. 2004 West Coast Meeting abstract 71. 7. Wolf, F. W. and Garriga, G. International meeting abstract 916.

Shen Z-Z et al. (1998) European Worm Meeting "Screen for body size mutants in C. elegans"

Leroi AM, Shen Z-Z, Patel MN

[European Worm Meeting, 1998]

Little is known about the molecular genetic specification of growth and body size in any metazoan. In C. elegans, a number of loci which influence the body size has been identified (Brenner, 1974). So far, these fall into three classes: First, genes involved in TGF-b signal transduction. Among them, daf-4 has been shown to encode a TGF-b receptor II (Estevez et al., 1993). Sma-2, sma-3 and sma-4 are TGF-b pathway components which are homologue to the Drosophila gene Mothers against Dpp (Savage et al., 1996). Second, genes involved in the control of cytoskeleton structure. he sequence from sma-1 has been shown to contain spectrin, a cytoskeleton component, and the sequence from lon-2 has been predicted to be regulatory proteins of cytoskeleton (Austin, pers comm.; Vellai pers comm). Finally, there are genes that affect cuticle structure. dpy-2, dpy-7 and dpy-10 encode collagen (Levy et al., 1993; Johnstone et al., 1992) and clearly are very small in size. How these pathways interact to control body size is unclear. In our attempt to understand the controls of body growth in C. elegans we conducted EMS mutagenesis for new body size loci. We are especially interested in giant mutants of which few have hitherto been found. We screened 50,000 genomes of Bristol N2 (table 1). We have begun measuring, backcrossing and mapping these mutants with a view towards cloning them. Some of the giants are 50-75% larger than wildtype (by volume). There may be a continuum between lons and giants.

Cunningham K A et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "The role of AMP-activated protein kinase in C. elegans feeding regulation"

Ashrafi K, Cunningham K A

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

The evolutionarily conserved AMP-activated protein kinase (AMPK) is a critical energy sensor in eukaryotes. In general, upregulation of AMPK leads to shutting down energy consuming processes and activation of energy generating processes. In C. elegans, the catalytic alpha subunit, encoded by aak-2, has been shown to have roles in regulating fat storage during the hibernating dauer state as well as longevity in both dauer and non-dauer animals.1,2 We found that aak-2 deficient animals have increased feeding rate relative to wild type animals, and reconstitution studies suggest that expression in pharyngeal muscle and the nervous system is sufficient for restoration of wild type feeding rate to aak-2 mutant animals. Moreover, using various neuron specific promoters, we found that neural sufficiency of aak-2 for feeding regulation can be restricted to the small subset of neurons defined by an hlh-34 promoter. Interestingly, hlh-34 encodes for a basic helix-loop-helix transcription factor whose mammalian counterpart, sim-1, has known developmental and regulatory roles in specific regions of the hypothalamus. Mammals deficient in SIM-1 develop obesity and exhibit increased feeding behavior.3 Thus, the role of AAK-2 in hlh-34/sim-1 expressing cells in modulating metabolism and feeding behavior may be conserved across evolution. References: 1 Narbonne, P. and Roy R., 2009. Nature. 457, 210-213. 2 Apfeld, J., O'Connor, G, McDonagh, T., DiStefano, P.S., Curtis, R., 2004. Genes and Dev. 18, 3004-3009. 3 Michaud, J.L., Boucher, F., Melnyk A., Gauthier, F., Goshu, E., Levy, E., Mitchell, G.A., Himms-Hagen, J., Fan, C.M., 2001. Hum. Mol. Genet. 10, 1465-73.

Kamat, Shaunak et al. (2013) International Worm Meeting "Modulation of mec-10(d)-induced necrosis by ER chaperone NRA-2."

Driscoll, Monica, Yeola, Shrutika, Bianchi, Laura, Kamat, Shaunak

[International Worm Meeting, 2013]

ER chaperones play a major role in cellular homeostasis by regulating the assembly of polypeptides, intracellular Ca2+ signaling, and degradation of misfolded proteins. Here we report on an ER - resident chaperone NRA-2 that modulates hyper-activated ion channel-induced necrosis by regulating surface expression of a death-inducing DEG/ENaC channel family member subunit MEC-10(d). nra-2 was previously identified in a screen for nicotinic acetylcholine receptor (nAChR) - interacting proteins and was found to regulate the subunit composition of the AChR receptor in C. elegans muscle1. We found that loss of function of nra-2 led to a significant increase in mec-10(d) - induced necrosis in C. elegans touch receptor neurons (TRNs), and this enhancement was rescued by TRN-specific transgenic expression of nra-2. We observed that loss of nra-2 led to a significant increase in surface localization of MEC-10(d)::GFP and a significant decrease in ER localization. Electrophysiological experiments in Xenopus oocytes revealed that NRA-2 suppresses amiloride-sensitive currents induced by hyperactivated MEC channels. Our study suggests a role for NRA-2 as an ER quality control protein that inhibits surface expression of mutant MEC-10(d) channels and is thus neuroprotective against hyperactivated ion channel-induced necrosis. 1. Ruta B. Almedom, Jana F. Liewald, Guillermina Hernando, Christian Schultheis, Diego Rayes, Jie Pan, Thorsten Schedletzky, Harald Hutter, Cecilia Bouzat, and Alexander Gottschalk. An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse. The EMBO Journal, 28(17):2636-2649, July 2009.

Smejkal Gary B et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Pressure mediated scheme for isolating native proteins from Caenorhabditis elegans under mild nondenaturing conditions"

Anderson Jobriah, Smejkal Gary B, Morris Krystalynne, Thomas W Kelley, Kuo Winston P, Strader Thomas E

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

The tough exterior cuticle of Caenorhabditis elegans makes this nematode very resistant to lysis and impedes proteomic and glycoproteomic analyses. So resilient is this organism, that specimens sent into space were the only survivors of the disastrous crash of the space shuttle Columbia [1]. Proteomic analysis is particularly challenging when preservation of the native conformation and biological activity of proteins is required, thus prohibiting the use of denaturing chaotropes and detergents to enhance lysis. The dilemma is that in mild physiological buffers, nematodes can withstand hydrostatic pressure hydrostatic pressure up to 20,000 psi with a 2.3% survival rate [2]. While bead beating and sonication are commonly used to disrupt nematodes, heat generated by these methods causes denaturation of proteins, and subsequently, their loss through aggregation and precipitation. By comparison, pressure cycling technology (PCT) generates minimal heat by rapidly oscillating between high and atmospheric pressure. The ProteoSOLVE CE Native Kit was developed specifically to maximize protein yields from live nematodes under mild nondenaturing conditions. In the optimized protocol, live nematodes collected from culture are concentrated on a filter and the resulting paste is mixed with the CE SiC abrasives and transferred to a PULSE Tube. The amalgam is frozen solid by placing the tube on dry ice for ten minutes, then ground with the serrated Shredder ram insert until completely extruded through perforations of the lysis disk. The Native Reagent is added, followed by 40 pressure cycles at 35,000 psi. Protein yields were six times greater with the Shredder ram than with high pressure alone, and an order of magnitude higher when the Shredder ram was used in combination with freezing and abrasives. [1] Szewczyk, N.J. et al. (2005). Astrobiology, 5, 690-705. [2] Smejkal, G.B. et al. (2008). Plant and Animal Genome XVII Conference, San Diego, CA.